drug discovery from marine cyanobacteria symploca …€¦ · drug discovery from marine...
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DRUG DISCOVERY FROM MARINE CYANOBACTERIA SYMPLOCA SPP. AND PHORMIDIUM SPP.: NOVEL STRUCTURES AND BIOACTIVITIES OF SECONDARY
METABOLITES
By
LILIBETH APO SALVADOR
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
2013
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© 2013 Lilibeth Apo Salvador
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To my mom, my brothers, and my husband
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ACKNOWLEDGMENTS
I am greatly indebted to my mentor Professor Hendrik Luesch, for giving me the
opportunity to be a part of his group and work on numerous research projects. I
appreciate his immense support, guidance and patience throughout my graduate
studies. I am also thankful to Dr. Luesch for helping me recognize my capabilities and
bringing out more than my best. I appreciate his insights and critiques, which all
contributed to the success of this study.
I also thank Professor Sixue Chen, Professor Margaret O. James and Professor
Xin Qi, for graciously agreeing to be members of my dissertation committee. I
appreciate their insightful comments in the preparation of this manuscript and their
timely response to my inquiries.
I am grateful to our collaborators, Dr. Valerie J. Paul and Dr. Jason S. Biggs, for
providing the cyanobacteria collections and lending their expertise. I thank them for their
support and fruitful discussions during the preparation of our manuscripts for
publication. I thank all the former and current members of Val and Jason’s group,
together with Ms. Gudrun Schelegel, who contributed to the collection and extraction of
cyanobacteria samples for screening and dereplication. I am also grateful to Diane
Littler for identifying several of the sample collections, the Fort Zachary Taylor State
Park and J. Quiñata of Cetti Bay Agat Station for permission to obtain the samples.
I acknowledge Dr. Jean Jakoncic, Dr. David A. Ostrov, and Ms. Kanchan Taori,
who conducted the experiments and data analysis for the cocrystallization of
lyngbyastatin 7–porcine pancreatic elastase. I thank them for their help in the discussion
and preparation of figures for the X-ray analysis. I also thank the Bioinformatics Core of
the Interdisciplinary Center for Biotechnology Research for assistance in the
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transcriptome data analysis and heat map generation. I am also grateful to Mr. James
Rocca for lending his expertise and passion for NMR. I thank Jim for his patience and
invaluable help in NMR training and troubleshooting. More so, I am grateful for his
friendship and constant reminder to smell the roses.
I would also like to acknowledge current and former members of the Luesch Lab
– Ms. Fatma Al-Awadhi, Ms. Michelle Bousquet, Ms. Weijing Cai, Dr. Qiyin Chen, Dr.
Jason C. Kwan, Dr. Yanxia Liu, Dr. Susan Matthew, Ms. Kamolrat Metavarayuth, Ms.
Rana Montaser, Dr. Ranjala Ratnayake, Dr. Rui Wang and Dr. Wei Zhang – for their
help at various stages of my graduate studies; from settling down and starting with my
projects, moving forward with my research and finishing up. I appreciate the technical
expertise that everyone has provided as well as insightful discussions and unforgettable
experiences during our collection trips. I also thank the staff of the Medicinal Chemistry
Department – Mr. David Jenkins, Ms. Jan Kallman and Mr. Brian Karcinski – for making
sure that all necessary requirements for my studies were taken care of.
I am also grateful to my friends and prayer warriors, Dr. Ma. Pythias Espino, Mr.
Krisnakanth Kondabolu, Dr. Francesca Diane Liu, Dr. Mario Edgar Moral and Dr.
Ranjala Ratnayake. They shared with me the joys and hardships of graduate school,
made Gainesville more memorable and lent their advice on embracing this endeavor,
working it through and taking the plunge to a new career. I also acknowledge my former
supervisors, Professor Gisela P. Concepcion and Professor Amelia P. Guevara, for
introducing me to natural products chemistry, for constantly believing in my capabilities,
for their support and encouragement.
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Finally, I thank my whole family for tons of love, patience, understanding and
faith throughout this journey. I thank my mother, Emilia, who serves as my inspiration
and role model for hardwork and perseverance. I am grateful to my husband, Joeriggo,
for sharing this dream with me and for being by my side from day one of graduate
school. Joeriggo has been one of my toughest critics, but he has also been the most
patient. And to my Shepherd, I am very much thankful; and all that is mine to give is for
your glory.
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TABLE OF CONTENTS
page
ACKNOWLEDGMENTS .................................................................................................. 4
LIST OF TABLES .......................................................................................................... 10
LIST OF FIGURES ........................................................................................................ 11
LIST OF ABBREVIATIONS ........................................................................................... 13
ABSTRACT ................................................................................................................... 19
CHAPTER
1 GENERAL INTRODUCTION .................................................................................. 21
Natural Products in Drug Discovery ........................................................................ 21
Drugs from the Sea ................................................................................................. 22 Marine Cyanobacteria: Source Organisms of Novel Molecules .............................. 24 Mechanism of Action of Bioactive Cyanobacterial Metabolites ............................... 25
Interference with Microtubule Dynamics ........................................................... 25 Inhibition of Histone Deacetylase ..................................................................... 26
Inhibition of Proteases ...................................................................................... 27 Objectives and Specific Aims of the Study .............................................................. 29
2 PROBING THE CHEMICAL SPACE AND ANTIPROLIFERATIVE ACTIVITIES OF CYANOBACTERIAL COLLECTIONS ............................................................... 35
Introduction ............................................................................................................. 35
Screening of Cyanobacteria Collections ................................................................. 37 Antiproliferative Assay as Preliminary Screening for Bioactivity ....................... 38
Dereplication using an HPLC-MS Approach ..................................................... 38 Prioritization of Sample Collections .................................................................. 39
Validation of the Dereplication Method ................................................................... 40
Conclusion .............................................................................................................. 40 Experimental Methods ............................................................................................ 41
General Experimental Procedures ................................................................... 41 Biological Material ............................................................................................ 41
HPLC-MS Profiling ........................................................................................... 42 Cell Viability Assay ........................................................................................... 42 Validation of Dereplication Method ................................................................... 43
3 POTENT ELASTASE INHIBITORS FROM CYANOBACTERIA: STRUCTURAL BASIS AND MECHANISMS MEDIATING CYTOPROTECTIVE AND ANTI-INFLAMMATORY EFFECTS IN BRONCHIAL EPITHELIAL CELLS ...................... 48
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Introduction ............................................................................................................. 48
Isolation and Structure Elucidation ......................................................................... 50 Enzyme Inhibition ................................................................................................... 54
Molecular Basis for Elastase Inhibition by Lyngbyastatins and Symplostatins........ 55 Biological Activity Evaluation .................................................................................. 57
Cytoprotective Effects of Symplostatin 5 (1) Against Elastase-Induced Antiproliferation and Apoptosis ...................................................................... 57
Cytoprotective Effects of Symplostatin 5 (1) Against Elastase-Induced Cell Detachment and Morphological Change ....................................................... 59
Attenuation of Global Transcript Changes Induced by Elastase ....................... 62 Conclusion .............................................................................................................. 65 Experimental Methods ............................................................................................ 65
General Experimental Procedures ................................................................... 65
Biological Material ............................................................................................ 66 Extraction and Isolation .................................................................................... 66
Enantioselective Analysis ................................................................................. 67
In Vitro Protease Assay .................................................................................... 69 Cocrystallization of Lyngbyastatin 7 with Porcine Pancreatic Elastase ............ 70 In Vitro Cellular Assays .................................................................................... 71
General cell culture procedure ................................................................... 71 Cell viability assay ...................................................................................... 71
Cell detachment and morphology change .................................................. 71 Caspase activation measurement .............................................................. 72 Measurement of sICAM-1 levels ................................................................ 72
Immunoblot analysis of mICAM-1 levels .................................................... 73 Isolation of nuclear and cytoplasmic proteins ............................................. 73
Measurement of IκBα degradation and NF-B p65 translocation............... 74 RNA isolation and reverse transcription ..................................................... 75
Real-time quantitative polymerase chain reaction (qPCR) ......................... 75 Transcriptome profiling .............................................................................. 76
4 VERAGUAMIDES A–G: CYTOTOXIC CYCLIC HEXADEPSIPEPTIDES WITH A C8-POLYKETIDE-DERIVED β-HYDROXY ACID MOIETY FROM CETTI BAY, GUAM ................................................................................................................... 100
Introduction ........................................................................................................... 100
Isolation and Structure Elucidation ....................................................................... 101 Biological Activity Studies ..................................................................................... 106 Conclusion ............................................................................................................ 108
Experimental Methods .......................................................................................... 108 Biological Material .......................................................................................... 108 Extraction and Isolation .................................................................................. 109 Hydrogenation of 7 ......................................................................................... 110
Acid Hydrolysis of Veraguamides and Enantioselective Analysis ................... 111 Methanolysis of 7 ........................................................................................... 113 Preparation of MTPA Esters of 15 .................................................................. 114
Biological Activity Assays ............................................................................... 115
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Cell viability assay .................................................................................... 115
Cell cycle analysis by flow cytometry ....................................................... 115
5 CAYLOBOLIDE B AND AMANTELIDES A AND B: ANTIPROLIFERATIVE POLYKETIDES FROM MARINE CYANOBACTERIA ........................................... 132
Introduction ........................................................................................................... 132 Isolation and Structure Elucidation ....................................................................... 133
Caylobolide B (18) .......................................................................................... 133 Amantelides A and B (19, 20) ......................................................................... 136
Configurational Analysis ....................................................................................... 138 Biological Activity Studies ..................................................................................... 139
Antiproliferative Activity .................................................................................. 139 Elucidation of the Mechanism of Action of Cyanobacterial Polyketides .......... 140
Conclusion ............................................................................................................ 142 Experimental Methods .......................................................................................... 142
General Experimental Procedures ................................................................. 142 Biological Material .......................................................................................... 143
Extraction and Isolation .................................................................................. 143 Caylobolide B (18) ................................................................................... 143 Amantelides A (19) and B (20) ................................................................. 144
Acetylation of amantelide A (19) .............................................................. 145 ESIMS/MS Fragmentation of Caylobolide B (18) and Amantelide A (19) ....... 145
Cell Viability Assay ......................................................................................... 146
6 GENERAL CONCLUSION .................................................................................... 160
APPENDIX
A CELL MORPHOLOGY AT 3 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR) .......................................................................................................... 164
B CELL MORPHOLOGY AT 6 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR) .......................................................................................................... 165
C CELL MORPHOLOGY AT 12 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR) .......................................................................................................... 166
D CELL MORPHOLOGY AT 24 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR) .......................................................................................................... 167
E ICAM1 TRANSCRIPT LEVELS AT 3 h AND 6 h .................................................. 168
F NMR SPECTRA OF ISOLATED COMPOUNDS .................................................. 169
LIST OF REFERENCES ............................................................................................. 257
BIOGRAPHICAL SKETCH .......................................................................................... 266
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LIST OF TABLES
Table page 2-1 Antiproliferative activity (IC50, nM) of known Symploca sp. metabolites ............. 47
3-1 NMR data of symplostatin 5 (1) and symplostatin 8 (4) in DMSO-d6 .................. 84
3-2 NMR data of symplostatin 6 (2) and symplostatin 9 (5) in DMSO-d6 .................. 87
3-3 NMR data of symplostatin 7 (3) and symplostatin 10 (6) in DMSO-d6 ................ 89
3-4 Antiproteolytic activity of Abu-containing cyclic depsipeptides from marine cyanobacteria ..................................................................................................... 92
3-5 Non-inflammatory elastase-inducible genes ....................................................... 93
3-6 Relevant genes involved in NOD- and MAPK- signaling pathways significantly modulated by elastase .................................................................... 94
3-7 Symplostatin 5 (1)-inducible genes potentially independent of elastase ............. 95
3-8 Reaction conditions for protease assays ............................................................ 96
3-9 Crystallography data and refinement statistics ................................................... 99
4-1 NMR data for veraguamide A (7) in CDCl3 ....................................................... 121
4-2 NMR data for veraguamide B (8) and veraguamide C (9) in CDCl3 .................. 123
4-3 NMR data for veraguamide D (10) and veraguamide E (11) in CDCl3 .............. 125
4-4 NMR data for veraguamide F (12) in CDCl3 ..................................................... 127
4-5 NMR data for veraguamide G (13) and tetrahydroveraguamide A (14) in CDCl3................................................................................................................ 129
4-6 Antiproliferative activity (IC50, µM) of natural and semisynthetic veraguamides 131
5-1 NMR data of caylobolide B (18) in DMSO-d6 .................................................... 155
5-2 NMR data of amantelide A (19) and amantelide B (20) in DMSO-d6 ................ 157
5-3 Cytotoxic activity (IC50, µM) of the isolated cyanobacterial polyketides (18–21) .................................................................................................................... 159
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LIST OF FIGURES
Figure page 1-1 Representative examples of natural products that influenced modern
medicine ............................................................................................................. 30
1-2 Marine natural products and analogs that have reached the clinic ..................... 31
1-3 The linear peptides symplostatin 1 and dolastatin 10 are potent antiproliferative agents that disrupt tubulin polymerization ................................. 32
1-4 Largazole is a cyclodepsipeptide prodrug that targets canonical histone deacetylases....................................................................................................... 33
1-5 Representative examples of non-cytotoxic metabolites from marine cyanobacteria that target proteases ................................................................... 34
2-1 Summary of chemical space and bioactivity profiles of Symploca spp. and Phormidium spp. collections ............................................................................... 44
2-2 Representative HPLC-MS profile of the simultaneous monitoring of largazole, dolastatin 10 and symplostatin 1 ........................................................................ 45
2-3 Prioritization scheme of cyanobacteria collections and the corresponding secondary metabolites isolated .......................................................................... 46
3-1 Elastase inhibitors from marine cyanobacteria and the clinically approved human neutrophil elastase inhibitor sivelestat .................................................... 78
3-2 Selectivity profile of Abu-containing cyclic depsipeptides from marine cyanobacteria ..................................................................................................... 79
3-3 Cocrystal structures of natural cyclic depsipeptide elastase inhibitors ............... 80
3-4 Changes in cell viability and caspase activation mediated by elastase and effects of inhibitors .............................................................................................. 81
3-5 Elastase acts as a sheddase and promotes cell morphology change and desquamation ..................................................................................................... 82
3-6 Elastase caused a global change in transcript levels via, in part, an NF-B dependent pathway ............................................................................................ 83
4-1 Structures of veraguamides A–G (7–13) and the semisynthetic tetrahydroveraguamide A (14) .......................................................................... 117
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4-2 MS/MS fragmentation of veraguamide A (7), veraguamide D (10), and veraguamide E (11) .......................................................................................... 118
4-3 Assignment of absolute configuration of veraguamide A (7) using methanolysis and subsequent Mosher’s analysis ............................................. 119
4-4 Cell cycle analysis of HT29 and HeLa cells treated with varying concentrations of veraguamide D (10) .............................................................. 120
5-1 Caylobolide B (18) and closely related compound caylobolide A ..................... 147
5-2 Key HSQC-TOCSY correlations for caylobolide B (18) .................................... 148
5-3 ESI-MS/MS of caylobolide B (18) ..................................................................... 149
5-4 Amantelides A and B (19, 20) and the semisynthetic derivative peracetylated amantelide A (21) ............................................................................................. 150
5-5 Partial structure of amantelide A (19) derived from NMR experiments in DMSO-d6 .......................................................................................................... 151
5-6 ESI-MS/MS fragmentation of amantelide A (19) ............................................... 152
5-7 Assignment of relative configuration of caylobolide B (18) based on Kishi’s Universal NMR Database (Database 2) ........................................................... 153
5-8 Time-course antiproliferative activities of amantelide A (19) and amphotericin B against cancer cells ....................................................................................... 154
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LIST OF ABBREVIATIONS
Å Angstrom
[α]20D
Specific optical rotation
Abu 2-amino-2-butenoic acid
Ac Acetyl
Ahp 3-amino-6-hydroxy-2-piperidone
Ala Alanine
ANOVA Analysis of variance
Arg Arginine
APCI/ESI Atmospheric pressure chemical ionization/electrospray ionization
ARID1B AT rich interactive domain 1B
Asp Aspartic acid
BCA Bicinchoninic acid
BEAS-2B Bronchial epithelial cell line
BEBM™ Bronchial epithelial basal medium
Br-Hmoya 8-bromo-3-hydroxy-2-methyl-7-octynoic acid
br q Broad quartet
c Concentration in g/100 mL
13C NMR Carbon-13 nuclear magnetic resonance spectroscopy
calcd Calculated
CDCl3 Deuterated chloroform
cDNA Complementary deoxyribonucleic acid
CE Collision energy
CEP Collision cell entrance potential
CH2Cl2 Methylene chloride
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CH3 Methyl
C=O Carbonyl
COPD Chronic obstructive pulmonary disease
COSY Correlation spectroscopy
CrO3 Chromium trioxide
CSNK1A Casein kinase 1, alpha
CUR Curtain gas
CuSO4 Copper (II) sulfate
CXP Collision cell exit potential
1D One-dimensional
2D Two-dimensional
δ Chemical shift (in ppm)
d Doublet
D- Configurational descriptor (Fisher system)
dd Doublet of doublets
dt Doublet of triplets
DAP3 Death associated protein 3
DDIT4 DNA-damage-inducible transcript 4
Dhoya 2,2-dimethyl-3-hydroxy-7-octynoic acid
DP Declustering potential
DMEM Dulbecco’s Modified Eagle Medium
DMSO Dimethyl sulfoxide
DMSO-d6 Deuterated dimethyl sulfoxide
ELISA Enzyme-linked immunosorbent assay
EP Entrance potential
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ESIMS Electrospray ionization mass spectrometry
EtOAc Ethyl acetate
EtOH Ethanol
FACS Fluorescence-activated cell sorting
FBS Fetal bovine serum
g Gravity
g Gram
GAPDH Glyceraldehyde 3-phosphate dehydrogenase
GAS1 Growth arrest-specific 1
Gln Glutamine
Glu Glutamic acid
Gly Glycine
GS1 Gas 1
GS2 Gas 2
h Hour
H2 Hydrogen gas
HCl Hydrochloric acid
HCOOH Formic acid
HDAC Histone deacetylase
1H NMR Proton nuclear magnetic resonance spectroscopy
Hiva 2-hydroxyisovaleric acid
HMBC Heteronuclear multiple-bond correlation spectroscopy
Hmoaa 3-hydroxy-2-methyl-7-octanoic acid
Hmoea 3-hydroxy-2-methyl-7-octenoic acid
Hmoya 3-hydroxy-2-methyl-7-octynoic acid
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Hmpa 2-hydroxy-3-methylpentanoic acid
HNE Human neutrophil elastase
HPLC-MS Tandem high pressure liquid chromatography-mass spectrometry
HPLC-UV Tandem high pressure liquid chromatography-ultraviolet spectroscopy
HRESIMS High-resolution electrospray ionization mass spectrometry
HSQC Heteronuclear single-quantum correlation spectroscopy
IC50 Half-maximal inhibitory concentration
ICAM-1 Intercellular adhesion molecule-1
IFN-γ Interferon γ
IBα NF-B inhibitor α
IL1A Interleukin 1A
IL1B Interleukin 1B
IL1R1 Interleukin receptor, type 1
IL8 Interleukin 8
Ile Isoleucine
i-PrOH Isopropanol
IS Ionspray voltage
IVT In vitro transcription
nJ Coupling constants via n bonds
λmax Wavelength maximum
LRESIMS Low-resolution electrospray ionization mass spectrometry
m Meter
m multiplet (NMR)
M Molar
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MALDI-TOF Matrix-assisted laser desorption/ionization-time of flight mass spectrometry
MAP2K5 Mitogen-activated protein kinase kinase 5
MAPK Mitogen-activated protein kinase
MeCN Acetonitrile
MeOH Methanol
MHz Megahertz
mICAM-1 Membrane-bound intercellular adhesion molecule-1
min Minute
MRM Multiple reaction monitoring
MTPA Methoxy(trifluorophenyl)phenylacetic acid
MTT 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
MyD88 Myeloid differentiation primary response gene (88)
Na Sodium
n-BuOH n-butanol
NH4OAc Ammonium acetate
nM Nanomolar
N-Me-Ile N-methyl isoleucine
N-Me-Phe N-methyl phenylalanine
N-Me-Val N-methyl valine
NF-B Nuclear factor kappa B
NFIB Nuclear factor I/B
NOD Nucleotide-binding oligomerization domain
OMe Methoxy
PAR Proteinase-activated receptor
PDB ID Protein Databank Identification
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Pd Palladium
PECAM Platelet endothelial cell adhesion molecule
Phe Phenylalanine
Pla Phenyllactic acid
PPE Porcine pancreatic elastase
PTK2 Protein tyrosine kinase 2
PVDF Polyvinylidene difluoride
RBM14 RNA binding motif protein 14
RNA Ribonucleic acid
RT-qPCR Reverse transcription followed by quantitative polymerase chain reaction
s singlet
SAR Structure-activity relationship
SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis
Ser Serine
sICAM-1 Soluble intercellular adhesion molecule-1
SIK2 Salt-inducible kinase 2
SV-40 Simian vacuolating virus-40
tR Retention time
TEM Temperature
Thr Threonine
TNF-α Tumor necrosis factor-α
TOCSY Total correlation spectroscopy
µM Micromolar
Val Valine
VCAM Vascular cell adhesion protein
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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
DRUG DISCOVERY FROM MARINE CYANOBACTERIA SYMPLOCA SPP. AND
PHORMIDIUM SPP.: NOVEL STRUCTURES AND BIOACTIVITIES OF SECONDARY METABOLITES
By
Lilibeth Apo Salvador
May 2013
Chair: Hendrik Luesch Major: Pharmaceutical Sciences – Medicinal Chemistry
Four marine cyanobacteria collections were prioritized for the discovery of novel
secondary metabolites, based on their antiproliferative activity against HT29 human
colorectal adenocarcinoma cells and unique HPLC-MS dereplication profiles.
Bioactivity- and 1H NMR-directed purification yielded the elastase inhibitors
symplostatins 5–10 (1–6), and the antiproliferative agents veraguamides A–G (7–13),
caylobolide B (18), and amantelides A and B (19, 20). Total structure elucidation was
done using 1D and 2D NMR spectroscopy, mass spectrometry and enantioselective
analysis.
Symplostatins 5–10 (1–6) are cyclic depsipeptides bearing the modified amino
acids 3-amino-6-hydroxy-2-piperidone and 2-amino-2-butenoic acid. Comprehensive
protease profiling of 1 indicated potent and selective elastase inhibition. Structure-
activity relationship (SAR) studies on 1–6, together with the related compounds
lyngbyastatins 4 and 7, identified critical and tunable structural elements. This was
corroborated by the X-ray cocrystal structure of lyngbyastatin 7–porcine pancreatic
elastase. The effects of symplostatin 5 (1) on the downstream cellular effects of
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elastase was probed using an epithelial lung airway model system. Compound 1
attenuated elastase-mediated receptor activation, proteolytic processing of adhesion
molecule ICAM-1, NF-B activation and global transcriptome changes, leading to
cytoprotection against elastase-induced cell death, detachment and inflammation.
Veraguamides A–G (7–13) are cyclic hexadepsipeptides bearing a C8-polyketide-
derived β-hydroxy acid, an invariant proline residue, multiple N-methylated amino acids
and an α-hydroxy acid. Compounds 7–13 together with the semisynthetic derivative
tetrahydroveraguamide A (14) displayed weak to moderate antiproliferative activity
against HeLa cervical carcinoma and HT29 cells, modulated by several sensitive
positions in the veraguamide scaffold. Flow cytometry indicated that veraguamide D
(10) caused a dose-dependent increase in cell populations at sub-G1 and G2.
Caylobolide B (18) and amantelides A and B (19, 20) are structurally-related
polyketides characterized by a polyhydroxylated macrolactone ring bearing an alkyl
pendant side chain. Amantelide A (19) displayed sub-micromolar IC50s against HT29
and HeLa cells, while 18 and 20 showed weaker activity. These cyanobacterial
polyketides potentially exert their cytotoxic effect through interaction with the cell
membrane.
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CHAPTER 1 GENERAL INTRODUCTION
Natural Products in Drug Discovery
Natural products are small molecules, typically less than 2,000 Da in size,
produced by terrestrial and marine macro- and microorganisms via enzymatically-
assisted biosynthesis. Also referred to as secondary metabolites, these compounds
have indirect and specialized function in the survival of producing organisms, but are
deemed nonessential in primary metabolic pathways. Natural products have evolved out
of functional necessity and are regarded to act as chemical defenses against predators,
parasites or diseases and may also fulfill intrinsic physiological functions for the
producing organisms.1 Similar to primary metabolites, natural products are derived from
ubiquitous precursor molecules such as acetyl-CoA and proteinogenic amino acids, but
differ from the latter by being species specific, rather than prevalent across
organisms.1,2 And while primary and secondary metabolites utilize the same precursor
molecules, higher structural diversity is observed in the latter due to the involvement of
evolutionary processes in the elaboration of biosynthetic enzymes of secondary
metabolites.1 Natural products are distinguished by the presence of a large number of
ring systems, functionalized mainly by oxygen and hydrogen bonding donor moieties.3
An unprecedented feature of secondary metabolites is sterical complexity – possessing
a high number of stereocenters – as these compounds are products of and target three-
dimensional protein systems.3 Comparison of natural products and synthetics indicated
that these compounds occupy complementary chemical spaces.3 Secondary
metabolites are also able to bind to different unrelated molecular targets and are thus,
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regarded as privileged structures.4 Hence, natural products represent structurally
diverse compounds which have been evolutionary optimized to their molecular targets.
It is for these reasons that man has relied on Nature to discover new drugs. The
earliest documented use of purified secondary metabolites as therapeutics dates back
to the early 19th century, with the discovery of morphine from opium poppy for the
alleviation of pain.5 Several centuries later, natural products continue to be recognized
as a validated source of new drugs and regarded as one of the most successful strategy
in the development of small molecule therapeutics. In a survey of agents introduced for
clinical use from 1980–2010, ~50% are derived from natural products.6 The secondary
metabolite itself may not be the final drug entity, but rather serve as template for the
design of best-in-class small molecule therapeutics. The majority of these are anti-
infectives and anticancer agents.6 Examples of these are the antibiotic penicillin,
antimalarials quinine and artemisinin, and antimitotics vinblastine and paclitaxel (Figure
1-1).
Drugs from the Sea
Terrestrial plants and microorganisms have been the traditional source of natural
products. Technological advancements in underwater exploration have paved the way
for the utilization of marine organisms as source organisms in drug discovery.7 Oceans
cover the majority of the Earth’s surface and harbor rich biodiversity. Each milliliter of
seawater is estimated to contain millions of viruses and bacteria, together with
thousands of fungi and microalgae.8,9 Complex ecological relationship also exists in
these environments, such as endosymbiosis,10 and there is intense competition for
space. These ecological factors can then be expected to impact the secondary
metabolite production in marine organisms.9
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Six marine natural products or their derivatives have successfully reached the
clinic, and several more at different stages of clinical trials.7,11 Clinically-approved
marine natural products include ziconotide for chronic pain management, antiviral agent
vidarabine conceived based on spongouridine from the sponge Tethya crypta and the
anticancer agents cytarabine, an analog of the spongothymidine also from the sponge
Tethya crypta, ecteinascidin-743 (ET-743), eribulin mesylate inspired by the sponge
compound halichondrin B and brentuximab vedotin designed based on the sea
hare/cyanobacterial metabolite dolastatin 10 (Figure 1-2). Ziconotide is a linear
polycationic peptide ω-conotoxin from the cone snail Conus magus, characterized by 25
amino acid residues including six Cys, that forms three disulfide linkages (Figure 1-2).12
This compound is utilized by the source organism to immobilize its prey, and in
mammalian system targets N-type voltage sensitive calcium channels.12 ET-743 from
the sea squirt Ecteinascidia turbinata was approved for use in the European Union for
refractory soft-tissue sarcoma. The core structure of ET-743 (Figure 1-2) consists of
fused tetrahydroisoquinoline rings that are deemed essential in binding to and
covalently modifying DNA.13 The clinically approved agent eribulin mesylate for breast
cancer treatment is a truncated version of halichondrin B (Figure 1-2).14 Halichondrin B
was initially isolated from the sponge Halichondria okadai, and subsequently from
several more sponge species such as Axinella and Phakellia carteri.15 Halichondrin B
binds to the Vinca domain of tubulin.16 The low yield and high structural complexity of
halinchondrin B, limited its clinical development. Simplified analogs of halichondrin B, as
in the case of eribulin mesylate, showed similar bioactivities as the natural product and
tapped for drug development.14 Brentuximab vedotin is an antibody-drug conjugate
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clinically approved for Hodgkin’s lymphoma and anaplastic large cell lymphoma.
Brentuximab vedotin consists of a CD33-targeting antibody, a cathepsin cleavable linker
and the drug monomethyl auristatin E (Figure 1-2).17 Monomethyl auristatin E is an
analog of the sea hare/cyanobacterial metabolite dolastatin 10 (Figure 1-2), which also
binds to and disrupt microtubule proteins.18
Marine Cyanobacteria: Source Organisms of Novel Molecules
Cyanobacteria or blue green algae are primitive organisms that have existed for
billions of years, despite lacking any morphological defense structures such as spines,
spicules or shell. Thus, these primitive prokaryotic organisms are thought to have
evolved an arsenal of bioweapons for chemical defense. Since the pioneering studies of
Professor Richard Moore, close to 1,000 secondary metabolites have been isolated
from these organisms.19–22 Marine cyanobacteria utilize polyketide synthases,
nonribosomal peptide synthetases and hybrids of these two biosynthetic pathways to
produce diverse secondary metabolites.23 The majority of these were isolated from the
genera Lyngbya, Oscillatoria, Phormidium and Symploca.
The complex ecological relationship among marine organisms and the production
of secondary metabolites can be observed in cyanobacteria – as the true producers of
bioactive natural products isolated from mollusks and ascidians. Sea hare-derived
dolastatins 10–15 were originally isolated from these herbivores, in low quantities.24 For
example, 1 mg of dolastatin 10 required 2 tons of sea hare.24 A comparable amount of
dolastatin 10 was isolated from a Guamanian Symploca sp., and required only 5 g of
dried cyanobacteria.25 The significantly enriched amounts of dolastatin 10 together with
the isolation of closely related compounds and other sea hare-derived metabolites from
marine cyanobacteria indicated that the true producers are marine cyanobacteria, and
25
are acquired by these herbivores through their diet.26 Cyanobacteria have also been
demonstrated to affect secondary metabolite production of other marine organisms
through endosymbiosis. For example, the production of patellamides by the Didemnidae
family of tunicate is dependent on the obligate cyanobacterial symbiont Prochloron
spp.27 It is then estimated that 35% of marine-derived anticancer agents are products of
cyanobacteria, based on structural similarity.21 The production of natural products from
microbes and microbe interaction with the host organism where the compound was
isolated has emerged as a pivotal concept in natural products discovery.6,8,10
Mechanism of Action of Bioactive Cyanobacterial Metabolites
Marine cyanobacteria are well-documented to be prolific producers of
antiproliferative agents.21 The majority of these are actin and tubulin poisons, with the
marine cyanobacteria Symploca sp. being the source organisms of the potent tubulin
poisons – dolastatin 10 and symplostatin 1.24,25,28,29 In addition, secondary metabolites
with atypical and remarkable mechanisms of action have also been isolated from this
marine cyanobacteria genus, such as largazole which inactivates histone deacetylases
(HDACs).30 Protease inhibition is perhaps the major theme among marine
cyanobacterial metabolites and are commonly encountered in various genera. 21
Interference with Microtubule Dynamics
Dolastatin 10 and symplostatin 1 are closely related linear pentapeptides
characterized by modified amino acids dolaphenine, dolaproline, and dolaisoleucine,
together with Val and a terminal N,N-dimethylated amino acid (Figure 1-3).24,25,28
Dolastatin 10 and symplostatin 1 are differentiated by their N-terminal amino acid
residue, N,N-dimethylVal and N,N-dimethylIle, respectively (Figure 1-3). These
compounds were both demonstrated to have broad spectrum cytotoxicity towards an
26
array of cancer cell lines, with pico- to nanomolar IC50s.29 A dose-dependent increase in
cell populations at G2 and concomitant formation of abnormal mitotic spindles were
observed in symplostatin 1- and dolastatin 10-treated cells. A10 and HeLa cells treated
with symplostatin 1 had disrupted cellular microtubule network as evidenced by
immunostaining using monoclonal β-tubulin antibody.29 Dolastatin 10 and symplostatin
1 were both shown to directly interact with tubulin, with the former demonstrated to
inhibit the binding of radiolabeled Vinca alkaloid.18,29 Molecular docking experiments
proposed that dolastatin 10 binds to a distinct region, close to the Vinca domain and
inhibited tubulin-dependent GTP hydrolysis and nucleotide exchange, processes that
are crucial for tubulin assembly.31
Symplostatin 1 retarded the growth of colon adenocarcinoma 38 and mammary
adenocarcinoma 16/C cells in vivo at dosages of 0.25–1.25 mg/kg.29 Symplostatin 1,
however, caused tissue damage at the site of injection and test animals showed 3–15%
body weight loss, depending on the dosing schedule.29 Dolastatin 10 reached Phase II
clinical trials for prostrate cancer treatment but was discontinued due to observed
peripheral neuropathy among patients and weak therapeutic activity as a single agent.32
Several analogs of dolastatin 10 were synthesized to improve the in vivo potency and
safety profile. On August 2011, FDA approved a dolastatin 10 analog, monomethyl
auristatin E conjugated to a CD33 targeting antibody for clinical use in Hodgkin’s
lymphoma and anaplastic large cell lymphoma treatment.17
Inhibition of Histone Deacetylase
Largazole is a cyclic depsipeptide that is characterized by several unique
structural features such as a 4R-methylthiazoline that is fused to a thiazole ring, and a
3S-hydroxy-7-mercapto-4-heptenoic acid linked to an n-octanoyl group that serves as
27
the prodrug moiety (Figure 1-4).30,33 Largazole requires a protein-assisted hydrolysis to
liberate the active species largazole thiol.34,35 Cytotoxicity testing showed potent activity
against cancer cell lines with superior selectivity index.30 Largazole is the first marine
cyanobacteria-derived agent demonstrated to target HDACs, with superior class I
isoform selectivity.34 The majority of known HDAC inhibitors were derived from
terrestrial microorganisms.36 The reported cocrystal structure of largazole and HDAC8
showed that the “warhead” thiol moiety is present as the thiolate and chelates the Zn2+
catalytic ion in a tetrahedral arrangement.37 This optimum interaction is facilitated by the
rigid depsipeptide macrocyle arising from the fused thiazole-thiazoline rings. NCI60
screening on largazole showed particular susceptibility of colon cancer cell lines to
treatment and an HCT116 xenograft mouse model was adopted.35 In this in vivo animal
model, largazole did not show significant toxic effects and was well-tolerated. Largazole
was able to retard tumor growth in test animals compared to control group, and caused
an upregulation of the cyclin-dependent kinase inhibitor p15 and pro-apoptotic effector
caspase 3, while prosurvival proteins HER2, cyclin D1, IRS-1, and pAKT were
downregulated in tumor sections.35
Inhibition of Proteases
From marine cyanobacteria, several non-cytotoxic metabolites have been
demonstrated to be potent protease inhibitors, particularly targeting the serine
proteases elastase, chymotrypsin and trypsin.21,38,39 The macrocycle of these
cyanobacterial serine protease inhibitors is distinguished by an N-methylated aromatic
amino acid residue, a small nonpolar amino acid such as Val or Ile and a characteristic
ester linkage formed by the condensation of the secondary hydroxy group of Thr. The
Thr residue is also modified on its N-terminus by one to three amino acid residues, and
28
capped by a terminal fatty or polar acid such as butanoic, hexanoic or glyceric acid,
giving rise to the pendant side chain of these cyclic depsipeptides. Lyngbyastatins 4–10
(Figure 1-5) and the related compounds somamide B and molassamide, which bear a
modified Thr residue, 2-amino-2-butenoic acid, adjacent to the Ahp residue on the N-
terminal showed potent elastase inhibition.40–44 A related compound, kempopeptin A,
from a Lyngbya sp. collection bears a Leu residue instead of Abu, and potently inhibited
elastase and chymotrypsin (Figure 1-5).45 Its analog kempopeptin B (Figure 1-5),
bearing a Lys residue, inhibited trypsin.45 Thus, it is evident that the residue on the N-
terminal side of the Ahp moiety modulates the activity of these inhibitors for different
serine proteases.38,41,46 These serine protease inhibitors have been demonstrated to
function as digestion inhibitors and feeding deterrents of herbivores, fishes and urchins
and may also possibly modulate the biosynthesis of other cyanobacterial secondary
metabolites.47–49
Statine (γ-amino-β-hydroxy acid)-containing modified linear peptides from marine
cyanobacteria on the other hand, are potent inhibitors of aspartic proteases.
Grassystatins A–C (Figure 1-5) isolated from a Floridian Lyngbya cf. confervoides
selectively inhibited the aspartic protease cathepsin E at pico- to nanomolar
concentrations and concurrently prevented cathepsin E-mediated antigen presentation
of dendritic cells.50 These compounds bear a leucine derived statine unit (4-amino-3-
hydroxy-6-methylheptanoic acid), critical for cathepsin inhibition while residues adjacent
to this moiety confer selectivity towards cathepsin E. The related linear peptide,
tasiamide B (Figure 1-5),51,52 on the other hand bears a phenylalanine-derived statine
moiety and has been demonstrated to inhibit β-site APP Cleaving Enzyme Type 1
29
(BACE1), an enzyme which has been shown to be central to the formation of amyloid
plaques and related to the progression of Alzheimer’s disease.53 Tasiamide B served as
the template in the design of new inhibitors of BACE1 with potent cellular activity and in
vivo efficacy.53
Objectives and Specific Aims of the Study
With marine cyanobacteria being validated source organisms of structurally and
pharmacologically diverse secondary metabolites, we aimed to utilize novel chemical
entities from these organisms for potential biomedical applications as antitumor agents
and modulators of elastase-mediated pathologies. This study focused on the under-
explored marine cyanobacteria genera of Symploca and Phormidium, which yielded
several of the best-in-class antitumor agents. This study aimed to:
1. Prioritize collections of Symploca and Phormidium using a preliminary profiling of bioactivity and chemical space
2. Perform a bioactivity-guided purification on cyanobacterial collections which demonstrated antiproliferative activity to isolate the bioactive constituent(s)
3. Perform a 1H NMR-guided purification to discover novel secondary metabolites from non-cytotoxic cyanobacterial collections
4. Determine the structure of isolated compounds from prioritized collections using combinations of spectroscopic techniques such as 1D and 2D NMR spectroscopy and mass spectrometry
5. Elucidate the biological activity and mechanisms of action of identified cyanobacterial secondary metabolites in mammalian cellular systems.
30
Figure 1-1. Representative examples of natural products that influenced modern
medicine.
31
Figure 1-2. Marine natural products and analogs that have reached the clinic.
32
Figure 1-3. The linear peptides symplostatin 1 and dolastatin 10 are potent
antiproliferative agents that disrupt tubulin polymerization.
33
Figure 1-4. Largazole is a cyclodepsipeptide prodrug that targets canonical histone
deacetylases.
34
Figure 1-5. Representative examples of non-cytotoxic metabolites from marine
cyanobacteria that target proteases.
35
CHAPTER 2 PROBING THE CHEMICAL SPACE AND ANTIPROLIFERATIVE ACTIVITIES OF
CYANOBACTERIAL COLLECTIONS*
Introduction
Filamentous marine cyanobacteria are a validated source of antiproliferative
agents, having yielded several of the best-in-class inhibitors of malignancies.20,21
Cytotoxins from marine cyanobacteria also display not just a variety in structure, but
mechanisms of action as well. Actin-targeting agents, with sub-nanomolar IC50s against
cancer cells, include lyngbyabellins,54–56 dolastatin 1157,58 and hectochlorin.59 The
marine cyanobacteria Lyngbya spp. afforded the cyclic depsipeptides apratoxins A–G
that are also potent cytotoxins,60–64 with apratoxin A preventing cotranslational
translocation leading to downregulation of receptors and growth factor ligands.65,66 The
marine cyanobacteria Symploca spp. and Phormidium spp. yielded several modified
linear peptides that target tubulin polymerization.25,28,29,67,68 The most potent among
these are the related dolastatin 1018 and symplostatin 1,29 with the former serving as the
template for the design of the clinically approved anti-Hodgkin’s and anaplastic large
cell lymphoma drug brentuximab vedotin. Another novel agent from Symploca sp. is the
histone deacetylase inhibitor largazole, which displayed potent activity in preclinical
evaluations.33 With the abundance of novel antitumor agents from marine
cyanobacteria, it is thus attractive to employ a primary screening of antiproliferative
activity against cancer cells for crude extracts. Measurements of cell viability can be
done using colorimetric or fluorometric reagents to measure cellular metabolism, protein
activity and interactions, membrane permeabilization and cellular respiration.36
*Reproduced with permission from J. Nat. Prod., submitted for publication. Unpublished work copyright
2013 American Chemical Society.
36
The isolation of a large number of antiproliferative agents from marine
cyanobacteria, however, also increases the possibility of reisolating known compounds
as bioactive components. Thus, it is advantageous to employ a screen of the chemical
space as well. Several dereplication methods – identification of known metabolites from
sample collections with the least effort and resources – have been developed for both
terrestrial and marine cyanobacteria, employing UV spectroscopy and mass
spectrometry.
To distinguish known bioactive compounds in a screen for phorbol debutyrate
receptor binding activity, a HPLC-UV dereplication was utilized.69 Members of the
aplysiatoxin class of compound are known to be phorbol debutyrate receptor binders,
and comparison of the retention time and UV profile of authentic debromoaplysiatoxin
allowed the identification of this compound as the active principle for several Lyngbya
majuscula collections.69 This method also accounted for debromoaplysiatoxin as the
bioactive constituent of seagrasses and macroalgae, possibly due to cyanobacterial
contamination.69 More compound-specific techniques emerged with the development of
new technologies in mass spectrometry such as MALDI-TOF and ESIMS. The initial
utilization of MALDI-TOF for dereplication was a serendipitous discovery, but
nonetheless, demonstrated the presence of microcystins, micropeptin and
anabaenopeptolin from collections of Microcystis, Anabaena and Oscillatoria.70 The
application of MALDI-TOF for dereplication has been extended to determine the spatial
distribution of secondary metabolites in cyanobacteria themselves and other marine
organisms, in addition to identification.71 Structure determination of nonribosomal
peptides have also greatly benefited from mass spectrometry, with tandem mass
37
spectrometry yielding the identity of these compounds via characteristic fragmentation
pattern. Recently introduced is comparative dereplication using tandem mass
spectrometry and spectral alignment algorithms to identify identical compounds and
related analogs.72 The requirement for minimal material to perform mass spectrometry
analysis and its amenability to high-throughput format makes this method an attractive
choice for dereplication.
Here, an HPLC-MS dereplication method utilizing multiple reaction monitoring
was developed to improve the resolution of known cytotoxins in collections of marine
cyanobacteria Symploca and Phormidium. This, together with antiproliferative screening
against HT29 colorectal adenocarcinoma cells, was utilized to prioritize cyanobacterial
collections for further studies.
Screening of Cyanobacteria Collections
A total of 38 marine cyanobacteria samples were collected in Florida, Guam and
the US Virgin Islands from 2007–2009. These collections were mainly Symploca spp.,
Phormidium spp. and several taxonomically unidentified organisms characterized by
puffy ball gross morphology characteristic for Symploca spp. Collected organisms were
lyophilized and extracted with either CH2Cl2–MeOH (1:1) or EtOAc–MeOH (1:1) to yield
the nonpolar extracts. These extracts were further subjected to a C18 solid phase
extraction (SPE) cleanup using a MeOH–H2O elution. Initial elution using 25% MeOH
removed the majority of the salts and ensured minimal non-specific bioactivity and
interference in HPLC-MS arising from these polar compounds. The fraction collected
from 100% MeOH elution was tested for antiproliferative activity against HT29 colorectal
adenocarcinoma cells and concurrently profiled by HPLC-MS.
38
Antiproliferative Assay as Preliminary Screening for Bioactivity
Antiproliferative activity was assessed based on the fractional survival of HT29
cancer cells, detected using the MTT reagent. Extracts which caused < 60% survival of
HT29 cells were considered bioactive at the specified concentration. From the 38
samples screened for antiproliferative activity, only two sample collections were inactive
at all concentrations tested (Figure 2-1A). Thirteen sample collections exhibited
moderate antiproliferative activity against HT29 cells at concentrations of 1,000 and
10,000 ng/mL (Figure 2-1A). The remaining 60% of the screened cyanobacteria
collections exhibited antiproliferative activity at concentrations of 10 and 100 ng/mL
(Figure 2-1A). With the large number of cyanobacterial collections showing
antiproliferative activity, additional information for prioritization of sample collections are
needed. Also, with potent cytotoxins such as dolastatin 10, symplostatin 1 and largazole
being produced by Symploca spp. and Phormidium spp. collections, determination of
the contribution of these known compounds to the bioactivity should be assessed at an
early stage of the discovery process.
Dereplication using an HPLC-MS Approach
The dereplication method for the known compounds largazole, dolastatin 10 and
symplostatin 1, consisted of a gradient HPLC run using CH3CN–H2O (+ 0.1% HCOOH)
and multiple reaction monitoring (MRM) as MS detection mode. This allowed for
sensitive, specific and high-throughput format for dereplication of previously isolated
metabolites from Symploca spp. and Phormidium spp. sample collections. The MRM
mode relies on the detection of both the parent ion mass (Q1) and a specific daughter
ion resulting from fragmentation (Q3), giving a significant reduction in background,
improvement in signal-to-noise ratio and limits of detection. This dereplication format
39
permitted automation, short run times per sample (< 20 min) and simultaneous
monitoring of largazole, symplostatin 1 and dolastatin 10 (Figure 2-2). This method does
not have specific structural requirements and can be done using commonly available
mass spectrometers. However, authentic standards are needed for optimization of the
HPLC-MS parameters. Since MRM is also a compound-specific detection, no
information on the presence of related congeners may be derived using this method.
Based on the HPLC-MS dereplication, the majority of the sample collections with
antiproliferative activity at 10 and 100 ng/mL contained combinations of dolastatin 10,
largazole or symplostatin 1 (Figure 2-1A, B). Except for one sample collection, all other
bioactive cyanobacterial collection at concentration of 10 ng/mL contained these three
antiproliferative agents at biologically relevant concentrations (Figure 2-1A). Extracts
containing symplostatin 1 or dolastatin 10 alone or lower concentrations of these
metabolites in combination showed activity at a higher concentration of 100 ng/mL.
Interestingly, largazole was consistently detected in combination with dolastatin 10 and
symplostatin 1, whenever present (Figure 2-1A, B). Samples without detectable levels
of largazole, symplostatin 1 or dolastatin 10 showed varied antiproliferative activity and
thus presented as prioritized candidates for both bioactivity- and 1H NMR-guided
purification (Figure 2-3).
Prioritization of Sample Collections
The bioactivity data together with the dereplication results and available material
of the cyanobacteria collection were considered in the prioritization of sample
collections for further purification (Figure 2-3). Bioactive collections at concentrations <
10,000 ng/mL with sufficient amounts of lyophilized cyanobacteria and/or nonpolar
extract were given highest priority. Non-cytotoxic or weakly cytotoxic samples were
40
further subjected to a silica SPE and 1H NMR profiling to check for relevant
functionalities such as N-CH3, O-CH3, -NHs and α-hydrogens. From the 38 profiled
cyanobacteria collections, four priority samples were further pursued (Figure 2-3). A
bioactivity-guided purification was undertaken to obtain the antiproliferative agent(s),
while cyanobacteria collections with weak cytotoxic activity were purified via a 1H NMR-
guided purification.
Validation of the Dereplication Method
To validate our current HPLC-MS dereplication method, largazole and dolastatin
10 were isolated from a Symploca sp. collection from Pickles Reef in Florida using a
HPLC-MS-guided purification. Monitoring by HPLC-MS required minimal amounts of
sample, while still permitting sensitive detection. Using this approach, sub-milligram
quantities of largazole and dolastatin 10 were isolated. The identities of the purified
compounds were verified using 1H NMR and LRESIMS measurements, and comparison
with literature values (Appendix F). The isolation of symplostatin 1 is presented in
Chapter 5. The antiproliferative activities of the purified largazole and dolastatin 10
against HeLa human cervical adenocarcinoma, HCT116 human colorectal carcinoma
and HT29 cells were also tested and in accordance with the literature values (Table 2-
1).
Conclusion
The bioactivity and chemical space of crude extracts of 38 cyanobacterial
collections, belonging mainly to Phormidium and Symploca cyanobacteria genera, were
screened using the MTT cell viability assay and HPLC-MS-based dereplication method,
respectively. The majority of the screened cyanobacterial collections with potent
bioactivity contained combinations of the cytotoxins largazole, dolastatin 10 and
41
symplostatin 1. These compounds were rapidly identified as the major cytotoxic
constituent through comparison of the HPLC-MS profiles with authentic standards,
using multiple reaction monitoring. The dereplication method was further validated using
large-scale isolation from a dolastatin 10- and largazole-containing cyanobacterial
collection. By combining dereplication information, antiproliferative activity profiles
against HT29 cancer cells, availability of material and/or initial 1H NMR profile, four
cyanobateria collections were prioritized for the discovery of novel bioactive secondary
metabolites.
Experimental Methods
General Experimental Procedures
1H NMR spectra were recorded in CDCl3 or CD2Cl2 on a Bruker Avance II 600
MHz spectrometer equipped with a 5-mm TXI cryogenic probe using residual solvent
signals [(CDCl3: δH 7.26), (CD2Cl2: δH 5.32)] as internal standards. LRESIMS
measurements, MRM analysis and MS/MS fragmentation were done on an ABI 3200Q
TRAP.
Biological Material
Symploca spp. or Phormidium spp. cyanobacteria collections were collected by
hand at various sites in Guam, Florida, and the US Virgin Islands. Samples were kept
frozen at –20 °C after collection. A voucher specimen, which is preserved in 100%
EtOH or formaldehyde, is deposited in the University of Guam Herbarium and at the
Smithsonian Marine Station, Fort Pierce, FL. Frozen cyanobacteria samples were
lyophilized prior to extraction. The freeze-dried cyanobacteria were extracted with
EtOAc–MeOH (1:1) or CH2Cl2–MeOH (1:1) to yield the nonpolar extracts.
42
HPLC-MS Profiling
Nonpolar cyanobacteria extracts (10–20 mg) were purified by a C18 SPE column
using a MeOH–H2O elution. The fraction from 100% MeOH was dried under N2,
weighed, and methanolic stock solution (1 mg/mL) was prepared. A dilution (10,000
ng/mL) of the stock solution was prepared in MeCN and spiked with the internal
standard harmine and was used as test solution. A 10 μL portion of the test solution was
injected for HPLC-MS analysis, using the following conditions: column, Kinetex (100
2.1 mm), Phenomenex; linear gradient of 0.1% HCOOH in MeCN–0.1% HCOOH in H2O
[50%–100% MeCN in 10 min and then 100% MeCN for 5 min, flow rate, 0.5 mL/min;
detection by ESIMS in positive ion mode (MRM scan)]. The retention times (tR, min;
MRM ion pair) of the analytes were as follows: harmine (1.5; 214→170.9), dolastatin 10
(2.2; 785.6→753.7), symplostatin 1 (2.4; 799.6→767.6), largazole (2.5; 623→497).
Cell Viability Assay
HT29 colorectal adenocarcinoma cells were cultured in Dulbecco’s modified Eagle
medium (DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS, Hyclone)
under a humidified environment with 5% CO2 at 37 °C. HT29 (12,500) cells were
seeded in 96-well plates. These were treated with varying concentrations (10, 100,
1,000, 10,000 ng/mL) of the nonpolar extract, dissolved in EtOH, 24 h post-seeding.
Cells were incubated for an additional 48 h before the addition of the MTT reagent. Cell
viability was measured according to the manufacturer’s instructions (Promega, Madison,
WI). Antiproliferative activity of purified largazole and dolastatin 10 was determined
using the same procedure, employing the cancer cell lines HT29 human colorectal
adenocarcinoma (12,500 cells/well), HeLa human cervival carcinoma (3,000 cells/well)
and HCT116 human colorectal carcinoma (10,000 cells/well).
43
Validation of Dereplication Method
A Symploca sp. collection from Pickles Reef, Florida was lyophilized and the
dried material (13.3 g) was extracted with EtOAc–MeOH (1:1) to yield the nonpolar
extract (1.7 g). The nonpolar extract was adsorbed on a Diaion HP-20 resin and eluted
with 100% H2O, 25%, 50%, 75% and 100% MeOH and 50% CH2Cl2 in MeOH. Each
fraction was monitored for the presence of largazole, symplostatin 1 and dolastatin 10
using the HPLC-MS method. The fraction eluting from 50% CH2Cl2 (33 mg) showed
peaks corresponding to largazole and dolastatin 10 and was applied onto a silica SPE
column, eluting with increasing gradients of i-PrOH in CH2Cl2, until 100% i-PrOH. The
fractions eluting from 10% i-PrOH and 20% i-PrOH contained largazole and dolastatin
10, respectively, based on HPLC-MS profiling. These fractions were further purified by
semipreparative HPLC (Phenomenex Synergi-HydroRP, 4 μm; flow rate, 2.0 mL/min)
using a linear gradient of MeOH–H2O (70%–100% MeOH in 60 min and then 100%
MeOH for 15 min). The 10% i-PrOH fraction yielded largazole (tR 41.7 min, 0.3 mg).
Using the same chromatographic condition, the 20% i-PrOH fraction afforded dolastatin
10 (tR 40.0 min, 0.2 mg). The 1H NMR and LRESIMS of the isolated compounds were
identical to those of the literature values.
Largazole: colorless, amorphous solid; 1H NMR spectrum is identical to that of an
authentic sample,30 see Appendix F; LRESIMS m/z 623.0 [M + H]+.
Dolastatin 10: colorless, amorphous solid; 1H NMR spectrum is identical to that of
an authentic sample,28 see Appendix F; LRESIMS m/z 785.6 [M + H]+.
44
Figure 2-1. Summary of chemical space and bioactivity profiles of Symploca spp. and
Phormidium spp. collections. (A) The majority of the cyanobacteria collections displayed antiproliferative activity against HT29 human colorectal adenocarcinoma cells as assessed using the MTT reagent. The majority of potent bioactive extracts showed combinations of dolastatin 10, largazole and symplostatin 1. (B) Distribution of the three known antiproliferative agents in profiled cyanobacterial collections.
45
Figure 2-2. Representative HPLC-MS profile of the simultaneous monitoring of
largazole, dolastatin 10 and symplostatin 1.
46
Figure 2-3. Prioritization scheme of cyanobacteria collections and the corresponding
secondary metabolites isolated.
47
Table 2-1. Antiproliferative activity (IC50, nM) of known Symploca sp. metabolitesa
Compound HT29 HCT116 HeLa
Dolastatin 10 0.4 ± 0.01 1.8 ± 0.02 0.2 ± 0.01 Largazole 10 ± 0.6 7.0 ± 1.4 12 ± 1.1 aData are presented as mean ± SD (n = 2).
48
CHAPTER 3 POTENT ELASTASE INHIBITORS FROM CYANOBACTERIA: STRUCTURAL BASIS AND MECHANISMS MEDIATING CYTOPROTECTIVE AND ANTI-INFLAMMATORY
EFFECTS IN BRONCHIAL EPITHELIAL CELLS
Introduction
Cyanobacteria, whether of marine, terrestrial or freshwater origin, have
consistently yielded serine protease inhibitors characterized by a conserved 19-
membered cyclic hexadepsipeptide core bearing the modified glutamic acid residue 3-
amino-6-hydroxy-2-piperidone (Ahp) and a highly variable pendant side chain.21,38,39
The isolation of over 100 members of this group of cyanobacterial metabolites, together
with antiproteolytic activity data primarily against the serine proteases elastase,
chymotrypsin, and trypsin, has provided insights into the importance of the Ahp moiety
and the adjacent residue on its N-terminal side, which confer selectivity.38,46 The role of
these moieties was elegantly demonstrated through X-ray cocrystallization of A90720A–
trypsin and scyptolin–elastase complexes.73,74 Not found in terrestrial or freshwater
cyanobacteria is the 2-amino-2-butenoic acid (Abu) moiety, which is hypothesized to
contribute to higher potency.41 The majority of the marine-derived cyanobacterial
metabolites in this class bears the Abu moiety adjacent to the Ahp residue. These
compounds, which include lyngbyastatins 4–10, showed potent antiproteolytic activity
against elastase with low nanomolar IC50s, and are perhaps among the most potent
small molecule inhibitors of elastase.40–42 Therefore, these small molecules are
attractive therapeutics for elastase-mediated pathologies, as well as molecular probes
to elucidate critical interactions for effective enzyme inhibition and to interrogate specific
Reproduced with permission from Salvador, L.A.; Taori, K.; Biggs, J. S.; Jakoncic, J.; Ostrov, D. A.; Paul, V. J.; Luesch, H. J. Med. Chem. 2013, 56, 1276–1290. Copyright 2013 American Chemical Society.
49
intracellular and extracellular molecular targets of elastase. However, limited SAR and a
lack of information beyond enzymatic assay data hinder further development of these
compounds as small molecule therapeutics.
Elastase is a broad-spectrum enzyme that preferentially cleaves on the C-
terminus of small hydrophobic amino acids such as Gly, Ala, and Val and degrades
collagen, elastin, fibronectin and components of the extracellular matrix.75 Elastase has
been linked to several diseases involving chronic inflammatory conditions such as
chronic obstructive pulmonary disease (COPD), asthma, cystic fibrosis, and systemic
inflammatory response syndrome, where there is a protease–antiprotease
imbalance.75,76 The canonical role of elastase in degrading the extracellular matrix has
been documented, as have the stimulating effects of elastase on signaling pathways
through direct or indirect receptor activation. The resulting changes in transcript and
protein levels have been linked to possible disease progression.77 Current therapies for
these diseases are aimed at alleviating the symptoms but not disease progression,
which may be related to the role of elastase.78 Sivelestat is the only approved drug
targeting elastase;79 however, clinical approval in the United States and Europe has
been stalled due to marginal clinical effects.80 Finding new small molecule therapeutics
for COPD is of importance since the disease has been recognized as a major public
health problem and the fourth leading cause of death worldwide.81 Intratracheal
instillation of elastase in animal models showed changes such as enlargement of
alveolar space, thickening of alveolar septae and mucus hypersecretion, comparable to
clinical observations.82 This enzyme has also been implicated in cell death,
transcriptional and translational modulation and processing of pro-inflammatory
50
cytokines, chemokines and adhesion molecules, which also dictate downstream cellular
effects.76 Development of elastase inhibitors has been particularly challenging because
of overlapping functions of elastase with those of other serine proteases, as well as
limited information on the role of elastase in the progression of disease. Here we aimed
to determine the potential utility of symplostatin 5 (1) and related compounds in
alleviating the cellular effects downstream of elastase release and compared the cellular
potency to sivelestat.
Isolation and Structure Elucidation
The lyophilized red cyanobacterium collected from Cetti Bay, Guam was
extracted with EtOAc–MeOH (1:1) to afford the nonpolar extract. Liquid-liquid
partitioning of the nonpolar extract yielded the hexanes-, n-BuOH- and H2O-soluble
fractions. The 1H NMR spectrum of the n-BuOH fraction showed characteristic
resonances for peptides and modified peptides. This fraction was further purified by
silica column chromatography and reversed-phase HPLC to give six new Ahp-
containing cyclic depsipeptides, termed symplostatins 5–10 (1–6) (Figure 3-1).
The major compound, symplostatin 5 (1) (Figure 3-1), showed a
pseudomolecular ion of 1044.3981 [M + Na]+, suggesting a molecular formula of
C47H64N7O15SNa. LRESIMS using negative ionization showed a loss of 46 amu (m/z
998.5 [M – Na]–) relative to the pseudomolecular [M + Na]+ ion. This corresponds to loss
of 2 Na+ ions and supported that 1 was present as a sodium salt. The 1H NMR
spectrum of symplostatin 5 (1) showed characteristic signals for peptides and modified
peptides such as secondary amide protons (δH 8.18, 7.71, 7.40, 7.34), N-CH3 protons
(δH 2.77), and α-protons for amino acids (δH 3.80–5.10). Analysis of the COSY, TOCSY,
51
HSQC and HMBC data acquired in DMSO-d6 established the presence of Val, Thr, Ile,
N-Me-Phe, Phe and the modified amino acids Ahp and Abu (Table 3-1).
Among the three remaining spin systems, one is a distinctive methine quartet (δH
6.50) that showed a COSY correlation to a CH3 doublet (δH 1.47) (Table 3-1). HMBC
correlations of the latter to a carbonyl at δC 162.9 and a quaternary sp2 C (δC 130.0),
together with a TOCSY correlation to a broad NH singlet (δH 9.24), established this unit
as Abu. The observed low-field methine signal at δC/δH 73.4/5.03 together with a
hydroxy proton resonating at δH 6.05 in 1 are distinctive for the Ahp unit. The presence
of this cyclized amino acid residue was further supported by COSY and HMBC
correlations (Table 3-1). The remaining spin systems consisted of a low-field methine
(δC/δH 79.9/3.98), an oxygenated diastereotopic methylene (δC/δH 66.1/3.90, 3.73) and
an –OCH3 group (δC/δH 57.1/3.33). From COSY and HMBC analysis, this moiety
corresponds to a modified glyceric acid, where the C-2 and C-3 positions are
methoxylated and sulfated, respectively (Table 3-1). The linear sequence of 2-O-CH3
glyceric acid sulfate–Val–Thr–Abu–Ahp–N-Me-Phe–Phe–Ile was established using
HMBC and NOESY correlations. In order to fulfill the molecular formula requirements
and to account for the low-field 1H NMR chemical shift of the vicinal methine of Thr (δH
5.52), additional anisotropic effect from a carbonyl group must be present, and this
indicated cyclization of symplostatin 5 (1) via the carbonyl group of Ile and the hydroxy
group of Thr.
Comparison of the 1H NMR spectrum of 1, 2 and 3 revealed differences in the
splitting pattern of signals in the methyl region (δH 0.75–0.90). No methyl triplet arising
from Ile was observed in symplostatin 6 (2). Instead, two pairs of methyl doublets were
52
present, suggesting the presence of 2 Val units, which was corroborated by the HSQC
spectrum of 2. Hence, the Ile unit present in the core ring structure of 2 is replaced by
Val, where the vicinal methine (δC/δH 30.7/2.00) showed COSY and HMBC correlations
to two methyl groups (δC/δH 19.0/0.89, 17.1/0.76) (Table 3-2), in agreement with
molecular formula of C46H62N7O15SNa deduced from HRESIMS. Symplostatin 6 (2) is
reminiscent of dolastatin 1383 with the primary difference being the modification of the 2-
O-CH3 glyceric acid unit, and is the sulfated analog of dolastatin 13 (Figure 3-1).
Symplostatin 7 (3) showed 14 amu mass difference with symplostatin 5 (1) and has a
molecular formula of C48H66N7O15SNa. The 1H NMR spectrum of 3 showed 2 × CH3
triplets (δH 0.92, 0.80) which correlated to two high-field carbons (δC 11.3, 10.7) based
on the HSQC spectrum (Table 3-3). Hence, 3 has Ile moieties in both the pendant and
macrocycle (Figure 3-1). Comparison of the 1H NMR spectrum of 1 and 3 corroborated
this result. Except for 1H NMR resonances belonging to the additional Ile unit, no
significant differences were observed between the two spectra.
The 1H NMR spectra of 1 and 4, 2 and 5, and 3 and 6 were highly similar, except
for the splitting pattern and chemical shifts of aromatic protons (δH 6.77–7.40). These
pairs also showed a difference of 16 amu in their HRESIMS spectra, corresponding to
an additional oxygen atom in 4–6. Comparison of the HSQC spectra of these
compounds showed an upfield shifted sp2 C at δC 115.2, which correlates to a proton at
δH 6.77. COSY correlation between δH 6.77 and δH 6.99, together with their doublet
splitting pattern and 3JH,H of 7.8 Hz, indicated a 1,4-disubstituted phenyl ring (Tables 3-
1–3-3). The upfield shifted 1H and 13C NMR resonances and the presence of a broad
singlet at δH 9.34 for a hydroxy group in the 1H NMR spectrum of 4–6 supported the
53
presence of an N-Me-Tyr instead of an N-Me-Phe in the macrocycle. Hence,
symplostatins 8–10 (4–6) are the N-Me-Tyr congeners of 1–3 (Figure 3-1), consistent
with the molecular formula requirements.
Enantioselective HPLC-MS analysis of the acid hydrolysates of 1–6 established
the configuration of the amino acids (Val, Phe, N-Me-Phe, N-Me-Tyr, Thr) as L, by
comparison to authentic standards. L-allo-Ile was detected for compounds having this
unit in the macrocycle alone (1, 4), while 3 and 6, which have Ile in both the macrocycle
and pendant chain showed peaks corresponding to L-allo-Ile and L-Ile at ~1:1 ratio.
Comparison of the 1H and 13C NMR chemical shifts of the macrocyclic Ile of 1, 3, 4, and
6 showed no significant differences and suggested the same configuration. Hence, the
pendant side chain Ile moiety would account for the peak corresponding to L-Ile. The
presence of L-allo-Ile in the macrocycle is also supported by comparison of the 13C NMR
chemical shifts of C-5 and C-6 of the macrocyclic Ile with similar compounds bearing the
same amino acid residue. Zafrir and Carmeli reported that the 13C NMR chemical shifts
of C-5 and C-6 of L-allo-Ile are distinctive, 11.4 and 14.3 ppm, respectively.84 1, 3, 4 and
6, which are proposed to bear an L-allo-Ile in the macrocycle, also displayed these
characteristic 13C NMR resonances (Tables 3-1, 3-3). Oxidation of 1 using CrO3 prior to
acid hydrolysis converted the Ahp unit to Glu. Enantioselective analysis of the acid
hydrolysate of the oxidation product showed a peak corresponding to L-Glu and hence,
the Ahp unit would have the same configuration at C-3. The configuration at C-6 of Ahp
is deduced to be R in comparison with the NMR chemical shifts with the related
compounds symplostatin 2 and lyngbyastatins 4–10.40,41,85 The 2-O-CH3 glyceric acid
liberated from the acid hydrolysate of symplostatin 5 (1) is proposed to have an R
54
configuration, based on comparison with authentic standards of 2-O-CH3 glyceric acid
synthesized from D- and L-Ser by a modified diazotization procedure.86 The other
analogs of symplostatin 5 (1) are proposed to have the same configuration of the Ahp
and 2-O-CH3 glyceric acid moieties based on similar 1H and 13C NMR chemical shifts.
Enzyme Inhibition
We tested the antiproteolytic activity of symplostatins 5–10 (1–6) against porcine
pancreatic elastase. Compounds 1–6 potently inhibited porcine pancreatic elastase with
IC50s of 37–89 nM (Table 3-4), which was comparable to the activity of the related
compounds lyngbyastatins 4 and 7. Symplostatins 8–10 (4–6), containing N-Me-Tyr, are
slightly more potent than their N-Me-Phe congeners (1–3) in inhibiting elastase. In
contrast, Ile to Val substitution in the macrocycle and pendant side chain did not affect
activity. To demonstrate that these compounds also inhibit the disease-relevant human
neutrophil elastase, we determined the antiproteolytic activity against this enzyme.
Symplostatins 8–10 (4–6) and lyngbyastatins 4 and 7 potently inhibited human
neutrophil elastase, while symplostatins 5–7 (1–3) gave higher IC50s (Table 3-4).
Compounds 4–6 and lyngbyastatins 4 and 7 showed higher potency than the drug
sivelestat, a selective human neutrophil elastase inhibitor, while 1–3 had similar activity
as sivelestat. Symplostatins 5–10 (1–6) and lyngbyastatins 4 and 7 were analogously
tested for antiproteolytic activity against human and bovine pancreatic chymotrypsin. All
the compounds tested were less potent inhibitors of chymotrypsin than elastase (Table
3-4).
To determine the selectivity of the cyanobacterial elastase inhibitors, we
screened the most potent inhibitor, lyngbyastatin 7, at a single concentration against a
panel of 68 proteases (Figure 3-2A). Lyngbyastatin 7 showed preferential inhibition for
55
serine proteases at 10 µM, completely inhibiting the serine proteases elastase,
chymotrypsin and proteinase K. The serine proteases cathepsin G, kallikrein 8,
kallikrein 12 and plasma kallikrein, the dipeptidyl peptidase cathepsin C and the
cysteine proteases caspases 1, 9 and 11 were partially inhibited. Lyngbyastatin 7 did
not inhibit any member of the cysteine carboxypeptidases, metalloproteases or aspartic
family of proteases. To validate the serine protease selectivity profile for this class of
inhibitors, a dose-response study against the same panel of 26 serine proteases was
undertaken for the most abundant compound, symplostatin 5 (1) (Figure 3-2B).
Symplostatin 5 (1), like lyngbyastatin 7, preferentially inhibited elastase over
chymotrypsin. Aside from these enzymes, the majority of the serine proteases including
proteinase K was less potently inhibited by 1 than by lyngbyastatin 7, with IC50s of 10
µM or higher.
Molecular Basis for Elastase Inhibition by Lyngbyastatins and Symplostatins
In order to understand the potent and selective inhibitory activity of the Abu-
bearing cyclic depsipeptides against elastase, we cocrystallized the most potent
inhibitor, lyngbyastatin 7, with porcine pancreatic elastase using the hanging drop vapor
diffusion method. The structure of the lyngbyastatin 7–porcine pancreatic elastase
complex was solved at a resolution of 1.55 Å, the best reported for an elastase–cyclic
depsipeptide inhibitor complex. The elastase complexes with the natural products
scyptolin (no Abu) and FR901277 (bicyclic) were previously cocrystallized and analyzed
at resolutions of 2.8 Å and 1.6 Å, respectively.74,87 Porcine pancreatic elastase, despite
sharing only 40% amino acid sequence homology to human neutrophil elastase, is an
accepted model system to understand key enzyme–inhibitor interactions.88 They are
structurally comparable and share analogous residues that compose the enzyme active
56
site. The improved resolution of the lyngbyastatin 7–elastase complex provided better
insights into key molecular interactions with the enzyme. The cocrystal structure of
porcine pancreatic elastase and lyngbyastatin 7 indicated that these compounds act as
substrate mimics, with the Abu moiety and the N-terminal residues occupying subsites
S1 to S4. They exploit the same binding sites occupied by FR901277 and scyptolin, and
the orientation of the macrocyle of these three compounds is also comparable (Figure
3-3A–C). The ethylidene moiety of the Abu unit in subsite S1 contributes a non-bonded
interaction with Ser203 and within distances for CH/π interaction (Figure 3-3D, E), as
previously hypothesized for FR901277.87 It also forms hydrogen bonds with Gly201 and
Ser222, and an indirect hydrogen bond with Thr44 via a water molecule. The cocrystal
structure did not show covalent bond formation between the Abu moiety of lyngbyastatin
7 and elastase or hydrolytic cleavage of the macrocyle. Lyngbyastatin 7 showed
extensive hydrogen bonding and van der Waals interactions with elastase and several
water molecules in the active site (Figure 3-3D). The difference in antiproteolytic activity
between the N-Me-Phe containing symplostatins 5–7 (1–3) with their corresponding N-
Me-Tyr congeners (4–6) was evaluated in the context of the cocrystal structure. The OH
group of N-Me-Tyr forms hydrogen bonds with three water molecules. Val to Ile
substitution in the macrocycle did not cause a significant difference in antiproteolytic
activity and, based on the cocrystal structure, this moiety is indeed not close to any
amino acid residues of elastase for interaction. Comparison of the antiproteolytic activity
of symplostatin 9 (5), lyngbyastatins 4 and 7–9, which all bear exactly the same
macrocycle, indicated the contribution of the pendant side chain in modulating the
antiproteolytic activity of these elastase inhibitors. Lyngbyastatins 8 and 9 are less
57
potent, with IC50s of 120–210 nM,42 suggesting that the presence of mainly hydrophobic
residues in the pendant side chain is unfavorable. The preference for a polar group in
the pendant side chain is supported by the cocrystal structure, wherein the Gln moiety
of lyngbyastatin 7 participates in indirect hydrogen bonding with Gln200 and Ser225, via
a water molecule; a molecular interaction is not possible with nonpolar moieties in the
pendant side chain. The side chain carbonyl of the Gln moiety of lyngbyastatin 7 also
participates in a network of inter- and intramolecular hydrogen bonding interaction
involving an active site water molecule, C=O (Thr) and C=O (Ahp) (Figure 3-3F). This
interaction has not been previously demonstrated and suggests the novelty of having a
Gln or related moiety at this position. A linear terminal unit in the pendant side chain
appears to be preferable, as the hexanoic acid of lyngbyastatin 7 displays a perfect fit to
the elastase binding pocket and also participates in nonbonded interactions with Val103
and Arg226.
Biological Activity Evaluation
Cytoprotective Effects of Symplostatin 5 (1) Against Elastase-Induced Antiproliferation and Apoptosis
We utilized the bronchial epithelial cell line BEAS-2B, a SV-40 transformed cell
line that maintains epithelial cell characteristics, as a model system.89 We challenged
these cells with disease-relevant concentrations of exogenous elastase and tested if
compound 1 was able to prevent the toxicity by elastase, which showed both a dose-
and time-dependent antiproliferative effect based on MTT assay, with an IC50 value of
77.5 ± 4.9 nM at 24 h (Figure 3-4A). Symplostatin 5 (1) dose-dependently protected the
cells, causing a shift in the IC50 of elastase (Figure 3-4B). The ordinarily toxic
concentrations of elastase had little effect on cell viability when 1 was coadministered.
58
At concentrations of 1 or 10 µM symplostatin 5 (1), cell viability in elastase co-treated
cells was >75%. Sivelestat was also protective but required higher concentration (100
µM) to completely negate elastase-induced cytotoxicity (Figure 3-4C). In addition,
symplostatin 5 (1) did not significantly affect the proliferation of BEAS-2B cells even up
to a concentration of 100 µM (Figure 3-4D) when co-administered with either the solvent
control or 100 nM elastase, thus providing a wide therapeutic window at least in cultured
cells. To determine the possible role of apoptosis in the observed antiproliferative effect
of elastase, we assessed caspase 3/7 activity of BEAS-2B cells. Increased pro-
apoptotic activity was observed upon 12–24 h incubation with 100 nM elastase (Figure
3-4E), paralleling with the onset of cell viability changes associated with elastase
(Figure 3-4A). Addition of a caspase 3 inhibitor abrogated the observed increase in
caspase 3/7 activity from elastase treatment (Figure 3-4E). Furthermore, treatment of
BEAS-2B cells with the caspase 3 inhibitor also caused significant protection from
elastase-induced antiproliferation. However, protection was incomplete, which suggests
that elastase also reduces cell viability through mechanisms other than apoptosis
(Figure 3-4C). Addition of ≥100 nM symplostatin 5 (1) lowered the caspase 3/7 activity
in elastase-treated cells and shifted the EC50 of elastase in activating caspases (Figure
3-4F). Thus, symplostatin 5 (1) counteracted both pro-apoptotic and antiproliferative
effects of elastase. Recent reports demonstrated that elastase can activate apoptosis
through a proteinase-activated receptor-1 (PAR-1)-dependent pathway that culminates
in the upregulation of NF-B and p53 and subsequent changes in mitochondrial
permeability and caspase activation.90,91 PARs are seven-transmembrane G-protein
coupled receptors that are activated by proteases following cleavage of the extracellular
59
N-terminus, which triggers a change in conformation and coupling to the G-protein.92
Thrombin is a canonical activator of PARs, while elastase has been reported to have
varied effects and PAR substrates, depending on the cell type.93 It is unclear whether
elastase directly or indirectly activates PARs. Furthermore, the antiproliferative effects
of elastase may be mediated by other pathways as well as its cytostatic effect based on
the partial cytoprotection using the caspase inhibitor when compared to 1 and
sivelestat. It is then evident that the key to maximum abrogation of elastase-mediated
antiproliferative effect is disarming its proteolytic activity.
Cytoprotective Effects of Symplostatin 5 (1) Against Elastase-Induced Cell Detachment and Morphological Change
A morphological change of BEAS-2B cells from an epithelial to a rounded and
retracted appearance was the most obvious and immediate cellular event that occurred
following elastase treatment (Figure 3-5A). This effect of elastase was observed within
2–3 h, and the early onset suggests that this was independent of cell death. Cells
incubated with elastase remained viable after 3 and 6 h, as assessed by MTT and
trypan blue staining, despite the obvious change in cell morphology. Furthermore,
caspase 3 inhibitor pre-treated cells showed the same rounded appearance
(Appendices A–D). Symplostatin 5 (1) and sivelestat both dose-dependently prevented
elastase-induced cell morphology change (Figure 3-5A), although sivelestat required a
higher concentration during longer incubation periods (12 and 24 h) (Appendices C and
D), consistent with results from cell viability assays (Figure 3-4C). Elastase caused a
three-fold increase in cell detachment from the collagen base matrix and neighboring
cells after 12 h, which was dose-dependently prevented by 1 (Figure 3-5B). At a
concentration of 10 µM of symplostatin 5 (1), elastase was unable to cause
60
desquamation. Sivelestat also showed the same cytoprotective effect but again only at
higher concentration (100 µM). The ability of elastase to induce cell detachment and
morphology change reflects its canonical role in degrading components of the
extracellular matrix such as collagen, fibronectin, and elastin and also implicates its
effects on cell adhesion molecules. This role of elastase is also dependent on its
proteolytic activity as evidenced by abrogation via small molecule inhibition using
symplostatin 5 (1) and sivelestat, but not with the caspase inhibitor.
Adhesion molecules such as the immunoglobulin-like cell adhesion molecules
(ICAM-1, -2, -3, VCAM, PECAM), integrins, selectins and cadherins are located on the
cell surface, are involved in cell and extracellular matrix attachment and also function to
modulate leukocyte adhesion and migration, a process essential to progression of
inflammation.94 ICAM-1 is a key regulator of cell-cell adhesion and exists as a
membrane-bound protein (mICAM-1) that can be cleaved to generate soluble ICAM-1
(sICAM-1) which is liberated into the medium.95 sICAM-1 is increased with inflammation
and cardiovascular disease and serves as a biomarker.96,97 To determine the possible
effects of elastase on total ICAM-1 levels in bronchial epithelial cells, culture medium
and whole cell lysates were collected after 6 h. mICAM-1 in whole cell lysates was
assessed by immunoblotting (Figure 3-5C) and provides a snapshot of the remaining
membrane-bound form at the specific timepoint. sICAM-1 in culture supernatants was
quantified by AlphaLisa® and reflects accumulated amount over time (Figure 3-5D).
Media from elastase-challenged cells contained significantly increased sICAM-1 level,
which was dose-dependently decreased by cotreatment with ≥1 µM symplostatin 5 (1)
(Figure 3-5D). Inhibition of the proteolytic activity of elastase by cotreatment with
61
symplostatin 5 (1) caused retention of mICAM-1, thus confirming the role of elastase
activity on this cellular event (Figure 3-5C). Sivelestat also showed a similar effect on
sICAM-1 and mICAM-1 levels in response to elastase. This inverse relationship is
consistent with sICAM-1 levels in the culture medium and provided internal validation of
the direct effects of elastase with the proteolytic cleavage of ICAM-1. Conversely,
ICAM1 transcript levels were not significantly modulated in this cell type as assessed by
reverse transcription followed by real-time quantitative polymerase chain reaction (RT-
qPCR) (Appendix E). Taken together, this data further supported the role of elastase as
a sheddase, which posttranslationally modifies the membrane-bound form by proteolytic
processing to the soluble form.
While elastase-mediated activation of caspases has been related to cell surface
receptors, we additionally demonstrated that it can proteolytically process ICAM-1, a
critical cell surface receptor that controls cell-cell adhesion, known to be affected by
elastase at both the transcript and protein levels in endothelial cells.98 Purified elastase
and sputum samples from cystic fibrosis patients with significant proteolytic activity were
shown to induce cleavage of ICAM-1, independent of cell surface expression.99,100
Aside from controlling cell-cell adhesion, mICAM-1 also binds to leukocytes via the LFA-
1 receptor, and its normal expression is required for immune defense.95 Shedding of
mICAM-1 is proposed to serve as a rapid mechanism to regulate leukocyte adhesion
and/or promote signal transduction, although it has not been fully elucidated.101
The observed cellular effects of elastase on cell detachment and cell death are
important clinical hallmarks of asthma and COPD.102 Neutrophils mainly cause cell
detachment, with elastase and cathepsin G degrading a variety of substrates.103 In a
62
cellular model system, TNF-α and IFN-γ were also shown to induce desquamation and
may function together with serine proteases.104 Furthermore, lung biopsies of patients
indicated that detachment and apoptosis may be related, with the initial sites of cell
detachment showing increased apoptotic cells.104 Cell death has been linked, in addition
to persistent inflammation, to contribute to the severity of COPD.105 Excessive apoptosis
is proposed to exacerbate lung disease by preventing re-epithelialisation, development
of apoptotic resistance leading to fibrosis and ineffective removal of apoptotic cells,
resulting in a persistent inflammatory state.106
Attenuation of Global Transcript Changes Induced by Elastase
Elastase has been demonstrated to induce changes in transcript levels of pro-
inflammatory cytokines, adhesion molecules and chemokines in vitro, mostly mediated
by an NF-B-dependent pathway.98,107,108 The expression of NF-B-inducible genes is
preceded by degradation of cytosolic IB and nuclear translocation of p65.109 To
determine the possible changes in transcript levels in elastase and
elastase+symplostatin 5 (1) treatments, the amount of cytosolic IB and nuclear p65
was assessed by immunoblotting and ELISA, respectively. Elastase caused a strong
decrease in IB level, which was prevented by 1 (Figure 3-6A). In accord, a significant
increase in nuclear translocation was observed 3 h after elastase treatment and
attenuated by cotreatment of 1. This data is indicative of possible transcript changes
associated with elastase treatment that may also be modulated by 1. Microarray
profiling using the Affymetrix GeneChip® Human Genome U133 plus 2.0 arrays was
performed to comprehensively determine global changes in transcript levels in bronchial
epithelial cells following elastase treatment. Elastase caused a significant change in
63
expression (P < 0.05, fold change > 1.5) of 364 transcripts corresponding to 348 genes
(Figures 3-6B, Tables 3-5, 3-6). Elastase affected the expression of signaling molecules
including chemokines, cytokines, and receptors, as well as components of the
spliceosome, transcription machinery, cell cycle and ubiquitin-mediated proteolysis. In
addition, 13% of elastase-inducible genes currently have no annotation of identity and
function, suggesting that our analysis may have identified novel target genes of elastase
signaling (Table 3-5). Also, of the other 87% of genes with known identity, 30% do not
have a clear function in cellular signaling. Aside from the members of the NOD- and
MAPK-signaling pathways (Table 3-6), the contribution of other elastase-inducible
genes to inflammation or downstream cellular effects of elastase has not been clearly
established. Upregulation of kinases (e.g., PTK2, MAP2K5, SIK2, CSNK1A) and
transcription factors (e.g., ARID1B, NFIB, RBM14) may suggest that elastase is
promoting cellular signaling by affecting signaling molecules and/or their activation. The
contribution of caspase-independent pathways to elastase-mediated cell death may
also be discerned, as several positive modulators of the cell cycle were also
upregulated by elastase (e.g., GAS1, DAP3, DDIT4). Importantly, the transcriptional
response to elastase was attenuated by co-administration of 10 µM symplostatin 5 (1).
Comparison of the heat map of significantly modulated transcripts indicated that 1
potently prevented the global effects of elastase (Figure 3-6B). Symplostatin 5 (1)
caused a 20–68% reduction in transcript levels of elastase-inducible genes including
those involved in NOD- and MAPK- signaling pathways which are relevant to
inflammation (Table 3-6). Microarray results were validated by measuring expression
levels of important pro-inflammatory cytokines IL1A, IL1B and IL8 using RT-qPCR. IL1B
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showed the greatest increase in transcript levels at 3 h, which was strongly abrogated
by cotreatment with 1 (Figure 3-6C). Similar results were obtained for IL1A and IL8. The
effects of elastase on mRNA levels of these three pro-inflammatory cytokines were also
assessed at 6 h (Figure 3-6C). The same trend was observed for IL1B and IL8, while
IL1A was not significantly affected at this time point. Transcriptome profiling of BEAS-2B
cells treated with symplostatin 5 (1) alone enabled us to characterize possible off-target
genes of 1 that are independent of elastase (Table 3-7). This analysis identified only
nine significantly upregulated transcripts corresponding to nine genes, suggesting high
specificity of symplostatin 5 (1) for elastase also in cells.
Our profiling of the transcriptome of bronchial epithelial cells in response to
elastase, with or without 1 and vehicle control treatments, indicated that this enzyme
upregulates the expression of specific genes. Comprehensive profiling enabled us to
identify IL1B as the major pro-inflammatory cytokine induced by elastase. Although IL8
has been reported to be upregulated by elastase in vitro,107,110,111 our microarray
analysis indicated that this gene is less inducible compared to IL1B. IL-1β is a key pro-
inflammatory cytokine and has increased activity in both COPD and asthma, causing
significant airway remodeling and pulmonary inflammation in animal models, and thus
serves as an important biomarker for elastase-mediated cellular effects.112,113 The
expression of IL-1β in elastase-treated animals has been demonstrated to occur via an
IL1R1/MyD88 pathway, thus further implicating the role of this enzyme in receptor
activation.112 We also demonstrated that elastase has a broad effect on the
transcriptome, and our identification of other elastase target genes may open up new
65
avenues towards the understanding of the physiological and pathological roles of this
enzyme.
Conclusion
We have demonstrated that novel cyanobacterial cyclodepsipeptides can
potently inhibit the proteolytic activity of elastase, thereby preventing the downstream
cellular effects of this serine protease in a bronchial epithelial model system.
Symplostatin 5 (1) alleviated elastase-induced changes in cell viability, apoptosis, cell
detachment and alterations in levels of the adhesion molecule ICAM-1, activation of
transcription factor NF-B and global transcriptome changes. At the same time, 1 did
not show any cytotoxic effects on bronchial epithelial cells, offering a remarkable
therapeutic window. Symplostatin 5 (1) showed equipotent activity as sivelestat in
enzyme inhibition and short-term cellular assays. However, 1 showed higher potency in
longer-term assays and successfully alleviated several clinical hallmarks of chronic
inflammatory diseases such as excessive sICAM-1 production, expression of pro-
inflammatory cytokines IL1A, IL1B and IL8 and increased cell death and desquamation.
Establishment of the molecular basis and biomarkers for elastase inhibition can aid in
the design of second-generation inhibitors that are potent, selective and cytoprotective
against both short- and long-term effects of elastase.
Experimental Methods
General Experimental Procedures
Optical rotations were measured on a Perkin-Elmer 341 polarimeter. UV spectra
were recorded on SpectraMax M5 (Molecular Devices). 1H and 2D NMR spectra were
recorded in DMSO-d6 on a Bruker Avance II 600 MHz spectrometer equipped with a 5-
mm TXI cryogenic probe using residual solvent signals [(DMSO-d6: δH 2.50; δC 39.5)]
66
as internal standards. HSQC and HMBC experiments were optimized for 1JCH = 145 and
nJCH = 7 Hz, respectively. TOCSY experiments were done using a mixing time of 100
ms. HRESIMS data was obtained using an Agilent LC-TOF mass spectrometer
equipped with an APCI/ESI multimode ion source detector. LRESIMS measurements
and MRM analysis were done on an ABI 3200Q TRAP. Compounds have purity ≥ 95%
based on HPLC.
Biological Material
The red Symploca sp. cyanobacterium was collected by hand from Cetti Bay,
Guam. Samples were kept frozen at –20 °C after collection. A voucher specimen
preserved in formaldehyde is deposited in the University of Guam Herbarium and at the
Smithsonian Marine Station, Fort Pierce, FL. Frozen cyanobacterium samples were
lyophilized prior to extraction.
Extraction and Isolation
The freeze-dried cyanobacterium was extracted with EtOAc–MeOH (1:1) to yield
the nonpolar extract. This was partitioned between hexanes and 80% aqueous MeOH,
the latter concentrated under reduced pressure and further partitioned between n-BuOH
and H2O. The n-BuOH fraction was concentrated to dryness and chromatographed on
Si gel eluting first with CH2Cl2, followed by increasing concentrations of i-PrOH, while
after 100% i-PrOH, increasing gradients of MeOH were used.
The fraction collected from 50% i-PrOH elution (Si column) was purified by C18
column chromatography eluting with 25%, 50%, 75% and 100% MeOH in H2O. The
fraction from 50% MeOH was further purified using semipreparative reversed-phase
HPLC (Phenomenex Synergi-Hydro RP, 4 μm; flow rate, 2.0 mL/min) using a linear
gradient of MeCN–H2O (25%–100% MeCN in 30 min and then 100% MeCN for 10 min)
67
to yield compounds 5 (tR 17.8 min, 1.0 mg), 4 (tR 19.0 min, 1.0 mg) and 6 (tR 36.6 min,
0.3 mg). The 75% MeOH fraction was purified using the same HPLC conditions to yield
compounds 2 (tR 23.8 min, 3.5 mg), 1 (tR 24.0 min, 7.0 mg) and 3 (tR 25.3 min, 1.0 mg).
Enantioselective Analysis
Portions of 1–6 (100 μg) were acid-hydrolyzed (200 μL 6 N HCl, 110 °C, 20 h),
the product mixtures dried and reconstituted in 100 μL H2O. The absolute configurations
of the amino acids (Ile, Val, N-Me-Tyr, N-Me-Phe, Phe, Thr) were determined by
enantioselective HPLC-MS [column, Chirobiotic TAG (250 4.6 mm), Supelco; solvent,
MeOH–10 mM NH4OAc (40:60, pH 5.30); flow rate, 0.5 mL/min; detection by ESIMS in
positive ion mode (MRM scan)]. The retention times (tR, min; MRM ion pair) of the
authentic amino acids were as follows: L-Val (7.8; 118→72), D-Val (13.7); N-Me-L-Phe
(22.7; 180→134), N-Me-D-Phe (40.4); L-Phe (12.1; 166→103), D-Phe (17.5); N-Me-L-
Tyr (18.8; 196→77), N-Me-D-Tyr (35.4); L-Thr (6.8; 120→74), L-allo-Thr (7.2), D-Thr
(8.0), D-allo-Thr (10.2). In order to separate Ile isomers, the mobile phase was modified
to MeOH–10 mM NH4OAc (90:10, pH 5.65) while keeping the same chromatographic
conditions. The retention times of authentic standards were as follows: L-Ile (10.4;
132→86), L-allo-Ile (11.2), D-allo-Ile (20.1), D-Ile (22.2).
The acid hydrolysates of 1–6 showed retention times at 6.8 and 12.1 min
corresponding to L-Thr and L-Phe, respectively. L-Val (tR 7.8min) was detected in the
acid hydrolysates of 1, 2, 4, and 5. The acid hydrolysates of 1, 3, 4 and 6 showed a
peak corresponding to L-allo-Ile (tR 11.2 min). 3 and 6 had an additional peak
corresponding to L-Ile (tR 10.4 min). N-Me-L-Phe (tR 22.7 min) was detected in the acid
68
hydrolysate of 1–3, while N-Me-L-Tyr (tR 18.8 min) was present in the acid hydrolysate
of 4–6.
The modified (2S)-2-O-Me glyceric acid residue was prepared using L-Ser (50
mg), isoamyl nitrite (70.5 μL), glacial CH3COOH (17.2 μL), MgSO4 (60 mg) and
anhydrous MeOH (1 mL). The mixture was heated at 110 °C for 4 h, cooled down to
room temperature and filtered. The filtrate was evaporated to dryness under N2 to yield
(2S)-2-O-Me glyceric acid. The same procedure was employed to prepare (2R)-2-O-Me
glyceric acid from D-Ser. LRESIMS and 13C NMR spectrum for (2S)-2-O-Me glyceric
acid and (2R)-2-O-Me glyceric acid were in agreement with reported literature values.86
The absolute configuration of Glu and 2-O-Me glyceric acid was also determined
using HPLC-MS [column, Chirobiotic TAG (250 4.6 mm), Supelco; solvent, MeOH–10
mM NH4OAc (40:60, pH 5.30); flow rate, 0.5 mL/min] with detection in the negative ion
mode (MRM). The retention times of the authentic standards (tR, min; MRM pair): L-Glu
(5.1; 146→102), D-Glu (6.1), (2S)-2-O-Me glyceric acid (6.1; 119→89), (2R)-2-O-Me
glyceric acid (6.6). Compound 1 was oxidized using CrO3 and hydrolyzed using 6 N HCl
(110 °C, 20 h) to convert Ahp to Glu. The oxidation product’s acid hydrolysate showed a
peak at 5.1 min corresponding to L-Glu. The acid hydrolysate of 1 yielded a peak for
(2R)-2-O-Me glyceric acid (tR 6.6 min).
Symplostatin 5 (1): colorless, amorphous solid; [α]20D
–3.6 (c 0.14, MeOH); UV
(MeOH); λmax (log ε) 210 (4.49); 1H NMR, 13C NMR, COSY, and HMBC data, see Table
3-1; HRESIMS m/z 1044.3981 [M + Na]+ (calcd for C47H64N7O15SNa, 1044.3971).
69
Symplostatin 6 (2): colorless, amorphous solid; [α]20D
–5.2 (c 0.26, MeOH); UV
(MeOH); λmax (log ε) 204 (4.49); 1H NMR and 13C NMR data, see Table 3-2; HRESIMS
m/z 1030.3815 [M + Na]+ (calcd for C46H62N7O15SNa, 1030.3815).
Symplostatin 7 (3): colorless, amorphous solid; [α]20D
–14 (c 0.15, MeOH); UV
(MeOH); λmax (log ε) 202 (4.48); 1H NMR and 13C NMR data, see Table 3-3; HRESIMS
m/z 1058.4104 [M + Na]+ (calcd for C48H66N7O15SNa, 1058.4128).
Symplostatin 8 (4): colorless, amorphous solid; [α]20D
–6.7 (c 0.09, MeOH); UV
(MeOH); λmax (log ε) 210 (4.13); 1H NMR and 13C NMR data, see Table 3-1; HRESIMS
m/z 1060.3941 [M + Na]+ (calcd for C47H64N7O16SNa, 1060.3920).
Symplostatin 9 (5): colorless, amorphous solid; [α]20D
–3.5 (c 0.10, MeOH); UV
(MeOH); λmax (log ε) 204 (3.94); 1H NMR and 13C NMR data, see Table 3-2; HRESIMS
m/z 1046.3747 [M + Na]+ (calcd for C46H62N7O16SNa, 1046.3764).
Symplostatin 10 (6): colorless, amorphous solid; [α]20D
–3.3 (c 0.03, MeOH); UV
(MeOH); λmax (log ε) 200 (5.24); 1H NMR and 13C NMR data, see Table 3-3; HRESIMS
m/z 1074.4060 [M + Na]+ (calcd for C48H66N7O16SNa, 1074.4077).
In Vitro Protease Assay
Porcine pancreatic elastase (Elastin Products Company, Owensville, MO) was
dissolved in Tris-HCl (pH 8.0) to give a concentration of 75 µg/mL. Test compounds (1
µL, DMSO), 5 µL elastase solution and 79 µL Tris-HCl (pH 8.0) were pre-incubated at
room temperature for 15 min in a 96-well microtiter plate. At the end of the incubation,
15 µL substrate solution were added [2 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide
(Sigma-Aldrich, St. Louis, MO) in Tris-HCl, pH 8.0] to each well, and the reaction was
monitored by recording the absorbance at 405 nm every 30 s. The inhibitory activity
against human neutrophil elastase was also determined using the same procedure with
70
minor modifications, using 100 µg/mL human neutrophil elastase (Elastin Products
Company) and 2 mM N-(OMe-succinyl)-Ala-Ala-Pro-Val-p-nitroanilide (Sigma-Aldrich),
both prepared in 0.1 M Tris-NaCl buffer (pH 7.5). Enzyme activity was determined by
calculating the initial slope of each progress curve, expressed as a percentage of the
slope of the uninhibited reaction. Antiproteolytic activity against bovine (100 μg/mL) and
human (50 μg/mL) pancreatic chymotrypsin (Sigma-Aldrich) were assessed using the
substrates N-succinyl-Gly-Gly-Phe-p-nitroanilide (Sigma-Aldrich) and N-succinyl-Ala-
Ala-Pro-Phe-p-nitroanilide (Sigma-Aldrich), respectively. In brief, the reaction buffer (39
μL), enzyme (10 μL) and inhibitor (1 μL) were incubated for 30 min at room temperature
before the addition of 50 μL of substrate. The absorbance was monitored at 405 nm.
Enzyme activity in each well was calculated based on the slope of the reaction curve
compared to that of the solvent control.
For high-throughput screening, enzyme and inhibitor [symplostatin 5 (1) or
lyngbyastatin 7] were incubated for 15 min in the reaction buffer before the addition of
the substrate. The reaction was monitored for 2 h and the initial linear portion of the
slope was analyzed. Detailed information on the enzymes, substrates, reaction buffers
and detection conditions are given in Table 3-8. High-throughput protease screening
was carried out by Reaction Biology, Inc.
Cocrystallization of Lyngbyastatin 7 with Porcine Pancreatic Elastase
A 10-µL aliquot of high purity porcine pancreatic elastase:lyngbyastatin 7 solution
(3:1) was incubated in a hanging drop setup equilibrated against a 0.42 M sodium
sulfate solution. Diffraction data was collected on beamline X6A at the National
Synchrotron Light Source (Upton, NY). Diffraction data was processed using
HKL200051 and the structure was solved by molecular replacement using 2V0B as
71
search model in MOLREP (CCP4).114,115 The model was refined using REFMAC and
COOT.116,117 Detailed information on the refinement statistics are provided in Table 3-9.
Coordinates are deposited in the Protein Databank with accession number 4GVU.
Cocrystallization experiments and data analysis were carried out by Ms. Kanchan Taori,
Dr. Jean Jakoncic and Dr. David A. Ostrov,
In Vitro Cellular Assays
General cell culture procedure
Bronchial epithelial cells (BEAS-2B, ATCC) were grown in bronchial epithelial
basal media (BEBM™) (Lonza, Walkersville, MD) supplemented with bronchial
epithelial growth factors (Lonza) under a humidified environment with 5% CO2 at 37 °C.
All culture plates and flasks were coated with collagen before use.
Cell viability assay
BEAS-2B (5,000/well) cells were seeded in collagen-coated 96-well plates and
treated with varying concentrations of elastase or vehicle (40% sodium acetate in
BEBM™) after 24 h of seeding. These were cotreated with varying doses of either
symplostatin 5 (1) or sivelestat (Sigma-Aldrich) or with DMSO. The cells were incubated
for an additional 24 h before the addition of the MTT reagent. Cell viability was
measured according to the manufacturer’s instructions (Promega, Madison, WI). IC50
calculations were done by GraphPad Prism® 5.03 based on duplicate experiments.
Cell detachment and morphology change
BEAS-2B cells were seeded in 6-cm dishes. The cells were treated with
vehicle+DMSO, elastase+DMSO and elastase+inhibitor. Brightfield photographs were
taken at 3 h, 6 h, 12 h and 24 h using a Nikon Eclipse Ti-U microscope (10
magnification). Media were collected after 12 h and the detached cells were pelleted by
72
centrifugation. Adherent cells were collected by trypsinization and pelleted afterwards.
Cell pellets were resuspended in fresh culture medium containing 0.04% trypan blue. A
10-µL aliquot was utilized for cell counting using a hemacytometer. Percent detachment
was calculated based on the ratio of the detached cells and total number of cells.
Graphs and data analysis were performed using the Prism® software and analyzed
using ANOVA followed by Dunnett’s t-test.
Caspase activation measurement
BEAS-2B cells were prepared similar to the cell viability assay. Cells were
treated 24 h post seeding with vehicle+DMSO, elastase+DMSO, elastase+symplostatin
5 (1). In addition, BEAS-2B cells were pre-incubated with 10 µM Z-
D(OMe)E(OMe)VD(OMe)-FMK, a caspase 3 inhibitor (Calbiochem, Billerica, MA), for 1
h prior to addition of varying concentrations of elastase. At the end of the 24 h
incubation period, the medium was replaced with fresh BEBM™ and incubated for 10
min at room temperature. The caspase reagent was prepared according to the
manufacturer’s instruction (Promega) and was added to each well and incubated for 10
min to ensure complete cell lysis. Luminescence was measured and the relative
caspase 3/7 activity of elastase and elastase+symplostatin 5 (1) treated cells were
compared to the control.
Measurement of sICAM-1 levels
BEAS-2B cells (60,000/well) were seeded in collagen-coated 24-well plates. After
overnight incubation, the medium was replaced with supplement-free media and the
cells were further incubated for 24 h. At the end of the incubation period, cells were
replenished with new supplement-free medium prior to treatment. Cells were treated
with elastase together with DMSO or varying concentrations of symplostatin 5 (1)
73
dissolved in DMSO. Control cells were treated with DMSO (1%) and sodium acetate in
supplement-free medium (4%). The cells were incubated and culture supernatants were
collected after 6 h. sICAM-1 levels were determined using ICAM-1 AlphaLisa® Kit
(PerkinElmer, Waltham, MA) according to the manufacturer’s instruction. Graphs and
data analysis were performed using the Prism® software and analyzed using ANOVA
followed by Dunnett’s t-test.
Immunoblot analysis of mICAM-1 levels
BEAS-2B cells (150,000/well) were grown in collagen-coated 6-cm tissue culture
dishes. The supplemented medium was replaced with BEBM™ after overnight
incubation and further left to acclimatize for 24 h in supplement-free medium. Cells were
replenished with fresh BEBM™ and treated with elastase together with DMSO or
varying concentrations of symplostatin 5 (1) or sivelestat. Cells were harvested and
lysed with PhosphoSafe™ lysis buffer (Novagen, Madison, WI) after 6 h. The protein
concentration of whole cell lysates was measured with the BCA Protein Assay kit
(Pierce Chemical, Rockford, IL). Equal amounts of protein were separated by SDS-
polyacrylamide gel electrophoresis (4–12%), transferred to polyvinylidene difluoride
(PVDF) membranes, probed with anti-ICAM-1 antibody (Abcam, Cambridge, MA) and
detected with the SuperSignal® West Femto Maximum Sensitivity Substrate (Pierce).
The immunoblots were stripped by heating in a water bath (90 °C) and reprobed with
anti-β-actin antibody (Cell Signaling, Danvers, MA) to confirm equal protein loading.
Isolation of nuclear and cytoplasmic proteins
Cells (150,000/well) were seeded in collagen-coated 10-cm dishes. Culture
media were replaced prior to treatment with elastase and/or elastase+symplostatin 5
(1). After 3 h, the culture supernatant was collected and phosphate-buffered saline
74
supplemented with 1.5 protease inhibitor cocktail (Roche, Indianapolis, IN) was added
to each dish. The cells were lifted from the culture dish using a cell scraper and pelleted
by centrifugation (300g) at 4 °C for 5 min. Cytoplasmic proteins were collected using the
NE-PER® Cytoplasmic Extraction Reagent (Pierce) according to the manufacturer’s
instruction. After collection of the cytoplasmic fraction, the insoluble pellet was washed
with PBS, centrifuged for 1 min and the supernatant was discarded. Nuclear proteins
were isolated from the insoluble pellet. All extracts were incubated on ice, and protein
concentration was determined using the BCA reagent (Pierce).
Measurement of IκBα degradation and NF-B p65 translocation
IBα degradation was assessed by immunoblotting of the collected cytoplasmic
proteins. Equal amounts of the cytoplasmic fraction was loaded and separated in a 4–
12% Bis-Tris HCl gel, transferred on a PVDF membrane and probed with an anti-IBα
antibody (Cell Signaling) and detected with SuperSignal® Femto Max reagent (Pierce).
The blots were stripped after detection by incubating at 90 °C and subsequently probed
with anti-β-tubulin (Cell Signaling) to assess protein loading.
NF-B p65 translocation was measured using the TransAM™ NF-B Chemi p65
kit (Active Motif, Carlsbad, CA) and done according to the manufacturer’s instruction. In
brief, equal amounts of the nuclear protein were prepared in the TransAM™ complete
lysis buffer. The nuclear extracts were added to oligonucleotide coated plates
containing complete binding buffer. This was allowed to incubate at room temperature
with mild agitation for 1 h, washed and incubated with NF-B p65 primary antibody for 1
h. The wells were washed and subsequently incubated for 1 h with anti-rabbit
horseradish peroxidase-conjugated antibody. The chemiluminescent reagent was
75
added after the incubation period and luminescence was measured. The relative NF-B
p65 translocation of elastase and elastase+symplostatin 5 (1) treated cells were
compared to the control. To ascertain the specificity of the measured activity, elastase
treatments were also incubated with wild-type oligonucleotide AM20 which prevented
NF-B p65 binding to the oligonucleotide probe immobilized on the plate. Each
experiment was performed in triplicate. Graphs and data analysis were performed using
the Prism® software and analyzed using ANOVA followed by Dunnett’s t-test.
RNA isolation and reverse transcription
A total of 1.2 106 BEAS-2B cells were seeded in 10-cm dishes and incubated
further for 24 h in supplement-free medium prior to treatment. RNA was isolated at 3
and 6 h post treatment using RNeasy® mini kit (QIAGEN, Valencia, CA). Total RNA was
quantified by UV absorbance. From 2 µg total RNA, cDNA synthesis was done using
SuperScript® II Reverse Transcriptase (Invitrogen, Carlsbad, CA) and oligo(dT)12–18
(Invitrogen).
Real-time quantitative polymerase chain reaction (qPCR)
qPCR after reverse transcription (RT-qPCR) was performed on a 25 µL reaction
solution containing a 1.5 µL aliquot of cDNA, 12.5 µL TaqMan® gene expression master
mix, 1.25 µL of 20 TaqMan® gene expression assay mix and 9.25 µL RNase-free
water. qPCR was carried out on an ABI 7300 sequence detection system using the
thermocycler program: 2 min at 50 °C, 10 min at 95 °C, and 15 s at 95 °C (40 cycles)
and 1 min at 60 °C. Each experiment was performed in triplicate. IL1A
(Hs00174092_m1), IL1B (Hs01555410_m1), and IL8 (Hs00174103_m1) were used as
target genes, while GAPDH (Hs02758991_g1) was used as endogenous control.
76
Graphs and data analysis were performed using the Prism® software and analyzed
using ANOVA followed by Dunnett’s t-test.
Transcriptome profiling
RNA was analyzed using a NanoDrop Spectrophotometer and Agilent 2100
Bioanalyzer to determine the RNA concentration and quality, respectively. RNA
samples were processed using the GeneChip® 3’ IVT Express kit (Affymetrix, Santa
Clara, CA) according to the manufacturer’s instruction. In brief, 250 ng RNA were used
for cDNA synthesis by reverse transcription and the cDNA was utilized as a template for
the biotin-labeled RNA prepared by in vitro transcription reaction. The labeled RNA was
further purified, fragmented and hybridized with rotation at 45 °C for 16 h to the
Affymetrix GeneChip® Human Genome U133 plus 2.0 arrays. The arrays were washed
and stained using the GeneChip® Hybridization Wash and Stain kit on an Affymetrix
Fluidics Station 450. The chips were scanned using a GeneChip® 7G Scanner. Analysis
of the microarray data was done according to the reported method.35 Raw data was
normalized using the Robust Multichip Analysis approach and statistical analysis was
done using the Bioconductor statistical software and R program. The probe set’s
detection call was estimated using the Wilcoxon signed rank-based algorithm. Probe
sets that are absent in all of the study samples were removed from further analyses.
Differential expression analysis was performed using a linear modeling approach and
the empirical Bayes statistics as implemented in the limma package of the R software.
The P values obtained were controlled for multiple testing (false discovery rate) using
the Benjamini-Hochberg method. P value and fold induction were calculated.
Differentially expressed transcripts were ranked by P values, and P < 0.05 and fold
induction >1.5 were considered at a statistically significant level. Hierarchical clustering
77
of the data was computed on log-transformed and normalized data by using complete
linkage and Pearson correlation distances. Computation and visualization were done
with R packages. Gene ontology was performed using the DAVID Bioinformatics
Resources 6.7.118,119 The transcriptome data is deposited in NCBI’s Gene Expression
Omnibus with accession number GSE41600.
78
Figure 3-1. Elastase inhibitors from marine cyanobacteria and the clinically approved
human neutrophil elastase inhibitor sivelestat.
79
Figure 3-2. Selectivity profile of Abu-containing cyclic depsipeptides from marine
cyanobacteria. (A) Screening of lyngbyastatin 7 (10 µM) against a panel of 68 proteases. (B) Selectivity profiling for symplostatin 5 (1) on a panel of 26 serine proteases. Assays were performed by Reaction Biology, Inc.
80
Figure 3-3. Cocrystal structures of natural cyclic depsipeptide elastase inhibitors. (A)
(Fo–Fc) plot for lyngbyastatin 7. (B) Comparison of lyngbyastatin 7 (yellow, PDB ID 4GVU) and scyptolin (white, PDB ID 1OKX) binding to elastase. (C) Comparison of lyngbyastatin 7 (yellow) and FR901277 (green, PDB ID 1QR3) binding to elastase. (D) Ligplot of the lyngbyastatin 7–porcine pancreatic elastase complex. The Abu moiety serves as the key residue for elastase inhibition. Chain designations are (A) elastase, (B) lyngbyastatin 7, (C) H2O. (E) Proposed CH-π interaction between the catalytic Ser203 and the Abu moiety (F) Network of inter- and intramolecular hydrogen bonding interaction in lyngbyastatin 7 mediated by a water molecule. Data courtesy of Ms. Kanchan Taori, Dr. Jean Jakoncic and Dr. David A. Ostrov.
81
Figure 3-4. Changes in cell viability and caspase activation mediated by elastase and
effects of inhibitors. (A) Elastase displayed both time- and dose-dependent decrease in cell viability, with substantial changes at 12–24 h. (B) Symplostatin 5 (1) attenuated the antiproliferative effects of elastase. (C) Sivelestat and the caspase 3 inhibitor Z-D(OMe)E(OMe)VD(OMe)-FMK also partially protected against the antiproliferative effects of elastase. (D) Symplostatin 5 (1) did not show any significant antiproliferative effect on BEAS-2B cells at 24 h. (E) Treatment with 100 nM elastase caused a time-dependent increase in caspase activation which was abrogated by the caspase 3 inhibitor. (F) Incubation of BEAS-2B cells with elastase for 24 h caused a dose-dependent increase in caspase 3/7 activity. Symplostatin 5 (1) attenuated the potency and efficacy of elastase to activate distal caspases. Data are presented as mean ± SEM (n = 2).
82
Figure 3-5. Elastase acts as a sheddase and promotes cell morphology change and
desquamation. (A) Elastase caused cell rounding after incubation for 3 h. Cotreatment with 10 µM symplostatin 5 (1) or sivelestat prevented this effect
of elastase (10 magnification). (B) Significant increase in cell detachment was observed after 12 h of incubation with elastase, which was abrogated by both symplostatin 5 (1) and sivelestat. (C) Levels of mICAM-1 in whole cell lysates in elastase-treated and elastase-inhibitor cotreated cells as assessed by immunoblotting (D) sICAM-1 in culture supernatants of elastase-treated and elastase-inhibitor cotreated cells.Data are presented as mean + SEM, * P < 0.05, ** P < 0.01, *** P < 0.001 compared to HNE-treated control cells using ANOVA, Dunnett’s t-test (n = 3).
83
Figure 3-6. Elastase caused a global change in transcript levels via, in part, an NF-B dependent pathway. (A) Symplostatin 5 (1) dose-dependently inhibited
elastase-induced IB degradation and p65 nuclear translocation at 3 h of cotreatment. (B) Heat map of differentially regulated transcripts by elastase with or without symplostatin 5 (1) cotreatment. Global transcriptome profiling (Affymetrix GeneChip® Human Genome U133 plus 2.0 arrays) was carried out using duplicate biological samples. (C) Validation of the microarray analysis using RT-qPCR. Data are presented as mean + SEM for A and mean + SD for C, * P < 0.05, ** P < 0.01, *** P < 0.001 compared to HNE-treated control cells using ANOVA, Dunnett’s t-test (n = 3).
84
Table 3-1. NMR data of symplostatin 5 (1) and symplostatin 8 (4) in DMSO-d6
Symplostatin 5 Symplostatin 8 unit C/H no δC
a δH (J in Hz)b COSYb HMBCb δCa δH (J in Hz)b
Ile 1 170.0,C e
2 54.0, CH 4.89, br NH 1 54.0, CH 4.87, d
(11.0) 3 37.5, CH 1.86, m H3-6 37.0, CH 1.86, m 4a 25.8, CH2 1.30, m H-4b, H3-5 2, 3 26.0, CH2 1.29, m 4b 1.11, m H-4a, H3-5 2, 3 1.12, m 5 11.2, CH3 0.92, t (7.2) H-4a, H-4b 3 11.4, CH3 0.91, t (7.3) 6 14.1, CH3 0.71, d (7.0) H-3 2, 3 14.3, CH3 0.70, d (6.8) NH 7.40, br H-2 7.40, br N-Me-Phec/ 1 172.7, C
e N-Me-Tyrd 2 60.2, CH 5.00, br H-3a, H-3b 1 60.7, CH 4.90, d
(10.6) 3a 33.4, CH2 3.23, brd
(–13.5) H-2, H-3b 4,5/9 32.7, CH2 3.11, d
(–14.2) 3b 2.84, m H-2, H-3a 5/9 2.70, dd
(–14.2, 10.6) 4 137.9, C
e 5/9 129.4, CH 7.23, d (7.5) H-6 130.3, CH 6.99, d (7.8) 6/8 128.4, CH 7.39, m H-5, H-7 4 115.2, CH 6.77, d (7.8) 7 126.5, CH 7.30, m H-6
e OH 8.13, br s N-Me 30.1, CH3 2.77, s 2, 1 (Phe) 30.3, CH3 2.75, s Phe 1 170.3, C 2 49.6, CH 4.70, dd
(11.4,4.7) H-3a, H-3b 1, 2 (Ahp) 50.0, CH 4.73, m
3a 34.8, CH2 2.84, dd (–14.7,11.4)
H-2, H-3b 4 34.6, CH2 2.87, dd (–14.2, 11.3)
3b 1.68, m H-2, H-3a 4 1.81, m 4 136.5, C
e
85
Table 3-1. Continued
Symplostatin 5 Symplostatin 8 unit C/H no δC
a δH (J in Hz)b COSYb HMBCb δCa δH (J in Hz)b
5/9 129.2, CH 6.77, d (7.5) H-6 7 129.3, CH 6.84, d (7.3) 6 127.6, CH 7.18, m H-5, H-7 4 127.7, CH 7.19, m 7 126.1, CH 7.15, m H-6 126.2, CH 7.15, m Ahp 2 168.7, C
e 3 47.8, CH 3.75, m H-4a, H-4b,
NH 2 48.0, CH 3.79, m
4a 21.7, CH2 2.38, m H-3, H-4b, H-5a
21.9, CH2 2.41, m
4b 1.56, m H-3, H-4a 1.58, m 5a 29.0, CH2 1.68, m H-4a, H-5b,
H-6 29.2, CH2 1.71, m
5b 1.50, m H-5a, H-6 1.56, m 6 73.4, CH 5.03, br s H-5a, H-5b,
OH 2 73.5, CH 5.07, m
OH 6.05, s H-6 6.07, br s NH 7.34, br H-3 7.33, br Abu 1 162.9, C
e 2 130.0, C
e 3 131.7, CH 6.50, q (7.2) H3-4 1, 4 131.5, CH 6.49, q (7.2) 4 12.8, CH3 1.47, d (7.2) H-3 1, 2 13.0, CH3 1.47, q (7.2) NH 9.24, br s 9.21, brs Thr 1
f
e 2 55.1, CH 4.67, br NH 55.2, CH 4.67, m 3 71.5, CH 5.52, br s H3-4
e 5.53, brs 4 17.5, CH3 1.22, d (6.5) H-3 2 17.7, CH3 1.22, d (6.2) NH 8.18, br s H-2 7.70, br s Val 1 172.2, C
e 2 56.4, CH 4.47, t (7.2) NH 1 56.7, CH 4.47, t (7.3) 3 30.7, CH 2.09, m H3-4, H3-5 30.6, CH 2.09, m
86
Table 3-1. Continued
Symplostatin 5 Symplostatin 8 unit C/H no δC
a δH (J in Hz)b COSYb HMBCb δCa δH (J in Hz)b
4 18.9, CH3 0.88, d (6.7) H-3 1 19.1, CH3 0.88, d (7.0) 5 17.5, CH3 0.83, d (6.7) H-3 1 17.5, CH3 0.83, d (7.0) NH 7.71, br s H-2 7.72, br s 2-O-CH3 1 168.9, C
e Glyceric Acid
2 79.9, CH 3.98, dd (7.4,3.4)
H-3a, H-3b 80.2, CH 3.97, dd (7.3, 3.4)
3a 66.1, CH2 3.90, dd (–10.8,3.4)
H-2, H-3b 66.2, CH2 3.89, dd (–10.7, 3.3)
3b 3.73, m H-2, H-3a 3.72, m OCH3 57.1, CH3 3.33g 2 57.3, CH3 3.32g aDeduced from HSQC and HMBC, 600 MHz. b600 MHz. cRefers to symplostatin 5 (1). dRefers to symplostatin 8 (4). eNot determined, predicted to have comparable chemical shifts based on highly homologous structures. fNo correlation observed from HMBC. gOverlapping with residual water.
87
Table 3-2. NMR data of symplostatin 6 (2) and symplostatin 9 (5) in DMSO-d6
Symplostatin 6 Symplostatin 9 unit C/H no δC
a δH (J in Hz)b δC a δH (J in Hz)b
Val 1 170.2, C e
2 56.0, CH 4.68, m 55.7, CH 4.70, m 3 30.7, CH 2.00, m 30.3, CH 2.08, m 4 19.0, CH3 0.89, d (6.8) 18.8, CH3 0.88, d (6.8) 5 17.1, CH3 0.76, d (6.8) 17.0, CH3 0.75, d (6.8) NH 7.51, d (8.8) 7.48, d (8.1) N-Me-Phec/ 1 169.3, C
e N-Me-Tyrd 2 60.3, CH 5.01, d (11.3) 60.5, CH 4.89, d (10.9) 3a 33.4, CH2 3.23, m 32.3, CH2 3.10, d (–14.2) 3b 2.85, m 2.71, m 4 137.9, C
e 5/9 129.4, CH 7.23, d (7.9) 130.1, CH 6.99, d (8.4) 6/8 128.4, CH 7.39, m 114.8, CH 6.77, d (8.4) 7 126.5, CH 7.30, m N-Me 30.2, CH3 2.79, s 29.9, CH3 2.76, s OH Phe 1 170.3, C
e 2 49.7, CH 4.71, dd (11.8,4.4) 49.8, CH 4.73, dd (11.4, 3.8) 3a 34.9, CH2 2.85, m 34.9, CH2 2.87, dd (–14.4, 11.4) 3b 1.69, m 1.81, dd (–14.4, 3.8) 4 136.5, C
e 5/9 129.1, CH 6.77, d (7.5) 129.1, CH 6.84, d (7.6) 6 127.6, CH 7.18, m 127.4, CH 7.19, m 7 126.0, CH 7.14, m 126.0, CH 7.14, m Ahp 2 168.7, C
e 3 47.9, CH 3.76, m 47.7, CH 3.78, m 4a 21.6, CH2 2.42, m 21.5, CH2 2.42, m 4b 1.56, m 1.57, m
88
Table 3-2. Continued
Symplostatin 6 Symplostatin 9 unit C/H no δC
a δH (J in Hz)b δCa δH (J in Hz)b
5a 29.1, CH2 1.70, m 29.0, CH2 1.71, m 5b 1.51, m 1.56, m 6 73.5, CH 5.04, s 73.3, CH 5.07, s OH 6.10, br s NH 7.23, br s 7.23, br s Abu 1 163.0, C
e 2 129.9, C
e 3 131.6,CH 6.51, q (7.0) 131.6, CH 6.51, q (7.1) 4 12.9, CH3 1.49, d (7.0) 12.9, CH3 1.49, d (7.1) NH 9.20, br s Thr 1
e
e 2 55.2, CH 4.65, m 54.8, CH 4.65, m 3 71.4, CH 5.54, br s 71.2, CH 5.54, br s 4 17.6, CH3 1.23, d (6.3) 17.4, CH3 1.23, d (6.4) NH 7.80, br 2 7.70, br s Val 1 171.7, C
e 2 56.5, CH 4.47, m 56.3, CH 4.46, m 3 30.4, CH 2.09, m 30.3, CH 2.09, m 4 19.0, CH3 0.89, d (6.3) 18.8, CH3 0.88, d (6.8) 5 17.5,CH3 0.82, d (6.7) 17.3, CH3 0.82, d (6.8) NH 8.13 br s 8.18, br s 2-O-CH3 1 168.9, C
e Glyceric Acid 2 80.0, CH 3.98, dd (7.3,3.4) 79.8, CH 3.98, dd (7.3, 3.4) 3a 66.0, CH2 3.90, dd (–11.1, 3.4) 65.8, CH2 3.89, dd (–10.9, 3.4) 3b 3.74, dd (–11.1, 7.3) 3.73, dd (–10.9,7.3) OCH3 57.4, CH3 3.32f 57.0, CH3 3.33f aDeduced from HSQC, 600 MHz. b600 MHz. cRefers to symplostatin 6 (2). dRefers to symplostatin 9 (5). eNot determined, predicted to have comparable chemical shifts based on highly homologous structures. fOverlapping with residual water.
89
Table 3-3. NMR data of symplostatin 7 (3) and symplostatin 10 (6) in DMSO-d6
Symplostatin 7 Symplostatin 10 unit C/H no δC
a δH (J in Hz)b δCa δH (J in Hz)b
Ile 1 e
e 2 54.1, CH 4.88, br d 53.9, CH 4.88, m 3 37.0, CH 1.88, m 36.9, CH 1.86, m 4a 26.0, CH2 1.30, m 25.8, CH2 1.29, m 4b 1.12, m 1.12, m 5 11.3, CH3 0.92, t (7.2) 11.9, CH3 0.91, t (7.4) 6 14.4, CH3 0.71, d (6.7) 14.2, CH3 0.70, d (6.8) NH 7.44, br s 7.38, br s N-Me-Phec/ 1
e e
N-Me-Tyrd 2 60.3, CH 5.01, br d 60.6, CH 4.90, m 3a 33.6,CH2 3.24, br d (–13.4) 32.5, CH2 3.11, d (–14.2) 3b 2.85, m 2.70, dd (–14.2,11.8) 4
e e
5/9 129.5, CH 7.24, d (7.6) 130.2, CH 6.99, d (8.3) 6/8 128.4, CH 7.40, m 115.1, CH 6.76, d (8.3) 7 126.6, CH 7.31, m N-Me 30.2, CH3 2.79, s 30.0, CH3 2.74, s OH 9.31, br s Phe 1
e e
2 49.9, CH 4.72,m 49.9, CH 4.72, dd (11.4,4.8) 3a 35.0, CH2 2.85, m 35.0, CH2 2.87, (–14.3,12.3) 3b 1.69, m 1.79, m 4
e e
5/9 129.2, CH 6.83, d (7.4) 129.2, CH 6.83, d (7.2) 6 127.6, CH 7.19, m 127.6, CH 7.19, m 7 126.1, CH 7.16, m 126.0, CH 7.14, m Ahp 2
e e
3 47.9, CH 3.77, m 47.9, CH 3.77, m 4a 21.8, CH2 2.39, m 21.8, CH2 2.39, m
90
Table 3-3. Continued
Symplostatin 7 Symplostatin 10 unit C/H no δC
a δH (J in Hz)b δCa δH (J in Hz)b
4b 1.57, m 1.57, m 5a 29.1, CH2 1.71, m 29.1, CH2 1.71, m 5b 1.55, m 1.55, m 6 73.5, CH 5.06, s 73.5, CH 5.06, br s OH 6.04, s 6.03, s NH 7.35, br s 7.35, br s Abu 1
e e
2 e
e 3 131.4, CH 6.48, q (7.1) 131.4, CH 6.48, q (7.2) 4 12.8, CH3 1.46, d (7.1) 12.8, CH3 1.46, d (7.2) NH 9.22, br s 9.22, br s Thr 1
e e
2 55.0, CH 4.68, m 55.0, CH 4.68, m 3 71.6, CH 5.52, br s 71.6, CH 5.52, br s 4 17.5, CH3 1.21, d (6.5) 17.5, CH3 1.21, d (6.3) NH 8.24, br s 8.19, br s Ile 1
e e
2 55.8, CH 4.48, m 55.8, CH 4.48, m 3 36.9, CH 1.86, m 36.9, CH 1.85, m 4a 23.7, CH2 1.43, m 23.7, CH2 1.43, m 4b 1.06, m 1.06, m 5 10.6, CH3 0.80, t (7.4) 10.6, CH3 0.80, t (7.4) 6 15.0, CH3 0.85, d (6.5) 15.0, CH3 0.85, d (6.8) NH 7.75, br s 7.73, br s 2-O-CH3 1
e e
Glyceric Acid 2 79.9, CH 3.96, dd (7.5,3.4) 79.9, CH 3.96, dd (7.3,3.2) 3a 66.1, CH2 3.88, dd (–10.8,3.4) 66.1, CH2 3.88, dd (–10.7,3.2) 3b 3.72, dd (–10.8,7.5) 3.72, dd (–10.7,7.3)
91
Table 3-3. Continued
Symplostatin 7 Symplostatin 10 unit C/H no δC
a δH (J in Hz)b δCa δH (J in Hz)b
OCH3 57.1, CH3 3.31f 57.1, CH3 3.31 f aDeduced from HSQC, 600 MHz. b600 MHz. cRefers to symplostatin 7 (3). dRefers to symplostatin 10 (6). eNot determined, predicted to have comparable chemical shifts based on highly homologous structures. fOverlapping with residual water.
92
Table 3-4. Antiproteolytic activity of Abu-containing cyclic depsipeptides from marine cyanobacteriaa
Compound Porcine Pancreatic Elastaseb
IC50 (nM)
Human Neutrophil Elastasec
IC50 (nM)
Bovine Pancreatic Chymotrypsind
IC50 (nM)
Human Pancreatic Chymotrypsine
IC50 (nM) (% Activity at 10 µM)
Symplostatin 5 68 9.7 144 2.9 322 3.2 > 10000
(53.4 3.2) Symplostatin 6 89 11 121 12 503 65 > 10000
(90.6 7.6) Symplostatin 7 77 5.4 195 28 515 43 > 10000
(70.7 3.8) Symplostatin 8 43 3.2 41 9.0 268 11 > 10000
(69.0 2.0) Symplostatin 9 37 3.1 28 5.8 324 27 > 10000
(73.9 1.0) Symplostatin 10 44 1.5 21 2.9 222 5.1 > 10000
(79.4 3.2) Lyngbyastatin 4 41 2.0 49 1.4 614 6.3 > 10000
(72.2 3.3) Lyngbyastatin 7 30 6.8 23 1.1 314 37 2000 Sivelestat 2810 95 136 18 4084 37 > 10000
(55.1 3.3) aData are presented as mean SD (n = 3). b–eSubstrates. bN-succinyl-Ala-Ala-Ala-p-nitroanilide. cN-(methoxysuccinyl)-Ala-Ala-Pro-Val-p-nitroanilide. dN-succinyl-Gly-Gly-Phe-p-nitroanilide. eN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide.
93
Table 3-5. Non-inflammatory elastase-inducible genes
Probe ID Symbol Annotation Fold inductiona
% Reductionb
Transcription Factors 203394_s_at HES1 Hairy and enhancer of split 1
(Drosophila) 2.98 58c
215898_at TTLL5 Tubulin tyrosine ligase-like family, member 5
2.96 62c
215191_at KDM2A Lysine (K)-specific demethylase 2A
2.50 54
215470_at GTF2H2 General transcription factor IIH, polypeptide 2, 44kDa
2.32 47
243561_at YAF2 YY1 associated factor 2 2.18 47 230791_at NFIB Nuclear factor I/B 2.12 50 232865_at AFF4 AF4/FMR2 family, member 4 2.10 49c 1556462_a_at KLF12 Kruppel-like factor 12 1.93 47c 232431_at NR3C1 Nuclear receptor subfamily 3,
group C, member 1 (glucocorticoid receptor)
1.91 53c
240008_at ARID1B AT rich interactive domain 1B (SWI1-like)
1.89 54c
Other Targets 232528_at NA NA 3.67 59c 238774_at KIAA1267 KIAA1267 3.62 64 219995_s_at ZNF750 Zinc finger protein 750 3.62 68c 230332_at ZCCHC7 Zinc finger, CCHC domain
containing 7 3.30 67c
235847_at ZFAND3 Zinc finger, AN1-type domain 3 3.29 60c 23149_at EIF4G3 Eukaryotic translation initiation
factor 4 gamma 3 3.22 59
1564378_a_at EXT1 Exostoses (multiple) 1 3.20 67c 234989_at NEAT1 Non-protein coding RNA 84 3.08 48c 241457_at FBXL7 F-box and leucine-rich repeat
protein 7 3.06 64c
242476_at NA NA 2.95 64c 207746_at POLQ Polymerase (DNA directed),
theta 2.46 62c
1559360_at EFNA5 Ephrin-A5 2.43 63c 1554638_at ZFYVE16 Zinc finger, FYVE domain
containing 16 2.24 60c
1563075_s_at NA NA 2.21 59c 224917_at MIR21 MicroRNA 21 2.08 43c aRelative to control, P < 0.05. bIn response to inhibitor cotreatment. cSignificant difference with inhibitor treatment, P < 0.05.
94
Table 3-6. Relevant genes involved in NOD- and MAPK- signaling pathways significantly modulated by elastase
Probe ID Symbol Annotation Fold Inductiona
% Reductionb
39402_at IL1B Interleukin 1B 2.91 58c 241786_at PPP3R1 Protein phosphatase 3,
regulatory subunit B 2.42 56c
205207_at IL6 Interleukin 6 2.26 22 1569540_at NLK Nemo-like kinase 2.23 49 239409_at RAP1A RAP1A, member of RAS
oncogene family 2.21 53c
230337_at SOS1 Son of sevenless homolog 1 1.89 46c 210118_at IL1A Interleukin 1A 1.85 34 1565889_at TAB2 Mitogen-activated kinase
kinase 7 interacting protein 1.83 42
211506_at IL8 Interleukin 8 1.53 22 aRelative to control, P < 0.05. bIn response to inhibitor cotreatment. cSignificant difference with inhibitor treatment, P < 0.05.
95
Table 3-7. Symplostatin 5 (1)-inducible genes potentially independent of elastasea
Probe ID Symbol Annotation P-value Fold Inductionb
243598_at GPD2 Glycerol-3-phosphate dehydrogenase 2 (mitochondrial)
0.03 2.15
242824_at NFIA Nuclear factor I/A 0.03 2.02 229728_at NA NA 0.03 2.01 236545_at PPP3CA Protein phosphatase 3 (formerly
2B), catalytic subunit, alpha isoform
0.04 1.97
233037_at NA NA 0.03 1.96 242696_at NUDCD3 NudC domain containing 3 0.05 1.94 226840_at H2AFY H2A histone family, member Y 0.04 1.88 236685_at NA NA 0.04 1.79 1553145_at FLJ39653 Hypothetical FLJ39653 0.05 1.67 aThese genes were not significantly affected by elastase treatment. bRelative to control.
96
Table 3-8. Reaction conditions for protease assays
Protease Substrate [Sub] μM
Ex/Em or λmax
Buffera
ACE1 MCA-RPPGFSAFK(Dnp) 10 320/405 A Activated Protein C(H) in 50% gly
Boc-DVLR-ANSNH-C4H9 50 355/460 C
ADAM9 MCA-PLAQAV-Dpa-RSSSR-NH3 10 320/405 I ADAM10 MCA-PLAQAV-Dpa-RSSSR-NH3 10 320/405 I BACE1 MCA-SEVNLDAEFRK(Dnp)-RR-
NH2 10 320/405 J
Calpain 1 Biomol, N-Succinyl-Leu-Tyr-AMC 10 355/460 K Caspase 1 Ac-LEHD-AMC 5 355/460 G Caspase 2 Ac-LEHD-AMC 5 355/460 G Caspase 3 Ac-DEVD-AMC 5 355/460 F Caspase 4 Ac-LEHD-AMC 5 355/460 G Caspase 5 Ac-LEHD-AMC 5 355/460 G Caspase 6 Ac-LEHD-AMC 5 355/460 G Caspase 7 Ac-DEVD-AMC 5 355/460 F Caspase 8 Ac-LEHD-AMC 5 355/460 G Caspase 9 Ac-LEHD-AMC 5 355/460 G Caspase 10 Ac-LEHD-AMC 5 355/460 G Caspase 11 Ac-LEHD-AMC 5 355/460 G Caspase 14 Ac-LEHD-AMC 5 355/460 G Cathepsin B Z-FR-AMC 5 355/460 L Cathepsin C Z-FR-AMC 5 355/460 L Cathepsin D MCA-KPILFFRLK(Dnp)-D-R-NH2 10 320/405 P Cathepsin E MCA-KPILFFRLK(Dnp)-D-R-NH2 10 320/405 P Cathepsin G Suc-AAPF-AMC 10 355/460 C Cathepsin H R-AMC 10 355/460 M Cathepsin K Z-GPR-AMC 10 355/460 C Cathepsin S Z-FR-AMC 10 355/460 M Cathepsin V Z-FR-AMC 10 355/460 E Cathepsin X/Z MCA-RPPGFSAFK(Dnp) 10 320/405 D Chymase Suc-AAPF-AMC 10 355/460 C Chymotrypsin (Human Pancreatic)
Suc-AAPF-pNa 3 405 U
Chymotrypsin (Bovine Pancreatic)
Suc-GGF-pNa 1.5 405 T
Complement Component C1s (CCC1s)
Dabcyl-SLGRKIQI-EDANS 10 340/490 A
DPP IV H-GP-AMC 10 355/460 H DPP VIII H-GP-AMC 10 355/460 H DPP IX H-GP-AMC 10 355/460 H
97
Table 3-8. Continued
Protease Substrate [Sub] μM
Ex/Em or λmax
Buffera
Elastase (Human Neutrophil)
(OMeSuc)-AAPV-pNa 2 405 V
Elastase (Porcine Pancreatic)
Suc-AAA-pNa 2 405 S
Factor VIIa Z-VVR-AMC 10 355/460 A Factor Xa CH3SO2-D-CHA-Gly-Arg-AMC-
AcOH 10 355/460 N
Factor XIa (Boc-Glu(OBzl)-Ala-Arg)-MCA 10 355/460 A Granzyme B Ac-IEPD-AMC 10 355/460 A Hepatitis C virus NS3/4A protease
Anaspec EnzoLyte (Hylite Biosciences, Catalogue: 22991)
10 340/490 R
Kallikrein 1 Z-GPR-AMC 10 355/460 A Kallikrein 5 Z-VVR-AMC 10 355/460 A Kallikrein 8 VPR-AMC 10 380/460 E Kallikrein 12 VPR-AMC 10 380/460 B Kallikrein 13 VPR-AMC 10 380/460 A Kallikrein 14 VPR-AMC 10 380/460 A MMP1 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP2 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP3 (5-FAM/QXLTM) FRET peptide 5 485/520 H 3-7MMP7 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP8 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP9 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP10 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP11 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP12 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP13 (5-FAM/QXLTM) FRET peptide 5 485/520 H MMP14 (5-FAM/QXLTM) FRET peptide 5 485/520 H Papain Z-FR-AMC 10 355/460 M Plasma Kallikrein Z-FR-AMC 10 380/460 A Plasmin H-D-CHA -Ala-Arg-AMC.2AcOH 10 355/460 A Proteinase K H-D-CHA -Ala-Arg-AMC.2AcOH 10 355/460 A TACE MCA-PLAQAV-Dpa-RSSSR-NH2 10 320/405 I Thrombin alpha H-D-CHA -Ala-Arg-AMC.2AcOH 10 355/460 O Tissue Plasminogen Activator
Z-GPR-AMC 10 355/460 Q
Trypsin H-D-CHA -Ala-Arg-AMC.2AcOH 10 355/460 A Tryptase beta 2 Z-GPR-AMC 10 355/460 A Tryptase gamma 1 Z-GPR-AMC 10 355/460 A Urokinase Bz-b-Ala-Gly-Arg-AMC.AcOH 10 355/460 A
98
Table 3-8. Continued aBuffers A 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35 B 50 mM Tris pH 7.5, 10 mM CaCl2, 150 mM NaCl, 0.05% Brij35 C 25 mM Tris pH 9, 150 mM NaCl D 25 mM Sodium Acetate pH 3.5, 5 mM DTT E 25 mM Sodium Acetate pH 5.5, 0.1 M NaCl, 5 mM DTT F 50 mM HEPES pH 7.4, 100 mM NaCl, 0.01% CHAPS, 0.1 mM
EDTA, 10 mM DTT G 50 mM HEPES pH 7.4, 1 M sodium citrate, 100 mM NaCl, 0.01%
CHAPS, 0.1 mM EDTA, 10 mM DTT H 50 mM HEPES pH 7.5, 100 mM CaCl2, 0.01% Brij35, store at 4°C,
add 0.1 mg/mL BSA before use I 25 mM Tris pH 9.0, 25 μM ZnCl2, 0.005% Brij J 0.1 M Sodium acetate, pH 4.0 K 75 mM Tris pH 7.0, 0.005% Brij35, 3 mM DTT, 0.5 mM CaCl2 L 25 mM MES pH 6.0, 50 mM NaCl, 0.005% Brij35, 5 mM DTT M 75 mM Tris pH 7.0, 1 mM EDTA, 0.005% Brij35, 3 mM DTT N 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 0.25 mg/mL BSA O 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 2.5 mM CaCl2, 1.0
mg/mL BSA P 0.1 mM Sodium Acetate pH 3.5, 0.1 M NaCl Q 25 mM Tris pH 8.0, 100 mM NaCl, 0.01% Brij35, 1.0% BSA R Assay kit Buffer S 1.0 M Tris pH 8.0 T 50 mM Tris pH 7.8, 100 mM NaCl, 1 mM CaCl2 U 0.1 M Tris pH 8.3, 25 mM CaCl2 V 0.1 M Tris pH 7.5, 0.5 M NaCl
99
Table 3-9. Crystallography data and refinement statistics
PDB Title Elastase/Lyngbyastatin 7 complex
PDB ID 4GVU Data collection Space group P212121 Cell dimensions
A,b, c (Å) (α==γ= 90°) 53.40 57.42 74.33
Resolution (Å) 30.00-1.55 (1.56-1.55) Total reflections 257532 Unique reflections 33573 (699) Rmerge 0.044 (0.55) I/σI 41.1 (2.0) Completeness (%) 99.6 (84.0) Redundancy 7.7 (4.9) Mean Mosaicity (°) 0.47 Wilson B factor (A2) 20.6 Matthews coefficient (A3Da-1) 2.18 Solvent content (%) 43.7 Refinement Resolution (Å) 28.71-1.55 (1.60-1.55) No. reflections 33518 Rwork/ Rfree (%) 17.6 / 20.6 (27.2 / 33.0) No. atoms : All 2139
Protein 1822 SO4 / Ca / Lyngbyastatin7 5 / 1 / 68 Water 243
B-factors (Å2) : All 24.4 Protein : all / main / side 23.2 / 21.9 / 24.6 SO4 / Ca / Lyngbyastatin7 29.1 / 21.1 / 21.6 Water 34.0
RMSD Bond lengths (Å) 0.013 Bond angles (º) 1.709
Ramachandran plot % residues Favored 85.4 Additional 14.6 Generously allowed 0 Disallowed 0
Numbers in parentheses refer to the highest resolution shell.
100
CHAPTER 4 VERAGUAMIDES A–G: CYTOTOXIC CYCLIC HEXADEPSIPEPTIDES WITH A C8-
POLYKETIDE-DERIVED β-HYDROXY ACID MOIETY FROM CETTI BAY, GUAM
Introduction
Marine cyanobacteria have provided both structurally diverse and potent
antiproliferative compounds with varying mechanisms of action as lead structures for
drug discovery.20,21 Most of these are products of nonribosomal peptide synthetases or
mixed nonribosomal peptide synthetases and polyketide synthases to yield cyclic and
linear modified peptides and depsipeptides. Cyanobacteria utilize mainly nonpolar and
neutral proteinogenic amino acids as building blocks, commonly Val, Ala, Phe, Tyr, Pro,
and Ile.20 These proteinogenic amino acids may be further modified by N- or O-
methylation, halogenation, or epimerization to yield the unnatural D- amino acids. Amino
acids such as Cys or Ser can undergo cycloaddition with other amino acids to yield
heterocyclic moieties such as thiazoline/thiazole or oxazoline/oxazole rings.120,121
Marine cyanobacteria may also incorporate α-hydroxy acids, β-hydroxy acids and β-
amino acids as building blocks of the peptide–polyketide hybrid compounds. In addition,
fatty acid type moieties consisting of four to twelve carbons in length are also a
signature polyketide-derived residue in marine cyanobacteria, and oftentimes bear a
mono- or dimethylation at the α-carbon position and decorated by a terminal alkyne,
alkene, or halogenated alkyne functionality.20,122–125 A recent survey of cyanobacteria
metabolites containing a 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya) or a 3-
hydroxy-2-methyl-7-octynoic acid suggested that these lipopeptides are widely
Reproduced in part with permission from Salvador, L. A.; Biggs, J. S.; Paul, V. J.; Luesch, H. J. Nat.
Prod. 2011, 74, 917–927. Copyright 2011 American Chemical Society.
101
distributed and may be products of an ancient biosynthetic pathway common across
different cyanobacteria genera.126 Thus, this class of secondary metabolites from
marine cyanobacteria clearly depicts Nature’s superior peptidomimetic machinery.
Incorporation of unnatural amino acids as well as polyketide derived units, aside from
increasing the structural diversity, is also postulated to contribute to the stability of this
class of compound against hydrolytic cleavage.127
From the screening profile of several cyanobacteria collection, we prioritized a
Symploca cf. hydnoides from Cetti Bay, Guam for discovery of new antiproliferative
agents. This collection showed antiproliferative activity, with the active principle not
related to the known compounds dolastatin 10, symplostatin 1 or largazole. Presented
herein is the cytotoxicity-directed fractionation of this S. cf. hydnoides collection from
Cetti Bay, Guam, which afforded the known compound dolastatin 16,128 together with
seven new cyclic depsipeptides, given the trivial names veraguamides A–G (7–13). The
trivial names were assigned to conform with the naming by W. Gerwick and co-workers,
who concurrently isolated members of this compound class.129 Initial structure-activity
relationship studies and effects on cancer cell populations of the veraguamides are also
discussed.
Isolation and Structure Elucidation
The freeze-dried Symploca sp. cyanobacterium from Cetti Bay, Guam was
extracted with EtOAc–MeOH (1:1). This extract showed antiproliferative activity at a
concentration of 1 μg/mL and did not contain largazole, symplostatin 1 or dolastatin 10
from initial profiling data. This extract was further solvent-partitioned into hexanes-, n-
BuOH, and H2O-soluble fractions, with the n-BuOH-soluble fraction being the most
cytotoxic. This fraction was further purified by silica column chromatography, the
102
fraction eluting with 20% i-PrOH in CH2Cl2 showed characteristic 1H NMR resonances
for peptides and modified peptides and potent antiproliferative activity. Reversed-phase
HPLC purification of this silica fraction yielded veraguamides A–G (7–13) (Figure 4-1).
HRESIMS of the major compound in this series, veraguamide A (7) (Figure 4-1),
showed the distinctive 1:1 isotopic cluster for a Br-containing compound for the [M + H]+
peak at m/z 767.3675/769.3660, suggesting a molecular formula of C37H59BrN4O8. The
1H NMR spectrum of 7 displayed characteristic peptide resonances for a secondary
amide proton (δH 6.25), two tertiary amide N-CH3’s (δH 3.00, δH 2.94) and several α-
protons (δH 3.85–4.95). 2D NMR analysis (Table 4-1) in CDCl3 using HSQC, COSY,
TOCSY, and HMBC established the presence of four amino acids (Pro, Val, 2 × N-Me-
Val) and an α-hydroxy acid [(2-hydroxy-3-methylpentanoic acid (Hmpa)]. The last spin
system consisted of a CH3 doublet (δH 1.25) that showed a COSY correlation to a
methine (δH 3.11) and HMBC correlations to a carbonyl (δC 170.8) and an oxymethine
(δC 76.4). Further extension of this unit using HMBC and COSY established the
presence of a 8-bromo-3-hydroxy-2-methyl-7-octynoic acid (Br-Hmoya) moiety in 7. This
was supported by HMBC correlations of the methylene (δC-6 19.2/δH₂-6 2.23) with two
quaternary carbons at δC 38.4 and δC 79.3 and by the large difference in chemical shifts
between these quaternary carbons characteristic for an alkynyl bromide.130 The linear
sequence of N-Me-Val-1–Pro–Hmpa–N-Me-Val-2–Val–Br-Hmoya was established
based on HMBC correlations between α-protons and carbonyl groups (Table 4-1) and
was verified by MS/MS fragmentation (Figure 4-2). The deshielded C-3 methine of the
Br-Hmoya unit suggested acylation with the carbonyl of N-Me-Val-1 to form a cyclic
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hexadepsipeptide, corroborated by HMBC and consistent with the molecular formula
requirements based on HRESIMS.
Veraguamide B (8) showed a 1:1 isotopic pattern for the pseudomolecular ion [M
+ H]+ at m/z 753.3517/755.3508, suggesting the presence of a Br as in 7 with a
negative difference of 14 amu corresponding to one less CH2 unit and thus a molecular
formula of C36H57BrN4O8. Comparison of the 1H NMR spectrum of 7 and 8 showed
differences in the splitting pattern in the CH3 region at δH 0.93 ppm and the chemical
shift of the α-proton (δH 4.85) of the α-hydroxy acid (Table 4-2). The vicinal methine (δH
2.17) of the α-hydroxy acid showed COSY correlations to two methyl groups (δH 0.93,
δH 1.02) instead of COSY correlations to methylene and methyl protons in 7. Therefore,
8 possesses a 2-hydroxyisovaleric acid (Hiva) instead of the Hmpa unit as in 7 (Figure
4-1).
The HRESIMS spectrum of veraguamide C (9) showed a negative deviation of
79 amu compared with 7 which indicated the lack of Br and a molecular formula of
C37H60N4O8. This was supported by the absence of the 1:1 isotopic pattern for the [M +
H]+ peak when compared to 7. The 1H NMR spectrum of 9 showed an additional triplet
at δH 1.93 with JH,H = 2.5 Hz (Table 4-2); otherwise it was virtually identical to that of 7.
This proton correlated to a methine at δC 68.8 and a quaternary C (δC 83.6) in the
HSQC and HMBC spectra, respectively. These signals are indicative of a terminal
alkyne; hence 9 had to bear a 3-hydroxy-2-methyl-7-octynoic acid (Hmoya) moiety in
lieu of Br-Hmoya present in 7 and 8 (Figure 4-1).
Veraguamide D (10) appeared closely related to 9 as its 1H NMR spectrum
showed the acetylenic proton at δH 1.93 (Table 4-3). In comparison to 9, the HRESIMS
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of 10 showed a positive difference of 14 amu, corresponding to an additional CH2 unit
and in agreement with a molecular formula of C38H62N4O8. The 1H NMR and HSQC
spectra of 10 showed a high-field CH3 at δC 11.4/δH 0.73, characteristic of an Ile or Ile-
derived moiety. COSY (δH/δH 0.93/1.46, 1.46/2.02, 2.02/4.01) and HMBC correlations
(δC/δH 15.8/4.01, 28.7/4.01) correlations (Table 4-3) established that the N-Me-Val-1
residue is replaced by an N-Me-Ile in veraguamide D (10) (Figure 4-1).
Compound 11 (C39H64N4O8) exhibited a close relationship to both 9 and 10,
showing a positive deviation of 28 amu and 14 amu, respectively, and also having the
Hmoya moiety. The NMR data of 11 (Table 4-3) indicated the presence of two high-field
methyl (δC 11.7/δH 0.96, δC 11.5/δH 0.85) and additional methylene (δC 26.6/δH 1.57,
1.06; δC 23.9/δH 1.47, 1.05) groups in comparison with 9, which suggested that two
isopropyl groups in the latter are replaced with sec-butyl groups in the former (Figure 4-
1). This is further corroborated by HMBC and COSY correlations (Table 4-3), which
established the replacement of Val and N-Me-Val-2 moieties with Ile and N-Me-Ile,
respectively, in veraguamide E (11). The N-Me-Val residue that was replaced by N-Me-
Ile was located at different positions in 10 and 11; with N-Me-Val-1 replaced in the
former and N-Me-Val-2 in the latter. This NMR result was verified by MS/MS
fragmentation of both 10 and 11 (Figure 4-2).
The 1H NMR spectrum of veraguamide F (10) (Table 4-4) showed additional
resonances for aromatic protons at δH 7.2 – 7.4 ppm, upfield-shifted N-Me protons to δH
2.60 ppm presumably due to the shielding by the aromatic ring, and a low-field α-proton
of the hydroxy acid (δH 5.47), with the acetylenic proton still present (δH 1.93). COSY
correlations of δH 5.47 to diastereotopic CH2 protons at δH 3.17/δH 2.91, together with
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HMBC correlations of the latter to aromatic carbons at δC 136.2/δC 129.3 (Table 4-4)
established the presence of phenyllactic acid (Pla) as the α-hydroxy acid in 12 (Figure
4-1). These NMR-derived conclusions fulfilled the molecular formula requirements for
C40H58N4O8 based on HRESIMS of 12.
Veraguamide G (13) lacked the acetylenic signal (δC 68.8/δH 1.93) observed for
9–12 and instead showed downfield resonances of a terminal methylene (δC 114.9/δH
4.97) and a methine (δC 138.2/δH 5.74) (Table 4-5). These signals indicated that the
terminal alkyne group of the C8-polyketide derived moiety is replaced by a terminal vinyl
group (Figure 4-1). This conclusion was further supported by the positive deviation of 2
amu compared to 9 and a molecular formula of C37H62N4O8. Hence, the Hmoya unit
present in 9–12 was replaced by 3-hydroxy-2-methyl-7-octenoic acid (Hmoea) in 13.
Enantioselective HPLC analysis coupled with mass spectrometry or UV detection
of the acid hydrolysates of 7–12 allowed us to assign the absolute configuration of all
the amino acids and α-hydroxy acid components as L and S, respectively. To determine
the absolute configuration at C-2 and C-3 of the Br-Hmoya unit, veraguamide A (7) was
subjected to methanolysis to yield the linear fragment 15 (Figure 4-3). The observed
coupling constant of 3.2 Hz was characteristic for a syn configuration, whereas a
coupling constant near 6.3 Hz would have been expected for the anti configuration.131
The absolute configuration at C-3 and consequently for C-2 of the Br-Hmoya unit of 15
was determined using Mosher’s analysis. The derived Δδ values (Figure 4-3) predicted
an R configuration at C-3 and hence from the relative configuration, C-2 should have an
S configuration. Of note, comparison of the 3JH,H values of H-2 and H-3 with a model
system to assign the relative configuration could only be applied when C-3 bears a free
106
hydroxy group. This moiety is involved in intramolecular hydrogen bonding with the
adjacent carbonyl group, thus hindering free bond rotation across C-2 and C-3.132,133
Accordingly, the corresponding MTPA-esters (16–17) did not show the same 3JH,H
values for H-2 and H-3 as that of 15. The same absolute configuration at C-2 and C-3
for Hmoya, Hmoea and 3-hydroxy-2-methyl-octanoic acid (Hmoaa) is expected based
on virtually identical 13C NMR shifts and specific optical rotations observed for 7–13.
Biological Activity Studies
To gain insight into structure–activity relationships, veraguamide A (7) was
partially (Lindlar catalyst, H2) and fully (Pd/C, H2) hydrogenated to yield the
semisynthetic veraguamide G (13) and tetrahydroveraguamide A (14) (Figure 4-1),
respectively. The cytotoxic activities of 7–14 and semisynthetic veraguamide G (13)
were evaluated for effects on viability of HT29 colorectal and HeLa cervical
adenocarcinoma cells (Table 3-6). The IC50 values of the natural and semisynthetic
veraguamide G (13) were comparable suggesting the activities of these compounds
were not likely due to traces of highly biologically active impurities. The most active in
this series of compounds are veraguamides D (10) and E (11), with IC50 values more
than 5-fold lower than those for their related congener veraguamide C (9). This
suggested that increased hydrophobicity of specific units (II, IV, V, VI) increased the
cytotoxicity of this compound class, with the position having minimal effect on the
bioactivity as 10 and 11 showed comparable IC50s. However, modification with bulkier
groups is detrimental to the activity, as exemplified by a close to 10-fold decrease in the
cytotoxic activity of 12 (Table 4-6) compared to 9, where a phenyllactic acid (Pla) moiety
is introduced at position IV of the former instead of the Hmpa unit. The C8-polyketide
derived moiety also plays a role in the cytotoxicity of these compounds. Comparing the
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biological activities of related compounds 7, 9, 13, and 14, weaker cytotoxicity was
observed for compounds with a Br-Hmoya or Hmoaa unit, while compounds with
Hmoya or Hmoea were about equally potent. This then suggests the importance of a π-
system combined with the presence of acetylenic or vinylic protons in this moiety for
cytotoxic activity.
In order to gain insights on the possible mode of cell death mediated by
veraguamides, cell cycle analysis by flow cytometry was performed. HeLa and HT29
cells were treated with 1.0, 3.2, and 10 μM veraguamide D (10) for 24 h, permeabilized
with EtOH and stained with propidium iodide. A dose-dependent increase in cell
populations at sub-G1 and G2 were observed with veraguamide D (10) (Figure 4-4).
The observed change in cell populations was, however, incremental. This further
suggested that the veraguamides are not likely to act as antimitotics. Antimitotic agents
such as the dolastatins, paclitaxel and vinca alkaloids cause a dramatic increase in cells
at G2/M.134 The result of the cell cycle analysis also corroborates the moderate
antiproliferative activity observed using the MTT assay.
The veraguamides are reminiscent of other cyanobacterial compounds such as
hantupeptins,133,135 antanapeptins,124 and trungapeptins.125 These compounds are also
cyclic hexadepsipeptides with the characteristic C8-polyketide derived units as Hmoya,
Hmoea, or Hmoaa. Veraguamide F (12) is a constitutional isomer of antanapeptin D,124
where an N-Me-Phe and Hiva are present in the latter instead of Pla and N-Me-Val as in
12. It is interesting that subtle changes in structure of these compounds have a
profound effect on the cytotoxicity. Antanapeptins A–D (brine shrimp) as well as
trungapeptin A (KB and LoVo cells) did not display cytotoxicity at the reported
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concentrations (10 μg/mL),124,125 while hantupeptins A–C were cytotoxic against MOLT-
4 leukemia and MCF7 breast cancer cells, with hantupeptin A being the most active in
this series with IC50 of 32 nM and 4.0 μM, respectively.133,135 Trungapeptins B and C
were not tested for cytotoxicity.125
Conclusion
Cytotoxicity-directed purification of a Symploca cf. hydnoides sample from Cetti
Bay, Guam, afforded seven new cyclic depsipeptides, veraguamides A–G (7–13),
together with the known compound dolastatin 16. The planar structures of 7–13 were
elucidated using NMR and MS experiments, while enantioselective HPLC and Mosher’s
analysis of acid and base hydrolysates, respectively, were utilized to assign the
absolute configurations of the stereocenters. Veraguamides A–G (7–13) are
characterized by the presence of an invariant proline residue, multiple N-methylated
amino acids, an R-hydroxy acid, and a C8-polyketide-derived β-hydroxy acid moiety with
a characteristic terminus as either an alkynyl bromide, alkyne, or vinyl group. These
compounds and a semisynthetic analogue (14) showed moderate to weak cytotoxic
activity against HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cell
lines. Preliminary structure–activity relationship analysis identified several sensitive
positions in the veraguamide scaffold that affect the cytotoxic activity of this compound
class. Additional studies are required to elucidate the mechanism of action of the
veraguamides.
Experimental Methods
Biological Material
The Symploca cf. hydnoides cyanobacterium was collected by hand while
snorkeling in the shallow waters of the southern fore-reef (1–3 m) of Cetti Bay, Guam,
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on April 17, 2009. A voucher specimen, which is preserved in 100% EtOH, is deposited
in the University of Guam Herbarium (accession no. GUAM-GH11446). A voucher
specimen is also retained at the Smithsonian Marine Station, Fort Pierce, FL.
Extraction and Isolation
The freeze-dried cyanobacterium (142.0 g) was extracted with EtOAc–MeOH
(1:1) to yield the nonpolar extract (11.6 g). This was partitioned between hexanes and
20% aqueous MeOH, the latter concentrated under reduced pressure and further
partitioned between n-BuOH and H2O. The n-BuOH fraction was concentrated to
dryness (2.8 g) and chromatographed on Si gel eluting first with CH2Cl2, followed by
increasing concentrations of i-PrOH; after 100% i-PrOH, increasing gradients of MeOH
were used. The 20% i-PrOH fraction was subjected to a C18 SPE eluting with 25%,
50%, 75%, and 100% MeOH in H2O. The 100% MeOH fraction was purified by
semipreparative reversed-phase HPLC (Phenomenex Synergi-Hydro RP, 4 μm; flow
rate, 2.0 mL/min) using a linear gradient of MeOH–H2O (70%–100% MeOH in 60 min
and then 100% MeOH for 10 min) to yield dolastatin 16 (tR 32.6 min, 21.6 mg),
semipure veraguamide C (tR 36.4 min, 15.1 mg), semipure veraguamide F (tR 37.4 min,
10.0 mg), a mixture of veraguamides B and D (tR 40.0 min, 25.0 mg), veraguamide A (7)
(tR 42.6 min, 25.9 mg), and a mixture of veraguamides E and G (tR 43.7 min, 9.0 mg).
The final purification of the semipure veraguamide C (9) was achieved using
semipreparative HPLC (Phenomenex Phenyl-hexyl, 4 μm; flow rate, 2.0 mL/min) using
a linear gradient of MeOH–H2O (85%–100% MeOH in 40 min and then 100% MeOH for
5 min) to yield veraguamide C (9) (tR 19.9 min, 10.7 mg). Using the same
chromatographic conditions, purification of the semipure veraguamide F yielded
veraguamide F (12) (tR 21.7 min, 6.8 mg). The mixture of veraguamides B and D was
110
resolved using the same chromatographic condition with a different linear gradient
(70%–100% MeOH in 45 min and then 100% MeOH for 10 min) to yield veraguamide D
(10) (tR 36.6 min, 4.0 mg) and veraguamide B (8) (tR 37.3 min, 11.5 mg). The mixture of
veraguamides E and G was further purified using the same chromatographic conditions
to yield veraguamide G (13) (tR 39.9 min, 4.4 mg) and veraguamide E (11) (tR 40.5 min,
3.6 mg).
Hydrogenation of 7
A catalytic amount of 10% Pd/C was added to a methanolic solution of 7 (1.8
mg/mL). The reaction was left to stir for 6 h under a hydrogen balloon. The catalyst was
filtered through a Celite pad, and the filtrate, upon concentration, was purified by
semipreparative HPLC (Phenomenex Phenyl-hexyl, 4 μm; flow rate, 2.0 mL/min) using
a linear gradient of MeOH–H2O (70%–100% MeOH in 45 min and then 100% MeOH for
10 min) to yield 14 (tR 40.9 min, 1.2 mg).
Partial hydrogenation of 7 was carried out with Lindlar catalyst, using the same
reaction and chromatographic conditions stated above. This afforded the semisynthetic
veraguamide G (tR 39.9 min, 1.7 mg). The LRESIMS and 1H NMR spectra of the
semisynthetic veraguamide G were in good agreement with the spectra for the natural
product (13).
Veraguamide A (7): colorless, amorphous solid; [α]20D –44 (c 0.44, MeOH); UV
(MeOH); λmax (log ε) 202 (6.29); 1H NMR ,13C NMR, COSY, and HMBC data, see Table
4-1; HRESIMS m/z 767.3675 [M + H]+ (calcd for C37H6079BrN4O8, 767.3594), m/z [M +
H]+ 769.3660 (calcd for C37H6081BrN4O8, 769.3574) (100:100 [M + H]+ ion cluster).
Veraguamide B (8): colorless, amorphous solid; [α]20D –40 (c 0.16, MeOH); UV
(MeOH); λmax (log ε) 202 (4.30); 1H NMR and 13C NMR data, see Table 4-2; HRESIMS
111
m/z 753.3517 [M + H]+ (calcd for C36H5879BrN4O8, 753.3438), m/z [M + H]+ 755.3508
(calcd for C36H5881BrN4O8, 755.3418) (100:100 [M + H]+ ion cluster).
Veraguamide C (9): colorless, amorphous solid; [α]20D –44 (c 0.31, MeOH); UV
(MeOH); λmax (log ε) 202 (4.17); 1H NMR and 13C NMR data, see Table 4-2; HRESIMS
m/z 689.4486 [M + H]+ (calcd for C37H61N4O8, 689.4490).
Veraguamide D (10): colorless, amorphous solid; [α]20D –57 (c 0.11, MeOH); UV
(MeOH); λmax (log ε) 202 (4.30); 1H NMR and 13C NMR data, see Table 4-3; HRESIMS
m/z 703.4639 [M + H]+ (calcd for C38H63N4O8, 703.4646).
Veraguamide E (11): colorless, amorphous solid; [α]20D –56 (c 0.22, MeOH); UV
(MeOH); λmax (log ε) 202 (4.30); 1H NMR and 13C NMR data, see Table 4-3; HRESIMS
m/z 717.4799 [M + H]+ (calcd for C39H65N4O8, 717.4802).
Veraguamide F (12): colorless, amorphous solid; [α]20D –41 (c 0.13, MeOH); UV
(MeOH); λmax (log ε) 206 (4.33); 1H NMR and 13C NMR data, see Table 4-4; HRESIMS
m/z 723.4411 [M + H]+ (calcd for C40H59N4O8, 723.4333).
Veraguamide G (13): colorless, amorphous solid; [α]20D –48 (c 0.17, MeOH); UV
(MeOH); λmax (log ε) 202 (4.26); 1H NMR and 13C NMR data, see Table 4-5; HRESIMS
m/z 691.4649 [M + H]+ (calcd for C37H63N4O8, 691.4646).
Tetrahydroveraguamide A (14): colorless, amorphous solid; [α]20D –43 (c 0.05,
MeOH); UV (MeOH); λmax (log ε) 202 (4.33); 1H NMR and 13C NMR data, see Table 4-5;
HRESIMS m/z 693.4791 [M + H]+ (calcd for C37H65N4O8, 693.4802).
Acid Hydrolysis of Veraguamides and Enantioselective Analysis
Portions of 7–13 (100 μg) were acid-hydrolyzed (200 μL of 6 N HCl, 110 °C, 20
h), and the product mixtures dried, reconstituted in 100 μL of H2O, and analyzed by
enantioselective HPLC-UV and enantioselective HPLC-MS. The absolute configurations
112
of the amino acids N-Me-Ile, Ile, N-Me-Val, Val, and Pro were determined by
enantioselective HPLC-MS [column, Chirobiotic TAG (250 4.6 mm), Supelco; solvent,
MeOH–10 mM NH4OAc (40:60, pH 5.30); flow rate, 0.5 mL/min; detection by ESIMS in
positive ion mode (MRM scan)]. The acid hydrolysates of 7–10, 12, and 13 showed
retention times at 7.8, 11.6, and 13.6 min corresponding to L-Val, N-Me-L-Val, and L-
Pro, respectively. The acid hydrolysate of 10 in addition showed a retention time at 12.4
min, corresponding to N-Me-L-Ile. The acid hydrolysate of 11 showed retention times at
8.4, 11.6, 12.4, and 13.6 min, corresponding to L-Ile, N-Me-L-Val, N-Me-L-Ile, and L-Pro,
respectively. The retention times (tR, min; MRM ion pair) of the authentic amino acids
were as follows: N-Me-L-Val (11.6; 132→86), N-Me-D-Val (34.3), L-Val (7.8; 118→72),
D-Val (13.7), N-Me-L-Ile (12.4; 146→100), N-Me-L-allo-Ile (15.0), N-Me-D-Ile (49.0), N-
Me-D-allo-Ile (51.0), L-Ile (8.4; 132→86), L-allo-Ile (8.6), D-allo-Ile (17.6), D-Ile (20.2), L-
Pro (13.6; 116→70), D-Pro (36.0). Compound-dependent parameters used were as
follows: N-Me-Val: DP 29.4, EP 4.2, CE 17.4, CXP 2.7, CEP 10.6; Val: DP 5.7, EP 9.0,
CE 40.0, CXP 8.0, CEP 10.0; N-Me-Ile: DP 35.0, EP 7.0, CE 17.0, CXP 2.0, CEP 10.0;
Ile: DP 40.0, EP 9.0, CE 15.0, CXP 3.0, CEP 8.0; Pro: DP 35.0, EP 7.7, CE 22.7, CXP
5.0, CEP 10.3. Source gas parameters used were as follows: CUR 40, CAD Medium, IS
4500, TEM 750, GS1 65, GS2 65. The absolute configurations of the R-hydroxy acids
[2-hydroxyisovaleric acid (Hiva), 2-hydroxy-3-methylpentanoic acid (Hmpa), and
phenyllactic acid (Pla)] were determined using enantioselective HPLC [column,
CHIRALPAK MA (+) (50 4.6 mm); solvent, CH3CN–2 mM CuSO4 (10:90); flow rate,
1.0 mL/min; detection by UV (254 nm)]. The acid hydrolysates of 7, 9–11, and 13 each
showed peaks at 33.0 min, corresponding to (2S,3S)-Hmpa. The acid hydrolysate of 8
113
contained a component that had a retention time at 10.0 min, corresponding to (2S)-
Hiva, while 12 gave a peak at 51.0 min, corresponding to (2S)-Pla. The retention times
of the authentic standards were as follows: (2R)-Hiva (6.0), (2S)-Hiva (10.0), (2R,3S)-
Hmpa (16.0), (2R,3R)-Hmpa (19.0), (2S,3R)-Hmpa (26.0), (2S,3S)-Hmpa (33.0), (2R)-
Pla (33.5), (2S)-Pla (51.0). All other amino acid units eluted within less than 5.0 min
using this chromatographic condition.
Methanolysis of 7
Compound 7 (5.0 mg) was dissolved in 2.0 mL of 5% (w/w) methanolic KOH
solution and stirred for 24 h at room temperature. The solvent was evaporated and the
residue was partitioned between CH2Cl2 and H2O. The organic layer was collected,
dried over anhydrous MgSO4 and concentrated to dryness under nitrogen. The crude
methanolysis product was further purified by semipreparative reversed-phase HPLC
(Phenomenex Synergi-Hydro RP, 4 μm; flow rate, 2.0 mL/min) using a linear gradient of
MeOH–H2O (70%–100% MeOH in 60 min and then 100% MeOH for 10 min) to yield 15
(tR 19.3 min, 1.4 mg).
15: colorless, amorphous solid; 1H NMR (CDCl3) δ 6.30 (d, J = 8.1 Hz, 1H), 4.92 (d,
J = 10.5 Hz, 1H), 4.76 (dd, J = 9.1, 6.6 Hz, 1H), 3.78 (ddd, J = 8.6, 4.4, 3.2 Hz, 1H),
3.49 (s, 3H), 3.08 (s, 3H), 2.42 (qd, J = 6.8, 3.2 Hz , 1H), 2.23 (m, 2H), 2.03 (m, 1H),
1.74 (m, 2H), 1.56 (m, 1H), 1.46 (m, 2H), 1.01 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz,
3H), 0.93 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H); HRESIMS m/z 497.1640 [M +
Na]+ (calcd for C21H3579BrN2O5Na, 497.1627), m/z [M + Na]+ 499.1616 (calcd for
C21H3581Br N2O5Na, 499.1607) (100:100 [M + H]+ ion cluster).
114
Preparation of MTPA Esters of 15
The methanolysis product 15 was dissolved in 50 μL CDCl3, and was divided into
two equal portions and to each was added 0.75 mL triethylamine. To one portion was
added 10 μL of (R)-MTPA-Cl and to another was added 10 μL of (S)-MTPA-Cl to give
the (S)-MTPA ester (16) and (R)-MTPA ester (17), respectively. Each reaction was
allowed to stir for 24 h and 10 μL of N,N-dimethylaminopropylamine was added to
quench the reactions. The reaction products were dried under N2 and applied onto silica
SPE eluting with EtOAc–hexanes (1:1). The semipure product was further purified by
semipreparative HPLC (Phenomenex Phenyl-hexyl, 4 μm; flow rate, 2.0 mL/min) using
a linear gradient of MeOHH2O (70%100% MeOH in 45 min and then 100% MeOH for
10 min) to yield 16 (tR 38.0 min, 0.1 mg) or 17 (tR 37.8 min, 0.1 mg).
16: colorless, amorphous solid; 1H NMR (CDCl3) δ 7.57 (dd, J = 6.4, 2.7 Hz, 2H),
7.41 (m, 3H), 6.14 (d, J = 9.2 Hz, 1H), 5.28 (q, J = 6.9 Hz, 1H), 4.93 (d, J = 10.6 Hz,
1H), 4.68 (dd, J = 9.1, 7.6 Hz, 1H), 3.69 (s, 3H), 3.58 (s, 3H), 3.06 (s, 3H), 2.46 (quintet,
J = 7.1 Hz , 1H), 2.21 (m, 1H), 2.15 (t, J = 6.8 Hz, 2H), 1.95 (m, 1H), 1.68 (m, 1H), 1.47
(m, 2H), 1.09 (d, J = 7.0 Hz, 3H), 1.01 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.9 Hz, 3H), 0.86
(d, J = 6.9 Hz, 3H), 0.81 (d, J = 6.9 Hz, 3H); HRESIMS m/z 729.1746 [M + K]+ (calcd for
C31H4279BrF3N2O7K, 729.1759), m/z [M + K]+ 731.1747 (calcd for C31H42
81BrF3N2O7K,
731.1755) (100:100 [M + K]+ ion cluster); LRESIMS m/z 691/693 (100:100 [M + H]+ ion
cluster), 713/715 (100:100 [M + Na]+ ion cluster).
17: colorless, amorphous solid; 1H NMR (CDCl3) δ 7.56 (dd, J = 4.3, 3.6 Hz, 2H),
7.41 (m, 3H), 6.27 (d, J = 8.3 Hz, 1H), 5.27 (q, J = 6.0 Hz, 1H), 4.94 (d, J = 10.4 Hz,
1H), 4.73 (dd, J = 9.1, 7.5 Hz, 1H), 3.69 (s, 3H), 3.57 (s, 3H), 3.07 (s, 3H), 2.54 (quintet,
115
J = 6.7 Hz, 1H), 2.22 (m, 1H), 2.08 (td, J = 7.2, 2.9 Hz, 2H), 2.00 (m, 1H), 1.63 (m, 1H),
1.31 (m, 2H), 1.19 (d, J = 6.7 Hz, 3H), 1.01 (d, J = 6.3 Hz, 3H), 0.95 (d, J = 6.8 Hz, 3H),
0.90 (d, J = 7.0 Hz, 3H), 0.83 (d, J = 6.5 Hz, 3H); HRESI/APCIMS m/z 691.2202 [M +
H]+ (calcd for C31H4379BrF3N2O7, 691.2206), m/z [M + H]+ 693.2186 (calcd for
C31H4381BrF3N2O7, 693.2186) (100:100 [M + H]+ ion cluster).
Biological Activity Assays
Cell viability assay
HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells were
cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with
10% fetal bovine serum (FBS, Hyclone) under a humidified environment with 5% CO2 at
37 °C. HeLa (3,000) and HT29 (12,500) cells were seeded in 96-well plates and treated
with varying concentrations of test samples and solvent control (DMSO) after 24 h of
seeding. The cells were incubated for an additional 48 h before the addition of the MTT
reagent. Cell viability was measured according to the manufacturer’s instructions
(Promega). IC50 calculations were done by GraphPad Prism® 5.03 based on duplicate
experiments. Paclitaxel was used as positive control.
Cell cycle analysis by flow cytometry
HeLa (75,000) and HT29 (200,000) cells were seeded 24 h prior to treatment in
6-well dishes and kept under a humidified environment with 5% CO2 at 37 °C. At the
end of the incubation time, the growth medium was replaced prior to sample treatment.
Cells were incubated with increasing concentrations of 10 for 24 h, with DMSO and
paclitaxel as the solvent and positive control, respectively. Cells were harvested at the
end of the 24 h incubation by trypsinization, followed by centrifugation at 4 °C. The
supernatant was discarded and the cell pellet was recovered for further use. Single cell
116
suspensions were prepared in PBS and subsequently permeabilized by dropwise
addition of EtOH. Cells were centrifuged, resuspended in PBS, containing 1.0 mM
EDTA and 100 µg/mL RNAse A, and stained with propidium iodide. Cells were sorted
using a FACScan™ (Becton Dickson) based on the fluorescence of the propidium
iodide-DNA complex.
117
Figure 4-1. Structures of veraguamides A–G (7–13) and the semisynthetic
tetrahydroveraguamide A (14).
118
Figure 4-2. MS/MS fragmentation of veraguamide A (7), veraguamide D (10), and
veraguamide E (11).
119
Figure 4-3. Assignment of absolute configuration of veraguamide A (7) using
methanolysis and subsequent Mosher’s analysis. Δδ = δ(S-MTPA ester) – δ(R-MTPA ester).
120
Figure 4-4. Cell cycle analysis of HT29 and HeLa cells treated with varying
concentrations of veraguamide D (10). Dose-dependent increase in sub-G1 and G2 cell populations was observed.
121
Table 4-1. NMR data for veraguamide A (7) in CDCl3
unit C/H no δCa δH (J in Hz)b COSYb HMBCb
Br-Hmoya 1 170.8, C 2 42.4, CH 3.11, br q (7.4) H-3,H3-9 1, 3, 4, 9 3 76.4, CH 4.85, dt (10.2, 2.5) H-2, H-4a, H-4b 1, 1 (N-Me-Val-1) 4a 27.4, CH2 2.06, m H-3, H-4b, H-5a,
H-5b 5, 6
4b 1.59, m H-3, H-4a, H-5a, H-5b
5a 25.0, CH2 1.60, m H-4a, H-4b, H-5b, H2-6
7, 8
5b 1.41, m H-4a, H-4b, H-5a, H2-6
6, 8
6 19.2, CH2 2.23, m H-5a, H-5b 7, 8 7 38.4, C 8 79.3, C 9 14.1, CH3 1.25, d (7.4) H-2 1, 2, 3 N-Me-Val-1 1 170.6, C 2 65.0, CH 3.93, d (10.3) H-3 1, 3, 4, N-Me, 1 (Pro) 3 28.3, CH 2.30, m H-2, H3-4, H3-5 2, 4, 5 4 19.56, CH3 0.98, d (6.6) H-3 2 5 19.51, CH3 0.91, d (6.6) H-3 2 N-Me 28.6, CH3 3.00, s 2, 1 (Pro) Pro 1 172.1, C 2 57.3, CH 4.94 dd (8.9, 4.5) H-3a, H-3b 1, 3, 4, 1 (Hmpa) 3a 29.5, CH2 2.30, m H-2, H-3b, H-4a 1, 2, 5 3b 1.79, m H-2, H-3a, H-4a 1, 2, 5 4a 24.9, CH2 2.03, m H-3a, H-3b, H-4b,
H-5a, H-5b 2, 3, 5
4b 1.98, m H-4a, H-5a, H-5b 2, 3, 5 5a 47.3, CH2 3.84, dt (–17.0, 7.1) H-4a, H-4b, H-5b 2, 3, 4 5b 3.61, dt (–17.0, 7.1) H-4a, H-4b, H-5a 3, 4
122
Table 4-1. Continued
unit C/H no δCa δH (J in Hz)b COSYb HMBCb
Hmpa 1 165.9, C 2 76.1, CH 4.90, d (9.1) H-3 1, 3, 4, 5, 1 (N-Me-Val-2) 3 35.7, CH 1.97, m H-2, H3-6 4a 24.81, CH2 1.54, m H-4b, H3-5 4b 1.13, m H-4a, H3-5 5 10.5, CH3 0.87, t (7.3) H-4a, H-4b 3, 4 6 13.8, CH3 1.01, d (6.8) H-3 2, 3, 4 N-Me-Val-2 1 169.6, C 2 66.0, CH 4.15, d (9.5) H-3 1, 3, 5, N-Me, 1 (Val) 3 28.5, CH 2.27, m H-2, H3-4, H3-5 1 4 20.4, CH3 1.00, d (7.0) H-3 2 5 20.2, CH3 1.11, d (7.0) H-3 2, 3 N-Me 30.0, CH3 2.94, s 2, 1 (Val) Val 1 173.4, C 2 52.8, CH 4.70, dd (8.7, 6.5) H-3, NH 1, 3, 4, 5, 1 (Br-Hmoya) 3 32.1, CH 1.96, m H-2, H3-4, H3-5 5 4 20.3, CH3 0.95, d (6.7) H-3 2, 3, 5 5 17.5, CH3 0.88, d (6.7) H-3 2, 3 NH 6.25, d (8.7) H-2 1, 1 (Br-Hmoya) a100 MHz b600 MHz
123
Table 4-2. NMR data for veraguamide B (8) and veraguamide C (9) in CDCl3
Veraguamide B Veraguamide C unit C/H no. δC
a δH (J in Hz)b δCa δH (J in Hz)b
Br-Hmoyac/ 1 170.8, C 170.8, C Hmoyad 2 42.3, CH 3.13, br q (7.4) 42.4, CH 3.10, br q (7.2) 3 76.4, CH 4.85, d (8.7) 76.4, CH 4.86, dt (10.4, 2.5) 4a 27.5, CH2 2.07, m 27.4, CH2 2.07, m 4b 1.60, m 1.62, m 5a 24.93, CH2 1.61, m 25.2, CH2 1.62, m 5b 1.42, m 1.44, m 6 19.2, CH2 2.21, m 18.0, CH2 2.19, m 7 38.4, C 83.6, C 8 79.4, C 68.8, CH 1.93 t (2.5) 9 14.6, CH3 1.25, d (7.4) 14.5, CH3 1.25, d (7.2) N-Me-Val-1 1 170.7, C 170.7, C 2 65.0, CH 3.94, d (10.4) 65.0, CH 3.93, d (11.0) 3 28.26, CH 2.29, m 28.3, CH 2.28, m 4 19.57, CH3 0.98, d (6.5) 19.58, CH3 0.98, d (6.8) 5 19.55, CH3 0.92, d (6.5) 19.56, CH3 0.91, d (6.8) N-Me 28.7, CH3 3.00, s 28.7, CH3 3.00, s Pro 1 172.1, C 172.2, C 2 57.2, CH 4.95 dd (8.6, 4.8) 57.3, CH 4.94 dd (8.4, 5.0) 3a 29.4, CH2 2.28, m 29.5, CH2 2.28, m 3b 1.79, m 1.79, m 4a 24.99, CH2 2.04, m 24.89, CH2 2.03, m 4b 1.98, m 1.99, m 5a 47.3, CH2 3.80, dt (–16.4, 6.9) 47.3, CH2 3.84, dt (–16.8, 7.1) 5b 3.60, dt (–16.4, 6.9) 3.60, dt (–16.8, 7.1) Hivac/Hmpad 1 165.8, C 165.9, C 2 77.2, CH 4.85, d (8.7) 76.0, CH 4.89, d (9.4) 3 29.6, CH 2.17, m 35.7, CH 1.98, m
124
Table 4-2. Continued
Veraguamide B Veraguamide C unit C/H no. δC
a δH (J in Hz)b δCa δH (J in Hz)b
4 18.1, CH3 1.02, t (6.6) 24.81, CH2 1.54, m 1.13, m 5 18.5, CH3 0.93, d (6.6) 10.5, CH3 0.86, t (7.3) 6 13.8, CH3 1.01, d (6.7) N-Me-Val-2 1 169.6, C 169.6, C 2 66.1, CH 4.15, d (10.2) 66.0, CH 4.13, d (10.0) 3 28.34, CH 2.28, m 28.5, CH 2.28, m 4 20.3, CH3 0.99, d (6.8) 20.4, CH3 0.99, d (6.4) 5 20.1, CH3 1.11, d (6.8) 20.2, CH3 1.10, d (6.4) N-Me 30.0, CH3 2.94, s 30.0, CH3 2.93, s Val 1 173.5, C 173.4, C 2 52.8, CH 4.71, dd (8.6, 6.4) 52.8, CH 4.70, dd (8.6, 6.2) 3 32.2, CH 1.98, m 32.1, CH 1.96, m 4 20.3, CH3 0.94, d (6.4) 20.3, CH3 0.94, d (6.7) 5 17.5, CH3 0.88, d (6.4) 17.6, CH3 0.87, d (6.7) NH 6.26, d (8.6) 6.26, d (8.6) a100 MHz b600 MHz cRefers to veraguamide B dRefers to veraguamide C
125
Table 4-3. NMR data for veraguamide D (10) and veraguamide E (11) in CDCl3
Veraguamide D Veraguamide E unit C/H no. δC
a δH (J in Hz)b δCa δH (J in Hz)b
Hmoya 1 170.8, C 170.71, C 2 42.4, CH 3.13, br q (7.2) 42.4, CH 3.08, br q (7.4) 3 76.4, CH 4.86, dt (10.8, 2.6) 76.5, CH 4.85, d (9.0) 4a 27.5, CH2 2.07, m 27.5, CH2 2.06, m 4b 1.63, m 1.62, m 5a 25.2, CH2 1.63, m 25.2, CH2 1.61, m 5b 1.47, m 1.43, m 6 17.5, CH2 2.18, m 18.0, CH2 2.18, m 7 83.6, C 83.6, C 8 68.8, CH 1.93, t (2.5) 68.8, CH 1.93, t (2.3) 9 14.4, CH3 1.24, d (7.2) 14.5, CH3 1.23, d (7.4) N-Me-Ilec/ 1 170.7, C 170.69, C N-Me-Vald 2 64.0, CH 4.01, d (10.6) 64.9, CH 3.93, d (10.0) 3 34.6, CH 2.02, m 28.3, CH 2.29, m 4 25.7, CH2 1.46, m 19.59, CH3 0.91, d (6.4) 5 11.4, CH3 0.93, t (6.5) 19.56, CH3 0.98, d (6.4) 6 15.8, CH3 0.94, d (6.8) N-Me 28.7, CH3 2.99, s 28.6, CH3 3.00, s Pro 1 172.7, C 172.2, C 2 57.2, CH 4.94 dd (8.9, 4.8) 57.3, CH 4.94 dd (9.0, 5.3) 3a 28.8, CH2 2.26, m 29.5, CH2 2.29, m 3b 1.78, m 1.78, m 4a 24.9, CH2 2.03, m 24.89, CH2 2.01, m 4b 1.97, m 1.99, m 5a 47.2, CH2 3.82, dt (–17.0, 7.3) 47.3, CH2 3.86, dt (–17.0, 7.0) 5b 3.60, dt (–17.0, 7.3) 3.60, dt (–17.0, 7.0) Hmpa 1 165.9, C 166.0, C 2 76.0, CH 4.90, d (9.2) 76.1, CH 4.85, d (9.0)
126
Table 4-3. Continued
Veraguamide D Veraguamide E unit C/H no. δC
a δH (J in Hz)b δCa δH (J in Hz)b
3 35.7, CH 1.98, m 35.1, CH 1.98, m 4 24.8, CH2 1.53, m 24.86, CH2 1.54, m 1.12, m 1.13, m 5 10.5, CH3 0.86, t (7.6) 10.5, CH3 0.86, t (7.0) 6 13.9, CH3 0.99, d (6.9) 13.8, CH3 1.01, d (6.8) N-Me-Valc/ 1 169.6, C 169.7, C N-Me-Iled 2 66.0, CH 4.15, d (9.4) 65.2, CH 4.22, d (9.6) 3 28.4, CH 2.28, m 35.7, CH 1.98, m 4 20.3, CH3 1.10, d (6.8) 26.6, CH2 1.54, m 1.06, m 5 20.2, CH3 0.99, d (6.8) 11.7, CH3 0.96, t (7.2) 6 16.5, CH3 1.04, d (6.9) N-Me 30.0, CH3 2.92, s 30.1, CH3 2.93, s Valc/Iled 1 173.4, C 173.5, C 2 52.8, CH 4.70, dd (8.6, 6.6) 52.4, CH 4.70, dd (8.4, 6.7) 3 32.1, CH 1.98, m 38.6, CH 1.69, m 4 19.0, CH3 0.94, d (6.6) 23.9, CH2 1.47, m 5 17.6, CH3 0.87, d (6.6) 1.05, m 11.5, CH3 0.85, d (6.6) 6 16.3, CH3 0.91, d (6.6) NH 6.24, d (8.6) 6.26, d (8.4) a125 MHz b600 MHz cRefers to veraguamide D dRefers to veraguamide E
127
Table 4-4. NMR data for veraguamide F (12) in CDCl3
unit C/H no. δCa δH (J in Hz)b
Hmoya 1 170.9, C 2 42.2, CH 3.24 br q (7.3) 3 76.6, CH 4.89, dt (10.6, 2.4) 4a 27.5, CH2 2.08, m 4b 1.64, m 5a 25.2, CH2 1.65, m 5b 1.45, m 6 18.0, CH2 2.20, m 7 83.5, C 8 68.9, CH 1.93, t (2.5) 9 14.6, CH3 1.29, d (7.3) N-Me-Val-1 1 170.7, C 2 65.3, CH 3.95, d (10.4) 3 28.3, CH 2.32, m 4 19.6, CH3 1.00, d (6.5) 5 19.7, CH3 0.93, d (6.5) N-Me 28.7, CH3 3.06, s Pro 1 172.3, C 2 57.1, CH 4.97, dd (9.0, 4.5) 3a 29.1, CH2 2.27, m 3b 1.83, m 4a 25.0, CH2 2.07, m 4b 1.97, m 5a 47.0, CH2 3.62, m 5b 3.56, m Pla 1 165.4, C 2 72.8, CH 5.47, dd (9.8, 4.0) 3 36.7, CH2 3.17, m
128
Table 4-4. Continued
unit C/H no. δCa δH (J in Hz)b
2.91, m 4 136.2, C 5//9 129.3, CH 7.18, d (8.0) 6/8 128.5, CH 7.28, m 7 126.7, CH 7.20, m N-Me-Val-2 1 168.9, C 2 65.8, CH 4.05, d (10.5) 3 27.4, CH 2.08, m 4 19.8, CH3 0.89, d (6.8) 5 20.0, CH3 0.91, d (6.8) N-Me 29.0, CH3 2.60, s Val 1 173.4, C 2 52.7, CH 4.76, dd (8.5, 6.1) 3 32.3, CH 1.98, m 4 20.3, CH3 0.91, d (6.6) 5 17.7, CH3 0.88, d (6.6) NH 6.28, d (8.5) a100 MHz b600 MHz
129
Table 4-5. NMR data for veraguamide G (13) and tetrahydroveraguamide A (14) in CDCl3
Veraguamide G Tetrahydroveraguamide A unit C/H no. δC
a δH (J in Hz)b δCb,c δH (J in Hz)b
Hmoead/ 1 170.9, C 170.7, C Hmoaae 2 42.4, CH 3.10, br q (7.4) 42.1, CH 3.08, br q (7.4) 3 76.8, CH 4.85, dt (10.6, 2.4) 76.8, CH 4.86, dt (10.1, 2.1) 4a 27.9, CH2 1.98, m 31.0, CH2 1.21, m 4b 1.45, m 1.26, m 5a 25.5, CH2 1.48, m 28.2, CH2 1.39, m 5b 1.30, m 6a 33.2, CH2 2.05, m 25.8, CH2 1.39, m 6b 1.20, m 7 138.2, CH 5.74, m 22.2, CH2 1.26, m 8 114.9, CH2 4.97, m 13.6, CH3 0.85, t (6.9) 9 14.4, CH3 1.22, d (7.4) 14.0, CH3 1.23, d (7.4) N-Me-Val-1 1 170.7, C 170.7, C 2 65.0, CH 3.93, d (9.8) 64.9, CH 3.93, d (10.7) 3 28.3, CH 2.28, m 28.3, CH 2.28, m 4 19.59, CH3 0.98, d (6.4) 19.2, CH3 0.98, d (6.5) 5 19.54, CH3 0.92, d (6.4) 19.3, CH3 0.91, d (6.5) N-Me 28.6, CH3 3.01, s 28.4, CH3 3.00, s Pro 1 172.2, C 172.0, C 2 57.3, CH 4.95, dd (8.7, 5.0) 57.1, CH 4.94, dd (9.0, 5.0) 3a 29.4, CH2 2.29, m 29.1, CH2 2.28, m 3b 1.79, m 1.79, m 4a 24.9, CH2 2.03, m 24.6, CH2 2.03, m 4b 1.98, m 1.99, m 5a 47.3, CH2 3.84, dt (–16.7, 7.1) 47.0, CH2 3.84, dd (–17.0, 7.3) 5b 3.61, dt (–16.7, 7.1) 3.60, dd (–17.0, 7.3) Hmpa 1 165.9, C 165.7, C
130
Table 4-5. Continued
Veraguamide G Tetrahydroveraguamide A unit C/H no. δC
a δH (J in Hz)b δCb,c δH (J in Hz)b
2 76.0, CH 4.90, d (8.7) 76.6, CH 4.90, d (8.8) 3 35.7, CH 1.98, m 35.4, CH 1.98, m 4 24.8, CH2 1.54, m 24.5, CH2 1.54, m 1.13, m 1.13, m 5 10.5, CH3 0.86, t (7.3) 10.2, CH3 0.86, t (7.1) 6 13.8, CH3 1.00, d (6.0) 13.5, CH3 1.00, d (6.4) N-Me-Val-2 1 169.6, C 169.5, C 2 66.0, CH 4.15, d (10.2) 65.8 CH 4.14, d (9.6) 3 28.6, CH 2.28, m 28.1, CH 2.28, m 4 20.4, CH3 1.00, d (6.1) 20.0, CH3 0.99, d (6.6) 5 20.2, CH3 1.10, d (6.1) 19.9, CH3 1.10, d (6.6) N-Me 30.0, CH3 2.93, s 29.7, CH3 2.93, s Val 1 173.4, C 173.3, C 2 52.7, CH 4.70, dd (8.6, 6.7) 52.5, CH 4.70, dd (8.6, 6.2) 3 32.1, CH 1.98, m 31.7, CH 1.96, m 4 20.3, CH3 0.93, d (6.8) 19.3, CH3 0.93, d (6.3) 5 17.6, CH3 0.86, d (6.8) 17.2, CH3 0.87, d (6.3) NH 6.23, d (8.6) 6.26, d (8.6) a125 MHz b600 MHz cBased on HSQC and HMBC dRefers to veraguamide G eRefers to tetrahydroveraguamide A
131
Table 4-6. Antiproliferative activity (IC50, µM) of natural and semisynthetic veraguamidesa
Compound HT29 HeLa
Veraguamide A (7) 26 ± 3.1 21 ± 0.8 Veraguamide B (8) 30 ± 2.4 17 ± 1.0 Veraguamide C (9) 5.8 ± 0.8 6.1 ± 1.0 Veraguamide D (10) 0.84 ± 0.09 0.54 ± 0.01 Veraguamide E (11) 1.5 ± 0.09 0.83 ± 0.06 Veraguamide F (12) 49 ± 12 49 ± 1.4 Veraguamide G (13) 2.7 ± 0.7 2.3 ± 0.9 Tetrahydroveraguamide A (14) 33 ± 0.2 48 ± 2.5 aData are presented as mean ± SD (n = 2).
132
CHAPTER 5 CAYLOBOLIDE B AND AMANTELIDES A AND B: ANTIPROLIFERATIVE
POLYKETIDES FROM MARINE CYANOBACTERIA*,†
Introduction
Secondary metabolites assembled solely by polyketide synthases represent a
minor fraction of isolated compounds from the phylum Cyanobacteria. These usually
polyhydroxylated compounds are reminiscent of secondary metabolites from
dinoflagellates136 such as the cytotoxic amphidinolides, amphidinols, and luteophanols
as well as bacteria-derived antibiotics desertomycins137 and oasomycins.138 Polyketides
from marine and terrestrial cyanobacteria also possess interesting biological activities
and may be decorated with unusual moieties. Tolytoxin and the related scytophycins,
produced by terrestrial cyanobacteria are potent cytotoxins.139 Tolytoxins are
distinguished by an epoxide substituent in their backbone structure. Oscillariolide,140 a
polyketide isolated from the genus Oscillatoria, inhibited the development of fertilized
echinoderm eggs, suggestive of its effects on cell division. Phormidolide,141 a compound
related to oscillariolide, was isolated from the genus Phormidium and is also a potent
cytotoxin. Both oscillariolide and phormidolide macrocycles contain a tetrahydrofuran
ring and a terminal vinyl bromide appended to their ring structure. In addition, one
hydroxy group in phormidolide is esterified with a C-16 carboxylic acid. The well-studied
marine cyanobacterium Lyngbya majuscula afforded the polyketide caylobolide A, which
is characterized by its contiguous pentad of 1,5-diols.142
*Reproduced with permission from Salvador, L. A.; Paul V. J.; Luesch, H. J. Nat. Prod. 2010, 73, 1606–
1609. Copyright 2010 American Chemical Society. †Reproduced with permission from J. Nat. Prod., submitted for publication. Unpublished work copyright
2013 American Chemical Society.
133
The structure elucidation of polyketides is particularly challenging due to difficulty
in establishing the relative and absolute configuration of the multiple stereocenters and
substantial overlap in the methylene region. Their configurational assignment has
greatly benefited from the development of Kishi’s Universal NMR Database143–145 as
well as derivatization techniques, particularly Mosher’s analysis146 and extensions of
this method,147 although applications still have certain limitations, particularly for those
bearing 1,n-diol (n 5) moieties. Assignment of the configuration of 1,n-diols has so far
been demonstrated on model systems using exciton coupling CD after derivatization
with arylcarboxylate chromophores within liposomes.148
Here we report the isolation, structure elucidation, and antiproliferative activity of
three related polyketides characterized by a polyhydroxylated macrocyle bearing a
pendant alkyl side chain, given the names caylobolide B (18) and amantelides A and B
(19, 20), from Floridian Phormidium spp. and a Guamanian gray cyanobacterium
collections, respectively.
Isolation and Structure Elucidation
Caylobolide B (18)
A freeze-dried sample of an assemblage of Phormidium cf. dimorphum and
Phormidium inundatum from Key West, Florida was extracted with EtOAc–MeOH (1:1).
This extract was cytotoxic at a concentration of 100 ng/mL and contained symplostatin 1
based on the HPLC-MS profiling. The nonpolar extract was solvent partitioned to yield
the hexanes-, n-BuOH- and H2O- soluble fractions. The n-BuOH fraction was cytotoxic
and was subjected to a bioactivity-guided isolation using silica gel chromatography and
reversed-phase HPLC to yield caylobolide B (18) (Figure 5-1). The major cytotoxic
activity was attributed to the known compound symplostatin 1, based on comparison of
134
LRESIMS and 1H NMR with literature values. Symplostatin 1 gave an IC50 of ~1.5 nM
against HT29 cells. However, because our cyanobacterial collection was largely a
binary mixture of two different Phormidum species, it is unclear if caylobolide B (18) and
the co-isolated cytotoxin symplostatin 1 were produced by the same or both species.
Caylobolide B (18) was isolated as a colorless, amorphous solid with molecular
formula of C42H80O11 based on pseudomolecular ion peaks observed by
HRESI/APCIMS at m/z 761.5767 [M + H]+ and m/z 783.5594 [M + Na]+. Fragmentation
of the [M + H]+ peak using positive ionization showed repetitive loss of 18 amu,
corresponding to elimination of H2O typical for alcohols. The structure of 18 was
determined by NMR analysis in DMSO-d6. The presence of exchangeable hydroxy
protons was evident from the lack of HSQC correlations for nine protons which resonate
at δH 4.2–4.6 ppm. Detailed interpretation of HSQC, TOCSY, HSQC-TOCSY and HMBC
experiments with 18 (Table 5-1, Figure 5-1) established that the hydroxy groups are part
of methine carbinols that form a highly oxygenated backbone structure consisting of a
1,3-diol system (C-7, C-9), a 1,3,5-triol system (C-25, C-27, C-29) and repeating 1,5-
diol moieties. Degenerate 1H and 13C NMR chemical shifts were observed for three
oxygenated methines at δC 69.6 (C-13, C-17, C-21), seven methylenes at δC 37.3 (C-
12, C-14, C-16, C-18, C-20, C-22, C-24), and two methylenes at δC 21.6 (C-15, C-19)
that make up the contiguous chain of 1,5-diol.
The 13C NMR chemical shifts are in good agreement with reported values for 1,5-
diol units of luteophanol.149 These degenerate signals together with HSQC-TOCSY
correlations (Figure 5-2) between δC 37.3/δH 4.20 and δC 21.6/δH 4.20 supported the
1,5-diol substitution pattern. HSQC-TOCSY correlations (Figure 5-2) between C-15/9-
135
OH, C-23/25-OH suggested that the contiguous chain of 1,5-diol is flanked by the 1,3-
diol and 1,3,5-triol units. HSQC-TOCSY correlations between C-31/29-OH, C-32/29-OH,
C-32/33-OH, C-33/H-35 enabled the extension of the polyhydroxylated chain which
terminates to form an ester linkage with a carbonyl group at δC 165.4 (C-1). The low-
field chemical shift of H-35 (δH 5.00) – due to anisotropy from an unsaturated system –
and HMBC correlation between C-1/H-35 confirmed the presence of the ester linkage.
From HMBC and TOCSY correlations of C-35/H-35 (Table 5-1), it was evident that C-35
was modified by an isohexyl side chain substitution. An additional unsaturation is
present in 18 due to a carbon-carbon double bond between C-2 and C-3. HMBC
correlations (C-1/H-2, C-3/H-2) and the characteristic chemical shifts for C-2 (δC 116.5)
and C-3 (δC 159.4) were suggestive of a polarized carbon-carbon double bond,
consistent with an α,β-unsaturated ester functionality. HMBC correlations between C-
2/H3-42 and C-3/H3-42 indicated a methyl substitution at the β position.
The structure of 18 bears a close resemblance to the 36-membered
macrolactone ring present in the known compound caylobolide A142 (Figure 5-1) and
was therefore termed caylobolide B. The C-1 to C-9 portion of these compounds
presents a major difference, where an additional carbon–carbon double bond and a
different hydroxylation pattern are present in 18. The isolated 1,3-diol system (C-7 to C-
9) is a distinctive feature of 18, instead of a 1,5-diol unit from C-5 to C-9 chain in
caylobolide A. The structure of caylobolide B (18) was confirmed using ESIMS
fragmentation in the negative ionization mode (Figure 5-3). It was evident that
fragmentation occurred mainly at positions α- and β- to the hydroxy groups, similar to
fragmentation patterns observed for amphidinols.137
136
Amantelides A and B (19, 20)
A gray cyanobacterium collected at Amantes Point, Tumon Bay, Guam was
extracted with CH2Cl2–MeOH (1:1). The resulting nonpolar extract exhibited
antiproliferative activity against HT29 cells at a concentration of 10 μg/mL and did not
contain largazole, symplostatin 1 or dolastatin 10, based on the HPLC-MS profiling.
Solvent partitioning of the nonpolar extract gave the hexanes-, n-BuOH-and H2O-
soluble fractions. The antiproliferative n-BuOH fraction was further purified by silica
column chromatography, with the bioactivity concentrated in the fraction eluting from
70% i-PrOH in CH2Cl2. Reversed-phase HPLC purification, afforded two related
polyketide-derived compounds, amantelides A (19) and B (20), as bioactive
constituents.
The HRESIMS spectrum of amantelide A (19) suggested a molecular formula of
C44H84O11 based on the observed pseudomolecular [M + Na]+ ion at m/z 811.5927. The
three degrees of unsaturation was partially accounted for by an α,β-unsaturated ester
based on 1H and 13C NMR, HSQC, and HMBC spectra, suggesting the presence of one
ring system to fulfill the molecular formula requirements. HMBC correlations with the sp2
C (δC160.3) were observed for the CH3 singlet (δH 1.85) and a vinyl group (δH 5.62), with
the latter also having long-range correlations to a carbonyl group (δC 165.5), confirming
the presence of an α,β-unsaturated ester (Figure 5-4, Table 5-2). The presence of an
ester functionality was also corroborated by the presence of a low-field methine (δC/δH
76.6/4.93), which also showed HMBC correlations to C-1 (δC 165.5). In addition, two
other spin system consisting of a 1,3-methine carbinol and a tert-butyl moiety were also
deduced. Using COSY, TOCSY and HMBC correlations, a partial structure (Figure 5-5)
for amantelide A (19) was derived. This is reminiscent of the C-1 to C-9 and C-33 to C-
137
40 moieties of caylobolide B (18) (Figures 5-1, 5-5). However, instead of an isohexyl
pendant side chain, amantelide A (19) bears a tert-butyl moiety (Figures 5-4, 5-5). The
overlapping 1H and 13C NMR signals only allowed for partial assignment of the structure
of 19. Comparison of the 1H and 13C chemical shifts of 18 and 19 indicated that the
latter lacks the distinctive 1,3,5-triol system present in caylobolides A142 and B (18)
(Tables 5-1, 5-2). Based on the 1H and 13C NMR chemical shifts as well as the
remaining C27H52O6 to be accounted for from the partial structure and molecular formula
of 19, a contiguous chain of 1,5-diol is proposed to form the macrocyclic structure of
amantelide A (19). The observed degenerate 13C NMR shifts in amantelide A (19)
(Table 5-2) are in accordance with literature values for 1,5-diols in luteophanols149 and
caylobolides A142 and B (18) (Table 5-1). To verify the proposed structure, MS/MS
fragmentation of amantelide A (19) was done under negative ionization (Figure 5-6).
Fragmentations were observed at α- and β-positions to the methine carbinols (Figure 5-
6) and confirmed that amantelide A (19) has a closely related structure to the
caylobolides.
HRESIMS data for amantelide B (20) showed pseudomolecular ion [M + Na]+ at
m/z 853.6044, with a 42 amu mass difference with amantelide A (19), suggesting a
molecular formula of C46H86O12. 1H and 13C NMR, HSQC and HMBC spectra of
amantelide B (20) suggested that these compounds belong to the same structural class,
with an additional acetyl group in amantelide B (20). This was corroborated by a singlet
CH3 (δC/δH 20.7/1.97) that showed an HMBC correlation to a carbonyl group (δC170.0)
(Table 5-2). This acetyl group is proposed to modify a methine carbinol and is evident
from the appearance of a downfield shifted methine (δC/δH 73.3/4.73) that also showed
138
an HMBC correlation to the carbonyl at δC 170.0 (Table 5-2). C-7 and C-9 were
eliminated as possible sites of acetylation since the characteristic 1H and 13C NMR
shifts of this moiety can still be clearly discerned (Table 5-2). A TOCSY correlation
between δH 4.73 and δH 3.21 suggested that C-33 bears the additional acetyl group in
20. Hence, amantelide B (20) is the C-33 monoacetylated analog of amantelide A (19)
(Figure 5-4). Verification by MS/MS fragmentation was, however, not possible due to
the immediate loss of the acetyl group upon ionization, yielding a similar fragmentation
pattern as amantelide A (19).
Amantelides A and B (19, 20) showed similarities to caylobolides A and B (18),
with the presence of a polyhydroxylated macrolactone ring that is modified by a pendant
aliphatic side chain. The C-1 to C-21 portions of the macrolactone ring of 18–20 are
similar, with the characteristic 1,3-diol moiety (C-7 to C-9) flanked by a 1,5-diol moiety
(C-10 to C-24) and an α,β-unsaturated ester (C-1 to C-3). Compounds 19 and 20 are
distinguished by their 1,5-dihydroxylation pattern (C-25 to C-39), as well as a larger 40-
membered macrolactone ring instead of a 36-membered macrocycle in caylobolides.
Amantelides also possess a tert-butyl side chain instead of an isohexyl moiety as in the
caylobolides. Tert-butyl bearing-natural products are rare and present only a small
portion of secondary metabolites. Among the cyanobacterial metabolites, the cytotoxins
apratoxins,60–64 laingolides,150,151 madangolide152 and bisebromoamide153 and the Ca2+
blocker palmyrolide A,154 bear a tert-butyl moiety.
Configurational Analysis
The relative configuration of selected stereogenic centers of 18 was assigned by
independently considering the 1,3-diol and 1,3,5-triol moieties using Kishi’s Universal
NMR Database (Database 2).143–145 The 13C NMR chemical shift of C-7/C-9 was in good
139
agreement with syn arrangement of 1,3-diol model system (Figure 5-7). The 1,3,5-triol
system was assigned as either syn/anti or anti/syn between C-25/C-27, C-27/C-29
based on comparison of δC at C-27 with the characteristic δC of the central carbon of the
1,3,5-triol model system (Figure 5-7).
This method cannot differentiate between syn/anti or anti/syn orientation.
Unfortunately, Mosher’s analysis failed to give any conclusive result on the absolute
configuration and was limited by the low yield of 18. The lack of chemical shift
dispersion in the contiguous chain of 1,5-diols in caylobolide B (18) limits the
assignment of the absolute configuration of this moiety, as well as those for 19 and 20.
The absolute configuration of the stereocenters in amantelides A and B (19, 20) was not
determined. The relative configuration at C-7/C-9 of amantelides were assigned as syn,
based on comparison with caylobolide B (18) (Tables 5-1, 5-2) and also in agreement
with the Kishi’s Universal NMR Database for 1,3-diols.143
Biological Activity Studies
Antiproliferative Activity
Caylobolide B (18) exhibited moderate cytotoxic activity against HT29 colorectal
adenocarcinoma and HeLa cervical carcinoma cells with IC50 of 4.5 μM and 12.2 μM,
respectively (Table 5-3). The cytotoxic activity of 18 is comparable to that of caylobolide
A against the human colon carcinoma HCT116 cells (IC50 9.9 μM).142 Due to the limited
amount of caylobolide B (18) and its weak cytotoxic activity, it was not pursued for
further biological studies. Amantelide A (19) showed superior antiproliferative activity in
HT29 and HeLa cancer cell lines, with submicromolar IC50s, compared to the
caylobolides (Table 5-3). Monoacetylation of amantelide A (19) at C-33, however,
caused more than 10-fold decrease in antiproliferative activity, as observed for
140
amantelide B (20) (Table 5-3). This then suggested the role of acetylation and
hydroxylation in modulating the antiproliferative activity of cyanobacterial polyketides
belonging to the caylobolide class. In order to gain insight into the role of acetylation in
the antiproliferative activity of amantelides, a semisynthetic derivative of 19 was
prepared using acetic anhydride and pyridine to yield the peracetylated amantelide A
(21). Antiproliferative activity testing on 21 indicated that peracetylation caused a
dramatic decrease in potency, causing a 20-fold and 67-fold increase in IC50 in HeLa
and HT29 cells, respectively (Table 5-3). In addition to acetylation of the hydroxy
groups, the difference in antiproliferative activities of caylobolides and amantelides may
suggest that the size of the macrolide ring, hydroxylation pattern and aliphatic side
chain may contribute to the antiproliferative activity of these compounds.
Elucidation of the Mechanism of Action of Cyanobacterial Polyketides
The preliminary SAR for 18–21 suggested that the hydroxy groups of
cyanobacterial polyketides are important to the biological activity. Time-course cell
viability analysis of HeLa and HT29 cells treated with amantelide A (19) indicated that
the cellular effects of 19 are observed within 1 h post-treatment (Figure 5-8). This then
indicated that these compounds may be acting as cell membrane disrupting agents,
based on the rapid cellular effects of 19. This mechanism of action is observed for
amphotericin B, a natural product isolated from Streptomyces nodosus, where changes
in membrane permeability culminate in the leakage of mono- and divalent ions.155 Close
inspection of the structure of amphotericin B and the cyanobacterial polyketides 18–20
indicated several similarities. C-1 to C-11 of amphotericin B bears close resemblance to
C-1 to C-13 of 18–20. The C-35 to C-37 moiety of amphotericin B is homologous to the
C-37 to C-39 of amantelides A and B (19, 20) and C-33 to C-35 of caylobolide B (18)
141
(Figures 5-1, 5-4). In amphotericin B, C-35 is a methine carbinol, while C-36 bears a
methyl group, and C-37 is derivatized to an ester which forms the macrocycle. Recent
investigations on the mechanism of action of amphotericin B indicated that it binds to
ergosterol in yeast cells through the mycosamine moiety and also forms ion channels
via the polyhydroxylated portion of the molecule.155–157 The formation of ion channels by
amphotericin B is postulated to be through the formation of both monomeric and dimeric
structures, with C-1 to C-13 and C-35 to C-37 being criticial structural elements.155–157
The hydroxy group at C-35 is in particular important; suggested to bridge the
amphotericin backbone to the lipid bilayer.155 This critical structural element parallels the
observation for amantelides, where the presence of an acetyl group at C-33 caused a
decreased in activity.
In order to probe the mechanism of action of 18–20, we utilized amantelide A
(19) as the model compound since it gave the highest potency in the antiproliferative
assay and also present in sufficient amounts. To verify the proposed mechanism of
action of amantelide A (19), the antiproliferative activity and cellular phenotype of
amphotericin B- and amantelide A (19)-treated cells were compared (Figure 5-8).
Significant changes in cell viability were observed for both amantelide A (19) and
amphotericin B-treated cells after 1 h (Figure 5-8). Amphotericin B however, induced
cell death at a slower rate compared to amantelide A (19). This is in accordance with
the close to 10-fold higher potency of 19 compared to amphotericin B (Table 5-3) in
preventing the growth of HT29 and HeLa cancer cells. Based on visual inspection,
amantelide A (19) and amphotericin B also both induced rapid morphological changes
142
in HeLa cells, within 1 h of treatment. The morphology of amantelide A (19)- and
amphotericin B-treated cells were distinct from control treatments.
Conclusion
Bioactivity-guided purification of two cyanobacteria collections yielded the closely
related polyketide macrolactones caylobolide B (18) and amantelides A and B (19, 20).
The structures of 18–20 were assigned based on 1H and 13C NMR, HSQC, HMBC,
TOCSY and COSY experiments. Compounds 18–20 are characterized by a
polyhydroxylated macrocycle modified by an aliphatic pendant side chain. Caylobolide B
(18) is characterized by a 36-membered macrocycle consisting of 1,3- and 1,5-diol and
a 1,3,5,-triol systems and an isohexyl pendant side chain. Amantelides A and B (19, 20)
have a distinctive 40-membered macrocycle composed of 1,3- and 1,5-diol moieties and
a tert-butyl pendant side chain, with compound 20 additionally being acetylated at C-33.
Antiproliferative activity assays with 18–20 indicated the importance of the hydroxy
groups for bioactivity, and were verified by the loss of activity of the peracetylated
derivative of 19. Compounds 18–20 bear structural similarities with amphotericin B.
This, together with the results of time-course cell viability determination for amphotericin
B- and amantelide A (19)-treated cells suggested that the latter may also affect the
integrity of the cell membrane, similar to amphotericin B. Additional experiments to
visualize the effects of 19 on the cell membrane may be carried out.
Experimental Methods
General Experimental Procedures
Optical rotation was measured on a Perkin-Elmer 341 polarimeter. The UV
spectrum was recorded on SpectraMax M5 Molecular Devices. 1H and 2D NMR spectra
were recorded in DMSO-d6 on a Bruker Avance II 600 MHz spectrometer equipped with
143
a 5-mm triple-resonance high-temperature superconducting (HTS) cryogenic probe
using residual solvent signals (δH 2.50; δC 39.5) as internal standards. The 13C NMR
spectrum was recorded in DMSO-d6 on a Bruker 500 MHz spectrometer, operating at
125 MHz. HSQC and HMBC experiments were optimized for 1JCH = 145 and nJCH = 7
Hz, respectively. TOCSY and HSQC-TOCSY experiments were done using a mixing
time of 100 ms. HRMS data were obtained using an Agilent LC-TOF mass spectrometer
equipped with an APCI/ESI multimode ion source detector. ESIMS/MS data were
obtained on a 3200 QTRAP (Applied Biosystems) by direct injection using a syringe
driver.
Biological Material
The cyanobacteria, Phormidium spp., were hand collected on June 24, 2008, at
the breakwater at Fort Zachary Taylor State Park (Key West), Florida, by snorkeling in
shallow waters. The collection was later identified to consist primarily of P. cf.
dimorphum and P. inundatum. Voucher specimens (#VP_6_24_08_FZT1) are
maintained at Smithsonian Marine Station, Fort Pierce, FL.
The gray cyanobacterium belonging to the Family Oscilliatoriales was collected
from Amantes Point, Tumon Bay, Guam. Voucher specimens are maintained at
Smithsonian Marine Station, Fort Pierce, FL.
Extraction and Isolation
Caylobolide B (18)
The freeze-dried organism (54.2 g) was extracted with EtOAc–MeOH (1:1) to
yield 5.7 g of the nonpolar extract. Subsequent extraction of the freeze-dried material
with EtOH–H2O (1:1) gave 11.5 g of the polar extract. The nonpolar extract was further
partitioned between hexanes and 20% aqueous MeOH. The latter was concentrated
144
under reduced pressure and was further partitioned between n-BuOH and H2O. The n-
BuOH (0.56 g) fraction was concentrated and subjected to Si gel column
chromatography eluting first with CH2Cl2, followed by increasing concentrations of i-
PrOH. After 100% i-PrOH, increasing gradients of MeOH were used until 100% MeOH.
The fraction that eluted with 25% MeOH was subjected to reversed-phase HPLC
(semipreparative, Phenomenex Synergi-Hydro RP, 4 μm) using a linear gradient of
MeOH–H2O (40–100% MeOH in 40 min and then 100% MeOH for 10 min) to yield
caylobolide B (18) (tR 31.1 min, 2.1 mg). Purification of the fraction from 50% MeOH
using the same conditions yielded symplostatin 1 (tR 31.4 min, 1.5 mg).
Caylobolide B (18): colorless, amorphous solid; [α]20D –15 (c 0.15, MeOH); UV
(MeOH); λmax (log ε) 215 (4.09); 1H NMR, 13C NMR, TOCSY, and HMBC data, see
Table 5-1; HRESI/APCIMS m/z 761.5767 [M + H]+ (calcd for C42H81O11, 761.5779); m/z
783.5594 [M + Na]+ (calcd for C42H80O11Na, 783.5593).
Symplostatin 1: colorless, amorphous solid; [α]20D –98 (c 0.03, MeOH) {lit.28 [α]D
–45 (c 1.6, MeOH)}; UV (MeOH); λmax (log ε) 204 (3.52), 240 (2.94); 1H NMR spectrum
(Appendix F) is identical to that of an authentic sample,28; LRESIMS m/z 799.3 [M + H]+.
Amantelides A (19) and B (20)
The cyanobacteria collection (22.0 g) was extracted with CH2Cl2–MeOH (1:1) to
yield 3.4 g of the nonpolar extract. The lipophilic extract was further partitioned between
hexanes:80% aqueous MeOH. The latter was concentrated and further partitioned
between n-BuOH:H2O. The n-BuOH fraction (0.488 g) was further purified on a silica
column, eluting with increasing gradients of i-PrOH in CH2Cl2 until 100% i-PrOH,
followed by 100% MeOH.The fraction from 70% i-PrOH elution was subjected to
reversed-phase HPLC (semipreparative, Phenomenex Synergi-Hydro RP, 4 μm) using
145
a linear gradient of MeOH–H2O (40%–100% MeOH in 30 min and then 100% MeOH for
10 min) to yield amantelide A (19) (tR 29.4 min, 13.3 mg) and amantelide B (20) (tR 30.8
min, 5.7 mg).
Amantelide A (19): colorless, amorphous solid; [α]20D –5.0 (c 0.06, MeOH); UV
(MeOH); λmax (log ε) 220 (3.99); HRESI/APCIMS m/z 811.5927 [M + Na]+ (calcd for
C44H84O11Na, 811.5911).
Amantelide B (20): colorless, amorphous solid; [α]20D –68 (c 0.02, MeOH); UV
(MeOH); λmax (log ε) 218 (3.76); HRESI/APCIMS m/z 853.6044 [M + Na]+ (calcd for
C46H86O12Na, 853.6017).
Acetylation of amantelide A (19)
Acetic anhydride (0.5 mL), pyridine (0.5 mL) and 19 (6.0 mg) was left to stir
overnight. The reaction was terminated and dried under N2 to yield peracetylated
amantelide A (21).
Peracetylated amantelide A (21): oily liquid; []20D –58 (c 0.02, MeOH); UV
(MeOH); λmax (log ε) 214 (4.19); HRESI/APCIMS m/z 1169.6891 [M + Na]+ (calcd for
C60H102O20Na, 1169.6857).
ESIMS/MS Fragmentation of Caylobolide B (18) and Amantelide A (19)
Individual solutions of 18 and 19 in MeOH were directly infused into the mass
spectrometer using a syringe driver. MS fragmentation was obtained by positive and
negative ionization using the enhanced product ion (EPI) and MS2 scan. The [M + H]+
and [M – H]– ions were fragmented by ramping the collision energy through the possible
allowed range. Compound-dependent and source gas parameters used were as
follows: DP ±65.0, EP ±10.0, CUR 10.0, CAD High, IS ±4500, TEM 0, GS1 10, GS2 0.
146
Cell Viability Assay
HT29 colorectal adenocarcinoma and HeLa cervical carcinoma cells were
cultured in Dulbecco’s modified Eagle medium (DMEM, Invitrogen) supplemented with
10% fetal bovine serum (FBS, Hyclone) under a humidified environment with 5% CO2 at
37 °C. HeLa (3,000) and HT29 (12,500) cells were seeded in 96-well plates. Varying
concentrations of 18–21 and amphotericin B were added to each well 24 h post-
seeding, with treatments done in duplicate. The cells were incubated for an additional 1,
3, 6, 12 and 48 h before the addition of the MTT reagent. Cell viability was measured
according to the manufacturer’s instructions (Promega). IC50 calculations were done by
GraphPad Prism® 5.03 based on duplicate experiments.
147
Figure 5-1. Caylobolide B (18) and closely related compound caylobolide A. Absolute
configuration for C-25, C-27 and C-29 is proposed by analogy to caylobolide A. Only relative configuration is shown for C-7 and C-9, which could not be related to C-25–C-29.
148
Figure 5-2. Key HSQC-TOCSY correlations for caylobolide B (18).
149
Figure 5-3. ESI-MS/MS of caylobolide B (18).
150
Figure 5-4. Amantelides A and B (19, 20) and the semisynthetic derivative peracetylated
amantelide A (21). Only the relative configurations for C-7 and C-9 are indicated.
151
Figure 5-5. Partial structure of amantelide A (19) derived from NMR experiments in
DMSO-d6. COSY correlations are indicated by solid double-headed arrows. Protons showing HMBC correlations to indicated carbons are shown by single-headed arrows.
152
Figure 5-6. ESI-MS/MS fragmentation of amantelide A (19).
153
Figure 5-7. Assignment of relative configuration of caylobolide B (18) based on Kishi’s
Universal NMR Database (Database 2). δ values between the model system and 18 are shown. The relative configuration shown is based on the best fit
with the model system. The 1,3-diol is assigned as syn. The δ values for the characteristic central carbon of the 1,3,5-triol system suggest either anti/syn or syn/anti arrangement.
154
Figure 5-8. Time-course antiproliferative activities of amantelide A (19) and
amphotericin B against cancer cells. (A) HT29 and (B) HeLa cell viability after 1, 3, 6, 12 and 48 h incubation with amantelide A (19). (C) HT29 and (D) HeLa cell viability after 1, 3, 6, 12 and 48 h incubation with amphotericin B.
155
Table 5-1. NMR data of caylobolide B (18) in DMSO-d6
Position δCa δH (J in Hz)b HMBCb TOCSYb
1 165.4, C 2 116.5, CH 5.63, s 1,3,42 H-4a, H-4b, H-42 3 159.4, C 4a 4b
32.8, CH2 2.64, m 2.42, m
2, 3 ,5, 42 2, 3, 5, 42
H-2,H-4b,H-5a,H-5b,H-7, 7-OH H-2,H-4a,H-5a,H-5b,H-7, 7-OH
5a 23.6, CH2 1.52, m H-4a,H-4b,H-7,7-OH 5b 1.40, m 7 H-4a,H-4b 6 37.1, CH2 1.37, m 7-OH 7 68.8, CH 3.58, m 9 H-4a, H-4b,H-5a,7-OH 7-OH 4.52, d (4.4) 6,7,8 H-4a, H-4b, H-5aH-6,H-7 8 44.15, CH2 1.39, m 9 69.0, CH 3.54, m 10 9-OH,13-OH 9-OH 4.47, d (4.8) 8,9,10 H-9,H-13 10 37.6, CH2 1.28, m 11 21.3, CH2 1.21, m 12 37.3, CH2 1.28, m 13 69.6, CH 3.35, m 9-OH, 13-OH 13-OH 4.20, m 12,13,14 H-9, H-13 14 37.3, CH2 1.28, m 15 21.6, CH2 1.21, m 16 37.3, CH2 1.28, m 17 69.6, CH 3.37, m 17-OH 17-OH 4.20, m 16,17,18 H-17 18 37.3, CH2 1.28, m 19 21.6, CH2 1.21, m 20 37.3, CH2 1.28, m 21 69.8, CH 3.36, m 21-OH,25-OH 21-OH 4.20, m 20,21,22 H-21,H-25 22 37.3, CH2 1.28, m 23 20.9, CH2 1.32, m
156
Table 5-1. Continued
Position δC
a δH (J in Hz)b HMBCb TOCSYb
24 37.3, CH2 1.29, m 25 68.1, CH 3.54, m 23,27 21-OH,25-OH, H-27 25-OH 4.37, d (4.4) 24,25,26 H-21,H-25,H-27 26 44.4, CH2 1.42, m 27 65.8, CH 3.79, dq (13.5, 6.4) 25,26,29 H-25,27-OH, H-29 27-OH 4.47, d (4.9) 27 H-27 28 44.08, CH2 1.39, m 29 66.6, CH 3.61, m 28,31 H-27, 29-OH 29-OH 4.28, d (5.2) 28,30 H-27,H-29 30 37.5, CH2 1.31, m 31a 21.0, CH2 1.29, m 31b 1.22, m 32 38.1, CH2 1.29, m 33 66.5, CH 3.31, m 32,34 33-OH, H-35 33-OH 4.29, d (6.03) 33,34 H-33, H-35 34 37.3, CH2 1.49, m 35 H-35 35 73.6, CH 5.00, ddd (10.2,
4.3, 2.2) 1,33,41 H-33,OH-33,H-34,H-36,H-37b,H3-
40,H3-41 36 36.0, CH 1.66, m 35 H-35,H-37b,H-41 37a 31.6, CH2 1.31, m 41 H-37b,H-38a 37b 1.06, dd (17.7, 8.9) 38a 28.8, CH2 1.31, m H-37a,H-37b 38b 1.23, m 39 22.3, CH2 1.26, m 40 H3-40 40 13.6, CH3 0.86, t (7.0) 38,39 H-35, H-39 41 14.4, CH3 0.80, d (6.9) 35,36,37 H-36 42 24.5, CH3 1.85, s 2, 3, 4 H-2 a125 MHz. b600 MHz.
157
Table 5-2. NMR data of amantelide A (19) and amantelide B (20) in DMSO-d6
Amantelide A Amantelide B Position δC
a δH (J in Hz)b δCa δH (J in Hz)b
1 165.5, C 165.8, C 2 116.2, CH 5.62, s 116.1, CH 5.67, s 3 160.3, C 160.0, C 4 32.9, CH2 2.54, m 32.4, CH2 2.56, m 5a 23.6, CH2 1.54, m 23.4, CH2 1.52, m 5b 1.40, m 1.40, m 6 37.0, CH2 1.27, m 37.0, CH2 1.23, m 7 68.7, CH 3.58, m 68.6, CH 3.58, m 7-OH 4.54, br 4.50, br 8 44.1, CH2 1.37, m 44.2, CH2 1.37, m 9 68.5, CH 3.55, m 68.8, CH 3.55, m 9-OH 4.52, br 4.55, br 10 37.0, CH2 1.22, m 37.0, CH2 1.23, m 11 21.3, CH2 1.21, m 21.2, CH2 1.22, m 12 37.0, CH2 1.31, m 36.9, CH2 1.30, m 13 69.4, CH 3.34, m 69.4, CH 3.34, m 13-OH 4.23, m 4.23, m 14 37.0, CH2 1.31, m 36.9, CH2 1.30, m 15 20.8, CH2 1.31, m 20.8, CH2 1.32, m 16 37.0, CH2 1.31, m 36.9, CH2 1.30, m 17 69.4, CH 3.34, m 69.4, CH 3.34, m 17-OH 4.23, m 4.23, m 18 37.0, CH2 1.31, m 36.9, CH2 1.30, m 19 20.8, CH2 1.31, m 20.8, CH2 1.32, m 20 37.0, CH2 1.31, m 36.9, CH2 1.30, m 21 69.4, CH 3.34, m 69.4, CH 3.34, m 21-OH 4.23, m 4.23, m 22 37.0, CH2 1.31, m 36.9, CH2 1.30, m 23 20.8, CH2 1.31, m 20.8, CH2 1.32, m
158
Table 5-2. Continued
Amantelide A Amantelide B Position δC
a δH (J in Hz)b δCa δH (J in Hz)b
24 37.0, CH2 1.31, m 36.9, CH2 1.30, m 25 69.4, CH 3.34, m 69.4, CH 3.34, m 25-OH 4.23, m 4.23, m 26 37.0, CH2 1.31, m 36.9, CH2 1.30, m 27 20.8, CH2 1.31, m 20.8, CH2 1.32, m 28 37.0, CH2 1.31, m 36.9, CH2 1.30, m 29 69.4, CH 3.34, m 69.4, CH 3.34, m 29-OH 4.23, m 4.23, m 30 37.0, CH2 1.31, m 36.9, CH2 1.30, m 31 20.8, CH2 1.31, m
c c 32 37.0, CH2 1.31, m
c c 33 69.4, CH 3.34, m 73.3, CH 4.73, m 33-OH 4.23, m 34 37.0, CH2 1.31, m
c c 35 20.8, CH2 1.31, m
c c 36 37.0, CH2 1.31, m
c c 37 66.4, CH 3.21, m 66.4, CH 3.21, m 37-OH 4.26, m 4.27, br 38 37.1, CH2 1.43, m 37.1, CH2 1.41, m 39 76.6, CH 4.93, br d 76.2, CH 4.93, br d 40 34.1, C 34.2, C 41–43 26.3, CH3 0.82, s 25.7, CH3 0.83, s 44 24.6, CH3 1.85, s 24.3, CH3 1.86, s 45 170.0, C 46 20.7, CH3 1.97, s a125 MHz. b600 MHz. cCannot be assigned due to significant overlap of signals
159
Table 5-3. Cytotoxic activity (IC50, µM) of the isolated cyanobacterial polyketides (18–21)a
aData are presented as mean ± SD (n = 2).
Compound HT29 HeLa
Caylobolide B (18) 4.5 ± 1.2 12.2 ± 1.0 Amantelide A (19) 0.87 ± 0.02 0.87 ± 0.07 Amantelide B (20) 12 ± 1.6 9.9 ± 0.05 Peracetylated amantelide A (21) 58 ± 6.7 18 ± 1.6 Amphotericin B 10 ± 2.7 10 ± 4.4
160
CHAPTER 6 GENERAL CONCLUSION
In the last 30 years, marine cyanobacteria have been utilized as a source of
small molecule therapeutics. In this study, we aimed to exploit the diverse secondary
metabolites from the marine cyanobacteria belonging mainly to the genera of
Phormidium and Symploca for drug discovery. Cyanobacteria collections from Guam,
Florida, and the US Virgin Island were extracted and profiled in an antiproliferative
assay using the HT29 human colorectal adenocarcinoma cell line and an HPLC-MS-
based dereplication as a preliminary screening of bioactivity and chemical space,
respectively. Based on the profiling results, four cyanobacteria collections were
prioritized for further purification of secondary metabolites. Bioactivity- and 1H NMR-
directed approaches for the prioritized cyanobacteria collections yielded symplostatins
5–10 (1–6), veraguamides A–G (7–13), caylobolide B (18) and amantelides A and B
(19, 20). The planar structures of purified compounds were established using a
combination of 1D and 2D NMR spectroscopy and mass spectrometry. Absolute
configurations of stereocenters were assigned by enantioselective HPLC-MS and/or
HPLC-UV analysis by comparison with authentic standards as well as derivatization
with chiral reagents and J-based analysis.
A Guamanian Symploca sp. collection yielded the cyclic depsipeptides
symplostatins 5–10 (1–6), bearing the modified amino acid residue 3-amino-6-hydroxy-
2-piperidone (Ahp) and 2-amino-2-butenoic acid (Abu). The Ahp-bearing
cyclodepsipeptides from cyanobacteria constitutes a predominant class of metabolites,
with more than 100 members isolated to date from terrestrial, marine and freshwater
origins. These cyanobacterial metabolites are serine protease inhibitors, with the Abu-
161
bearing compounds such as symplostatins 5–10 (1–6) and the related lyngbyastatins 4–
10 being potent elastase inhibitors. Using the structural diversity of these agents, the
molecular basis for potent elastase inhibition was established using structure-activity
relationship (SAR) and X-ray cocrystallization studies. Aside from the Ahp and Abu
moieties, an N-Me-Tyr residue in the macrocyle and a polar functionality in the pendant
side chain are contributors to potent elastase inhibition. This was verified from the X-ray
cocrystal structure of lyngbyastatin 7–porcine pancreatic elastase, where the hydroxy
group of the N-Me-Tyr and the terminal amide group of Gln in lyngbyastatin 7 contribute
critical hydrogen bonding interactions with the enzyme and active site water molecules.
The involvement of the pendant side chain, which is highly variable among members of
this compound class, highlights Nature’s own combinatorial chemistry. Comprehensive
serine protease profiling for symplostatin 5 (1) and lyngbyastatin 7 demonstrated
preferential inhibition of elastase by these agents. The cellular effects of symplostatin 5
(1) against the downstream cellular effects of elastase in bronchial epithelial cells were
also interrogated. Symplostatin 5 (1) attenuated the effects of elastase on cell death,
detachment, genome-wide transcript changes as well as proteolytic processing of
adhesion molecules. Compound 1 alleviated key pro-inflammatory mediators stimulated
by elastase, such as NF-B activation and upregulation of interleukins IL1A, IL1B and
IL8. Compared to the clinically-approved elastase inhibitor sivelestat, symplostatin 5 (1)
has a long-lasting effect against the cellular effects of elastase to bronchial epithelial
cells, while having no cytotoxic effects. Therefore, key aspects in protease inhibitor
development – selectivity, potency and cellular activity – have been addressed in this
study. Additional investigations are warranted to determine the in vivo cellular effects of
162
this class of compounds in a COPD animal model system, as well as SAR studies to
further probe the effects of the highly divergent pendant side chain to selectivity and
potency of this class of compounds.
Antiproliferative agents constitute the majority of the purified secondary
metabolites in this study. A Guamanian Symploca cf. hydnoides collection yielded the
modified cyclic depsipeptides veraguamides A–G (7–13), characterized by a C8-
polyketide derived β-hydroxy acid, multiple N-methylated amino acids and an α-hydroxy
acid. Compounds 7–13 and a semisynthetic derivative tetrahydroveraguamide A (14)
showed moderate to weak antiproliferative activity against HT29 human colorectal
adenocarcinoma and HeLa cervical carcinoma cell lines. Preliminary structure-activity
relationship studies on veraguamides indicated that the α-hydroxy acid and the terminal
functionality of the C8-polyketide derived β-hydroxy acid moieties are major contributors
to the antiproliferative activity of this class of compounds. Veraguamide D (10) caused
an incremental change in cell populations at sub-G1 and G2. Additional studies are
needed to determine the mechanism of cell death induced by the veraguamides. Three
polyketide compounds, caylobolide B (18) and amantelides A and B (19, 20), were
isolated from Floridian Phormidium spp. assemblage and a Guamanian gray
cyanobacterium collections, respectively. These compounds bear a polyhydroxylated
macrolactone ring with an alkyl pendant side chain. Caylobolide B (18) bears a
contiguous chain of 1,3- and 1,5-diol and 1,3,5-triol moieties and an isohexyl side chain.
Amantelides A (19) and B (20) bear a contiguous chain of 1,3-diol and 1,5-diol systems
and a tert-butyl side chain. The C-33 of 20 bears an acetyl group instead of a hydroxy
group, which differentiates 19 and 20. Among the purified polyketides, amantelide A
163
(19) displayed the most potent antiproliferative activity, with sub-nanomolar IC50 against
HT29 and HeLa cells, indicating that acetylation of the hydroxy groups of this class of
compound is detrimental to the activity. This was corroborated by the weak
antiproliferative activity of the semisynthetic derivative of 19, peracetylated amantelide A
(21). Preliminary studies on the mechanism of action of amantelide A (19) indicated that
this class of cyanobacterial metabolites may target the cell membrane, leading to
cytotoxicity.
This study demonstrated that marine cyanobacteria are validated source
organisms of novel bioactive secondary metabolites, yielding both structurally and
pharmacologically diverse compounds that have potential applications as small
molecule therapeutics in malignancies and elastase-mediated pathologies.
164
APPENDIX A CELL MORPHOLOGY AT 3 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR)
165
APPENDIX B
CELL MORPHOLOGY AT 6 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR)
166
APPENDIX C CELL MORPHOLOGY AT 12 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR)
.
167
APPENDIX D CELL MORPHOLOGY AT 24 h POST TREATMENT WITH ELASTASE (+/- INHIBITOR)
168
APPENDIX E ICAM1 TRANSCRIPT LEVELS AT 3 h AND 6 ha
aData are presented as mean + SD (n = 3)
169
APPENDIX F
NMR SPECTRA OF ISOLATED COMPOUNDS
On the following pages are the NMR spectra of isolated compounds in this study,
which includes the known compounds largazole, dolastatin 10, symplostatin 1 and
dolastatin 16, as well as the new secondary metabolites symplostatins 5–10 (1–6),
veraguamides A–G (7–13), caylobolide B (18) and amantelides A, B (19, 20) and the
semisynthetic analogs tetrahydroveraguamide (14) and peracetylated amantelide A
(21).
170
1H NMR SPECTRUM OF LARGAZOLE IN CDCl3 (600 MHz)
171
1H NMR SPECTRUM OF DOLASTATIN 10 IN CD2Cl2 (600 MHz)
172
1H NMR SPECTRUM OF SYMPLOSTATIN 1 IN CD2Cl2 (600 MHz)
173
1H NMR SPECTRUM OF DOLASTATIN 16 IN CDCl3 (400 MHz)
174
13C NMR SPECTRUM OF DOLASTATIN 16 IN CDCl3 (100 MHz)
175
1H NMR SPECTRUM OF SYMPLOSTATIN 5 (1) IN DMSO-d6 (600 MHz)
176
HSQC SPECTRUM OF SYMPLOSTATIN 5 (1) IN DMSO-d6 (600 MHz)
177
COSY SPECTRUM OF SYMPLOSTATIN 5 (1) IN DMSO-d6 (600 MHz)
178
HMBC SPECTRUM OF SYMPLOSTATIN 5 (1) IN DMSO-d6 (600 MHz)
179
NOESY SPECTRUM OF SYMPLOSTATIN 5 (1) IN DMSO-d6 (600 MHz)
180
1H NMR SPECTRUM OF SYMPLOSTATIN 6 (2) IN DMSO-d6 (600 MHz)
181
HSQC SPECTRUM OF SYMPLOSTATIN 6 (2) IN DMSO-d6 (600 MHz)
182
COSY SPECTRUM OF SYMPLOSTATIN 6 (2) IN DMSO-d6 (600 MHz)
183
1H NMR SPECTRUM OF SYMPLOSTATIN 7 (3) IN DMSO-d6 (600 MHz)
184
HSQC SPECTRUM OF SYMPLOSTATIN 7 (3) IN DMSO-d6 (600 MHz)
185
COSY SPECTRUM OF SYMPLOSTATIN 7 (3) IN DMSO-d6 (600 MHz)
186
1H NMR SPECTRUM OF SYMPLOSTATIN 8 (4) IN DMSO-d6 (600 MHz)
187
HSQC SPECTRUM OF SYMPLOSTATIN 8 (4) IN DMSO-d6 (600 MHz)
188
COSY SPECTRUM OF SYMPLOSTATIN 8 (4) IN DMSO-d6 (600 MHz)
189
1H NMR SPECTRUM OF SYMPLOSTATIN 9 (5) IN DMSO-d6 (600 MHz)
190
HSQC SPECTRUM OF SYMPLOSTATIN 9 (5) IN DMSO-d6 (600 MHz)
191
COSY SPECTRUM OF SYMPLOSTATIN 9 (5) IN DMSO-d6 (600 MHz)
192
1H NMR SPECTRUM OF SYMPLOSTATIN 10 (6) IN DMSO-d6 (600 MHz)
193
HSQC SPECTRUM OF SYMPLOSTATIN 10 (6) IN DMSO-d6 (600 MHz)
194
COSY SPECTRUM OF SYMPLOSTATIN 10 (6) IN DMSO-d6 (600 MHz)
195
1H NMR SPECTRUM OF VERAGUAMIDE A (7) IN CDCl3 (600 MHz)
196
13C NMR SPECTRUM OF VERAGUAMIDE A (7) IN CDCl3 (100 MHz)
197
HSQC SPECTRUM OF VERAGUAMIDE A (7) IN CDCl3 (600 MHz)
198
COSY SPECTRUM OF VERAGUAMIDE A (7) IN CDCl3 (600 MHz)
199
HMBC SPECTRUM OF VERAGUAMIDE A (7) IN CDCl3 (600 MHz)
200
1H NMR SPECTRUM OF VERAGUAMIDE B (8) IN CDCl3 (600 MHz)
201
13C NMR SPECTRUM OF VERAGUAMIDE B (8) IN CDCl3 (100 MHz)
202
HSQC SPECTRUM OF VERAGUAMIDE B (8) IN CDCl3 (600 MHz)
203
COSY SPECTRUM OF VERAGUAMIDE B (8) IN CDCl3 (600 MHz)
204
HMBC SPECTRUM OF VERAGUAMIDE B (8) IN CDCl3 (600 MHz)
205
1H NMR SPECTRUM OF VERAGUAMIDE C (9) IN CDCl3 (600 MHz)
206
13C NMR SPECTRUM OF VERAGUAMIDE C (9) IN CDCl3 (100 MHz)
207
HSQC SPECTRUM OF VERAGUAMIDE C (9) IN CDCl3 (600 MHz)
208
COSY SPECTRUM OF VERAGUAMIDE C (9) IN CDCl3 (600 MHz)
209
HMBC SPECTRUM OF VERAGUAMIDE C (9) IN CDCl3 (600 MHz)
210
1H NMR SPECTRUM OF VERAGUAMIDE D (10) IN CDCl3 (600 MHz)
211
13C NMR SPECTRUM OF VERAGUAMIDE D (10) IN CDCl3 (125 MHz)
212
HSQC SPECTRUM OF VERAGUAMIDE D (10) IN CDCl3 (600 MHz)
213
COSY SPECTRUM OF VERAGUAMIDE D (10) IN CDCl3 (600 MHz)
214
HMBC SPECTRUM OF VERAGUAMIDE D (10) IN CDCl3 (600 MHz)
215
1H NMR SPECTRUM OF VERAGUAMIDE E (11) IN CDCl3 (600 MHz)
216
13C NMR SPECTRUM OF VERAGUAMIDE E (11) IN CDCl3 (125 MHz)
217
HSQC SPECTRUM OF VERAGUAMIDE E (11) IN CDCl3 (600 MHz)
218
COSY SPECTRUM OF VERAGUAMIDE E (11) IN CDCl3 (600 MHz)
219
HMBC SPECTRUM OF VERAGUAMIDE E (11) IN CDCl3 (600 MHz)
220
1H NMR SPECTRUM OF VERAGUAMIDE F (12) IN CDCl3 (600 MHz)
221
13C NMR SPECTRUM OF VERAGUAMIDE F (12) IN CDCl3 (100 MHz)
222
HSQC SPECTRUM OF VERAGUAMIDE F (12) IN CDCl3 (600 MHz)
223
COSY SPECTRUM OF VERAGUAMIDE F (12) IN CDCl3 (600 MHz)
224
HMBC SPECTRUM OF VERAGUAMIDE F (12) IN CDCl3 (600 MHz)
225
1H NMR SPECTRUM OF VERAGUAMIDE G (13) IN CDCl3 (600 MHz)
226
13C NMR SPECTRUM OF VERAGUAMIDE G (13) IN CDCl3 (100 MHz)
227
HSQC SPECTRUM OF VERAGUAMIDE G (13) IN CDCl3 (600 MHz)
228
COSY SPECTRUM OF VERAGUAMIDE G (13) IN CDCl3 (600 MHz)
229
HMBC SPECTRUM OF VERAGUAMIDE G (13) IN CDCl3 (600 MHz)
230
1H NMR SPECTRUM OF TETRAHYDROVERAGUAMIDE A (14) IN CDCl3 (600 MHz)
231
HSQC SPECTRUM OF TETRAHYDROVERAGUAMIDE A (14) IN CDCl3 (600 MHz)
232
1H NMR SPECTRUM OF LINEAR FRAGMENT 15 IN CDCl3 (500 MHz)
233
COSY SPECTRUM OF LINEAR FRAGMENT 15 IN CDCl3 (500 MHz)
234
1H NMR SPECTRUM OF R-MTPA ESTER 16 IN CDCl3 (600 MHz)
235
COSY SPECTRUM OF R-MTPA ESTER 16 IN CDCl3 (600 MHz)
236
1H NMR SPECTRUM OF S-MTPA ESTER 17 IN CDCl3 (600 MHz)
237
COSY SPECTRUM OF S-MTPA ESTER 17 IN CDCl3 (600 MHz)
238
1H NMR SPECTRUM OF CAYLOBOLIDE B (18) IN DMSO-d6 (600 MHz)
239
13C NMR SPECTRUM OF CAYLOBOLIDE B (18) IN DMSO-d6 (125 MHz)
240
HSQC SPECTRUM OF CAYLOBOLIDE B (18) IN DMSO-d6 (600 MHz)
241
COSY SPECTRUM OF CAYLOBOLIDE B (18) IN DMSO-d6 (600 MHz)
242
HMBC SPECTRUM OF CAYLOBOLIDE B (18) IN DMSO-d6 (600 MHz)
243
HSQC-TOCSY SPECTRUM OF CAYLOBOLIDE B (18) IN DMSO-d6 (600 MHz)
244
1H NMR SPECTRUM OF AMANTELIDE A (19) IN DMSO-d6 (600 MHz)
245
13C NMR SPECTRUM OF AMANTELIDE A (19) IN DMSO-d6 (125 MHz)
246
HSQC SPECTRUM OF AMANTELIDE A (19) IN DMSO-d6 (600 MHz)
247
COSY SPECTRUM OF AMANTELIDE A (19) IN DMSO-d6 (600 MHz)
248
HMBC SPECTRUM OF AMANTELIDE A (19) IN DMSO-d6 (600 MHz)
249
1H NMR SPECTRUM OF AMANTELIDE B (20) IN DMSO-d6 (600 MHz)
250
13C NMR SPECTRUM OF AMANTELIDE B (20) IN DMSO-d6 (125 MHz)
251
HSQC SPECTRUM OF AMANTELIDE B (20) IN DMSO-d6 (600 MHz)
252
COSY SPECTRUM OF AMANTELIDE B (20) IN DMSO-d6 (600 MHz)
253
HMBC SPECTRUM OF AMANTELIDE B (20) IN DMSO-d6 (600 MHz)
254
TOCSY SPECTRUM OF AMANTELIDE B (20) IN DMSO-d6 (600 MHz)
255
1H NMR SPECTRUM OF PERACETYLATED AMANTELIDE A (21) IN CDCl3 (600 MHz)
256
HSQC SPECTRUM OF PERACETYLATED AMANTELIDE A (21) IN CDCl3 (600 MHz)
257
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BIOGRAPHICAL SKETCH
Lilibeth Apo Salvador was born in Quezon City, Philippines. She received her
Bachelor of Science in chemistry at the University of the Philippines – Diliman in 2000.
On the same year, she became a qualified chemist and joined the Marine Science
Institute at the University of Philippines – Diliman as science research specialist, under
the supervision of Professor Gisela P. Concepcion and Professor Amelia P. Guevara.
She worked with the Antibody and Molecular Oncology Research (AMOR) program and
the National Cooperative Drug Discovery Group (NCDDG) on the discovery of
anticancer therapeutics from Philippine plants and marine sponges. During this time,
she developed a strong interest on natural products-intiated drug discovery. Lilibeth
finished her Master of Science in chemistry in 2006 and subsequently served as
research and development consultant for Euro-Med Laboratories Inc. and TEDA
Pharmaceuticals Inc. She joined the Department of Medicinal Chemistry, College of
Pharmacy at the University of Florida in 2008, under the mentorship of Professor
Hendrik Luesch. Lilibeth worked on the purification and structure determination of novel
secondary metabolites from marine cyanobacteria as well as elucidation of the
mechanisms of action and pharmacokinetics of cyanobacterial-derived compounds. She
received her Ph.D. in pharmaceutical sciences – medicinal chemistry from the
University of Florida in the spring of 2013.