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Natural Product-inspired Macrocyclic Toolbox for
“Undruggable Targets”: Our Wnt Journey
Prabhat AryaFounder and Chief Scientific Officer, SignMod
Chief Scientific OfficerSignMod-Transcell Oncologics, Joint Venture
www.signmod.org
prabhat.arya@signmod.org
June 25, 2019 1
June 25, 2019
Underestimated the complexity of human biology!
2
…Our current understanding of molecular pathways is insufficient as a platform for effective pharmaceutical discovery…
…Several biotechnology companies have focused on the known elements of a few key pathways to target them with new medicines. But for the genome to be translated into medicines with any reliability and regularity, far more work needs to be done. Defining the role of pathways in complex diseases will undoubtedly take many years…
Mark FishmanPresident
NIBR, 2002-2016
Defining the target (could be
enzymes or isolated
protein(s)
Structural information
on the target – finding the pocket
Structural guided chem
/med chem
Lipinski’s Rule of 5
Classical Approaches
• Complex, multiple protein-
protein interactions
• Dynamic and temporal
processes!
• Regulation (normal) and de-
regulation (disease)
tough journey From genes to pathways:
Science 2003, 300, 445-452
The sequencing of complete genomes provides a list that includes the proteins responsible for cellular regulation....
However, this does not immediately reveal what these proteins do, nor how they are assembled into the molecular machines and functional networks that control cellular behavior.
June 25, 2019 5
• Map large surface area• Shallow surface • Combination of several weak
interactions• Extended hydrophobic
interactions• Possible hot spots
2014
6
O
Me Me
H OH
O
O
Me
OMe
OH
OH
Me
Me
Me
MeO O
O N
O
H
FK506
O
O
O
OHH2N
O
OO O
H
H
O OO
H
Eribulin
7
• present complex 3D architectures • dense display of stereo-defined groups • challenging task in placing them on the drug
discovery path!
• excellent track record as small molecule modulators of protein-protein interactions
• can serve as a good source of inspiration for developing
novel scaffolds in the drug discovery arena!
Finding Molecules as Effective Modulators of Pathways!
BioactiveNatural
Products
Exploring MacrocyclicChemical Space!
• Cyclic compounds• Large surface area• Pre-organization• Less freely rotating bonds
• Enhanced cell permeation
Building A Chemical Toolbox for Challenging “Undruggable” Targets
Natural ProductInspired
Sub-structures
Natural Product Fragments as Sub-structures
Our working model
Key features in our design:
• 3D architectures • sufficient complexity• stereochemical and skeletal diversity • synthesis in a reasonable time-scale • easy to follow-up medicinal chemistry studies
From Arya Research Team:
Chem. & Biol.163 (2005)
Curr. Opin. Chem. Biol. 247 (2005)
Chem. Rev. 1999 (2009)June 25, 2019 8
June 25, 2019 9
Angew Chem 2001
Chem & Eng News 2004
June 25, 2019 10
Natural Product-Inspired, Functionalized 14- and 17-Membered Rings
Macrocyclic Toolbox
1
2
3
Indoline / Tetrahydroquinoline (Alkaloids) and Benzofuran (Flavonoids)-Inspired
Macrocyclic Toolbox
Glyco-based Macrocyclic Toolbox
Examples of Our Early Work
11
Org. Lett. (2013) Eur. J. Org. Chem. (2013)
Org. Lett. (2013)
Org. Lett (2008)Eur. J. Org. Chem. (2014)
Angew Chem (2005)Eur. J. Org. Chem. (2013)
12
Example 1: Rapamycin fragment-based Macrocyclic Toolbox
Rapamycin
from amino
alcohol der
Org. Lett. 480-483 (2015)
Recent Examples
13
• Isolated from Red Sea sponge
Negombata magnifica
• Potent G-actin Inhibitors
• Latrunculol A is more potent
compared to other family members
O
O
HN
S
H
HO H
O
O
H
O
O
HN
S
H
HO H
O
O
H
OH
O
O
OH
HN
S
H
HO H
O
O
H
Latrunculin B Latrunculin A Latrunculol A
Example 2: Latrunculin-derived Macrocyclic Toolbox
O
HN
O
O
OO
R1
O
MeO
PMBN
S
O
H
H HO
HN
O
O
OO
R1
O
MeO
PMBN
S
O
H
H H
13 15
17
13 15
17
15-Membered ring
Diversity
site
Org. Lett. 472-475 (2015)
O
O
O
OHH2N
O
OO O
H
H
O OO
H
2932 30
31
34
Eribulin
Practical and scalable synthesis
Precise
fragment-based
Diastereomeric
fragment-based
A
B
14Org. Lett. 468-471 (2015)
Example 3: Macrocyclic Toolbox based on Eribulin Sub-structures
Our ApproachOur Approach
Jimmidi, Krishna Reddy, Arya Org. Lett. 2015, 17, 468-471 15
single diastereomer
single diastereomer
Synthesis of Diastereomeric Fragment
16
June 25, 2019 17
Synthesis Special Issue (2017)
Different Macrocyclic Architectures
18
Leading to A New Family of Macrocyclic Architectures
Isomeric A
Precise
Isomeric B
O
OP1
HOOH
O
EtOCCH
H108
3
7
Submitted for publication
Growing Faith in Emerging Chemical Biology Model
Modern Organic Synthesis
novel chemical toolbox toexplore the scope of biologically relevant chemical space// natural product-inspired compounds
placing them onto the drug discovery path!
clinically-relevant questions
Cell Signaling Biology(biological questions/novel assays)
cellular/zebrafish studiesin vivo studies
Phenotypic Unbiased Screens
Discovery of functional small molecules
understanding the mode of action – biophysics, genomic,CRISPR-Cas9 tools
June 25, 2019 19
• Tumor heterogeneity
• Every patient is different!
• Cancer cells vs cancer stem
cells/tumor initiating cells
• Selective killing – understanding of
signaling pathways involved
• Tumor metastasis – Understanding of
signaling pathways involved
Challenges in Cancer Challenges in Cancer
Nat Rev Drug Discov 2014June 25, 2019 20
Cancer Stem Cell-derived Signaling Pathways
Cell Stem Cell 2019
our interest
our interest
our interest21
Cancer Patient-derived Banking Platform
• >900 Indian patient samples banking with primary cancer cells, tumorspheres (enriched with cancer stem cells).
• Patient-derived tumor xenograft (1st
generation, 10 samples as proof of concept).
• Novel procedure for obtaining tumorspheres that are rich in tumor initiating cells/cancer stem cells
June 25, 2019 22
Indian Patient-derived Transcriptomic Study
Tumor Biopsy Primary Cancer Cells Tumorspheres
• Oncogenic Signatures
(Broad Inst)
• Cancer Hall marks
(Broad Inst)
• Apoptosis
K-RAS
c-Myc
Wnt
Unpublished 23
Transcriptomic Data – Breast Cancer
TS PCC Control
TS: Tumorspheres
PCC: Primary Cancer
Cells
Down
UpKRAS
target genes
Myc target genesTS PCC Control
unpublished24
Breast Cancer
TS PCC Control
TS: Tumorspheres
PCC: Primary Cancer CellsDown
Up
Wnt target genes
TS PCC Control Anti apoptotic genes
Unpublished
25
Wnt Signaling and Stemness
• “Undruggable” domain
• Multiple cell surface interactions
• Multiple protein-protein interactions (Cytoplasm)
• Multiple DNA-protein-protein interactions (Transcription/Nucleus)
• Play a key role to maintain stemness / epithelial to mesenchymal transition / critical for cancer stem cells / metastasis
Nat Rev Drug Disc 2014
Transcription as the target
June 25, 2019 26
30,000 cells per well //
Incubate the plates
overnight in CO2 incubator
Add agonist (Wnt 3a), antagonist (DKK1), control (DMSO)
Add 100 μL luciferase substrate mixture
Incubate for 10 min and then read the chemi-luminescence signal
Primary Screening – WnT Inhibitors
IWR-1
XAV
ICG001
Controls
June 25, 2019 27
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
DMSO WNT3A IWR1 61 62 63 64 65 66 67 68
Luci
fera
se a
ctiv
ity
Con. 10 uM
Wnt Luciferase Screening Data
@10 µM
SM-2015-61/62/63/64/65SM-2015-66/67/68/69/70
open 17-memmacrocycles
Family 1 Hits
Five
Analogs
Single olefin geometry
The effect of small molecule on cytoplasmic protein complexes?
The effect of small molecule on transcriptional machinery – related to multiple protein-protein // DNA-protein-protein interactions?
Tools and Methods:
RT-PCRWestern blotsRNA sequencing Quantitative proteomicsFinally, the target pull-down plan
Our Chemical Biology Journey!
On the Functional Biology Front:
- The effect on tumorspheres from established cell lines?
- The effect on tumorspheres from patients-derived cells?
- The effect on organoids synthesized from patient organs/tissues?
- The effect on stemness / EMT / cancer stem cells?
RT-PCR Data – Transcription Genes
IWR-1 (control)
O
O
NH
O
OBnO
OO
O
O
O
H
H
NH
O
OBnO
OO
O
SM-61 SM-66 SM-110 SM-115
@ 10 uM
0
0.5
1
1.5
2
2.5
DMSO 61 66 IWR-1
Fold
Change
TCF4
0
0.5
1
1.5
2
2.5
3
DMSO 61 66 IWR-1
Fold
Change
LEF
0
0.2
0.4
0.6
0.8
1
1.2
1.4
DMSO 110 115 IWR-1
Fold
Change
TCF4
0
0.5
1
1.5
2
2.5
3
DMSO 110 115 IWR-1
Fold
Change
LEF
30
RT-PCR Data (contd.) IWR-1Selected genes related to…
Migration / Invasion
66: macrocycle
61: precursor
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Fo
ld c
ha
nge
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Fo
ld c
ha
nge
0
0.2
0.4
0.6
0.8
1
1.2
Fo
ld c
ha
nge
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6F
old
ch
an
ge
Metastasis Proliferation
00.20.40.60.8
11.21.41.6
Fo
ld c
ha
nge
With Family 1 hits 31
Western Blots
IWR-1
Moving on to proteins
O
O
NH
O
OBnO
OO
O
O
O
H
H
NH
O
OBnO
OO
O
SM-61 SM-66 SM-110 SM-115 32
Western Blots (contd)
GAPDH
TCF1
β-catenin
GAPDH
TCF1
β-catenin
GAPDH
Cyclin D1
c-Myc
p300
GAPDH
Cyclin D1
c-Myc
p300
June 25, 2019 33
34
Axin 2: 66 Showed Axin 2 stabilization compared to 61. 110 Showed Axin 2 stabilization compared to 115.
LEF 1: 61, 66, 110 and 115, all showed decreased expression levels of LEF1 but 110 was more potent.
CBP: 66 and 110 decreased the expression of CBP
Axin 2
LEF1
CBP
GAPDH
Western Blots (contd)
Source: http://www.jnsci.org/content/36
p300
CBP: (CREB, cAMP response element-binding protein)-binding protein
O
O
NH
O
OBnO
OO
O
O
O
H
H
NH
O
OBnO
OO
O
RNA Seq Study
SM-2015-61 vs SM-2015-66small molecule treated transcriptome study
SM-2015-66
SM-2015-61
2017
anti
pro
Bhatia’s lab, McMaster
What’s next
ICG001
35
RNA Seq Study
SM-2015-66
SM-2015-61Cell Migration Gene Set
IWR-1 (control)
down
up
Family 1 Hits
O
O
NH
O
OBnO
OO
O
O
O
H
H
NH
O
OBnO
OO
O
36
Metastasis Breast Cancer Gene Set
IWR-1 (control)
down
up
RNA Seq Study (contd)
SM-2015-66
SM-2015-61
Family 1 Hits
O
O
NH
O
OBnO
OO
O
O
O
H
H
NH
O
OBnO
OO
O
June 25, 2019 37
Epithelial to Mesenchymal Transition
down
up
RNA Seq Study (contd)Stemness
Transcription
down
up
Translation
Gene sets / pathways are taken from the public data bases:KEGG: https://www.genome.jp/kegg/genes.htmlGSEA: http://software.broadinstitute.org/gsea/msigdb/REACTOME: https://reactome.org/
open macrocycle
39
Cell Migration Gene SetEpithelial to Mesenchymal
Transition Gene Set
down
up
O
O
NH
O
MeOBnO
O
O
O
O
NH
O
MeOH
H
BnO
O
O
SM-110
SM-115
Family 2 Hits
Effect on Tumorspheres (G2*) from HCT 116
DMSO IWR1 SM-61SM-66
(Macrocycle)
48
Hr
72
Hr
96
Hr
12
0H
r1
68
Hr
G2* = 2nd generation
100 µm
SM-2015-66
SM-2015-61
Family 1 Hits
O
O
NH
O
OBnO
OO
O
O
O
H
H
NH
O
OBnO
OO
O
41
0
0.5
1
1.5
2
DMSO 110 115 IWR-1
Fo
ld C
ha
ng
e
LEF
0
1
2
3
4
5
6
7
DMSO 110 115 IWR-1
Fo
ld c
ha
ng
e
TCF4
Wnt Transcription-related Genes
RT-PCR Analysis: Small Molecule Treated Tumorsphere
(G2) from HCT 116 (contd)
0
1
2
3
4
5
DMSO 61 66 IWR-1
Fo
ld c
ha
ng
e
LEF
0
1
2
3
4
5
6
7
DMSO 61 66 IWR-1
TCF4
SM-61 SM-66
O
O
NH
O
MeOBnO
O
R1
O
O
O
NH
O
MeOH
H
BnO
O
Bn
O
SM-110
SM-115
June 25, 2019 42
RT-PCR Analysis: Small Molecule Treated Tumorsphere
(G2) from HCT 116
Metastasis
0
2
4
6
DMSO 110 115 IWR-1
Fo
ld c
ha
ng
e
VIMENTIN
0
2
4
6
DMSO 61 66 IWR-1
VIMENTIN
0
5
10
DMSO 110 115 IWR-1
Fo
ld c
ha
ng
e
CD44
0
5
10
DMSO 61 66 IWR-1
Fo
ld c
ha
ng
e
CD44
0
0.5
1
1.5
DMSO 110 115 IWR-1
Fo
ld c
ha
ng
e
CD133
0
0.5
1
1.5
DMSO 61 66 IWR-1
Fo
ld c
ha
ng
e
CD133
Stemness
0.000
0.500
1.000
1.500
2.000
2.500
Fo
ld c
ha
ng
e
SNAIL
0.000
0.500
1.000
1.500
2.000
Fo
ld C
ha
ng
e
SNAIL
0
1
2
3
4
DMSO 110 115 IWR-1
Fo
ld C
ha
ng
e
HEF1
0
1
2
3
4
DMSO 61 66 IWR-1
Fo
ld c
ha
ng
e
HEF1
Migration and Invasion
June 25, 2019 43
Effect on Tumorspheres (G2*) from Patient Buccal Mucosa (Indian Patient 001) G2 = 2nd generation
DM
SO
IWR
-1S
M-6
6S
M-1
10
SM
-115
0 Hr 72 Hr 144 Hr 168 Hr
O
O
H
H
NH
O
OBnO
OO
O
SM-66
SM-110
SM-115
44100 µm
Our Working Hypothesis
CBP
Our small molecules as transcription
inhibitors
RNA seq with 110/115
Quant proteomics with66, 110 and 115
Evaluation on patient-derived organoids
Target pull-down studies
Medicinal chemistry // Protein degradation direction
Ongoing Studies
June 25, 2019 45
Filing two US provisional patents related to macrocyclic inhibitors of Wnt pathway / b-catenin-CBP-TCF interactions
Academic (India) - Graduate Students (2009-2018): Madhu AeluriSrinivas Chamakuri Ravikumar Jimmidi Shiva Krishna Reddy Bhanudas Dasari Srinivas Jogula Saidulu Konda Mahender KhatravathNaveen KumarJagan Gaddam
Our Biotech Team:Cell signaling/stem cell tech: Raveendra Babu Vamshi KrishnaAnusha KolusuSamarpita TarafderManjushriSubhadra DravidaNGS: Madhu MohanChem toolbox/med chem: Jagan GaddamNaveen Kumar Mahender KhatravathAnand Neha KardamPrabhat Arya
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