STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES ACTING ON BACTERIAL MEMBRANES
Boštjan Japelj
Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia
Antibiotics – “miracle drugs”
Bacterial resistance is becoming a major problem in modern medicine
Cationic antimicrobial peptides:
- up to 50 aminoacids long
- a net positive charge of at least +2 (Arg, Lys)
- antimicrobial activity against G-, G+ bacteria, fungi, protozoa, viruses,
anticancer activity, effectors of innate immune response
- 4 structural classes:
ref:
ref.: Matsuzaki, K.,. Biochim Biophys Acta, 1999. 1462(1-2): p. 1-10.
- act on membranes and intracellular targets,
-advantage: fast acting, resistance is unlikely to develop, able to neutralize bacterial endotoxins and prevent development of sepsis
LACTOFERRIN
LF11 FQWQRNIRKVR-NH2
C12LF11 lauryl-FQWQRNIRKVR-NH2
P3-55 octanoyl-FWRIRIRR – NH2
Membrane models:-LPS (lipopolysaccharide): model for baterial membrane -SDS (sodium dodecyl sulphate): model for bacterial membrane-DPC (dodecyl phosphocholine): model for eucaryotic membrane
NMR study of LF11 + S-LPS, LF11 + SDS, LF11 + DPC
TRNOE between aromatic and aliphatic side chains in 2 mM LF11 upon addition of 1/20 of molar ratio of LPS (b) and LTA (c). The reference NOESY spectrum of LF11 is shown in (a). Spectra were recorded at a mixing time of 150 ms.
LF11 + S-LPS: trNOE
+koff
LF11 LPS
LF11-LPS
2tanh
21
2
022
cpex
cpexex
bfCPMG tk
tkk
ppRR
CPMG-T2 experiment:
1( )ex f offk p k
Family of 3D structures of LF11 in complex with LPS. ( basic, hydrophobic,
LF11 + S-LPS
Complex between LF11 and LPS441 Å2 of surface area buried
polar residues)
Comparison of LPS interaction motifs in FhuA (left)1, LF11 (center)2 and polymyxin B (right)3,4 in the same orientation with respect to LPS, which would be in front of the plane of the page
Binding motif:LF11 :Phe1 , Arg5 , Lys9 , Arg11 FhuA :Phe355, Lys439 , Arg384 , Lys351
PmxB :Phe6 , Dab8,9 , Dab3 , Dab1
1 Ferguson, A. D., Hofmann, E., Coulton, J. W., Diederichs, K., and Welte, W. (1998) Science 282: 2215–22202Japelj, B., Pristovšek, P., Majerle, A., Jerala, R. J Biol Chem, 2005. 280(17): p. 16955-61. 3Pristovšek, P. and J. Kidrič, J Med Chem, 1999. 42(22): 4604-13 4Pristovšek, P., Simčič, S., Wraber, B., Urleb, U. , J Med Chem, 2005. 48: 7911-7914
Comparison of structures LF11+S-LPS, LF11+SDS, LF11+DPC
LF11+SDS
LF11+S-LPS LF11+DPC
N-terminal part of LF11 is protected from fluoresscence quenching
Fluorescence quenching
LF11 FQWQRNIRKVR-NH2 1 2 3 4 5 67 8 91011
F0, F…Fluorescence emission intensity in the absence and presence of the quencher(Q)[Q]… concentration of the quencherKSV… Stern-Volmer quenching constant
C12LF11:-acylation enhances antimicrobial activity against G- and G+ bacteria
CD spectra of LF11 and C12LF11 inDPC micelles Family of structures of C12LF11 in DPC1
-acylation stabilizes secondary structure
1Japelj, B., Zorko, M., Majerle, A., Pristovšek, P.,et al.. JACS, (2007), 129: 1022-1023.
OCTANOYL-F W R I R I R R – NH21 2 3 4 5 6 7 8 9
P3-55:
Circular dichroism spectra
Backbone conformation of P3-55 in DPC
Structure of P3-55 in DPC Structure of P3-55 in SDS
Positioning and orientation of P3-55 in micelles(NMR experiments using paramagnetic probes 5-DSA and 16-DSA)
doxyl group
reference 5 - DSA
NOESY
TOCSY
Normalized I/Iref ratios of HN-H cross peaks in NOESY spectrum
P3-55 in SDS
P3-55 in DPC
Order parameter tensor elements of DPC micelle for the simulation of DPC micelle a) and DPC micelle + P3-55 b). –SCD=2/3Sxx + 1/2Syy
DPC
DPC + P3-55
Molecular dynamics of P3-55 in DPC
Total energy (left) and temperature (right) of the system (P3-55 + DPC + 14482 SOL + 4 Cl-) during first 2 ns of simulation
time [ps]
Ratios between princpal moments of inertia of DPC during simulation of P3-55 in DPC. Principal moments of inertia are shown in the table.
*moments of inertia in units 104 amu nm2. Asymetry parameter, , defined as = (2I1-I2-I3)/(I1+I2+I3)
*
Radial density of P3-55 in complex with DPC micelle.
DPC coordinates were taken fromTieleman, D.P., et al. J. Phys Chem. B. 2000, 104:6380-6388
LPS
zunanjamembrana
citoplazemskamembrana
a)
b) c)
d) e)
f)
++ +
++
+ ++
++ +
+
++ +
+
++++
+ +++
++++
++++
Outer membrane
Cytoplasmic membrane
Mechanism of interaction of ANEPID peptides with the membrane of Gram-negative bacteria.
Acknowledgements:
Andreja Majerle, Primož Pristovšek, Mateja Zorko, Roman Jerala (NIC, Ljubljana)
Miha Kotnik, Katja Kristan, Drago Kuzman, Andrej Preželj, Jan Humljan, Petra Igličar, Vjekoslava Car, Uroš Urleb (Lek, Drug Discovery, Ljubljana)
co-workers from the EU project ANEPID (Antimicrobial Endotoxin-neutralazing
Peptides to Combat Infectious Deseases):
Dagmar Zweytick, Karl Lohner (Graz)
Guillermo Martinez de Tejada, Ignacio Moriyon, Susana Sanchez-Gomez (Pamplona)
Sylvie E. Blondelle (San Diego, CA)
Klaus Brandenburg, Jörg Andrä (Borstel)