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)