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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:.

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slide1

STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES ACTING ON BACTERIAL MEMBRANES

Boštjan Japelj

Lek Pharmaceuticals, Drug Discovery, Ljubljana, Slovenia

slide2

Antibiotics – “miracle drugs”

Bacterial resistance is becoming a major problem in modern medicine

slide3

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:

slide4

- act on membranes and intracellular targets,

ref.: Matsuzaki, K.,. Biochim Biophys Acta, 1999. 1462(1-2): p. 1-10.

-advantage: fast acting, resistance is unlikely to develop, able to neutralize bacterial endotoxins and prevent development of sepsis

slide5

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
slide6

NMR study of LF11 + S-LPS, LF11 + SDS, LF11 + DPC

LF11 + S-LPS: trNOE

CPMG-T2 experiment:

+

LF11

LPS

koff

LF11-LPS

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.

slide7

LF11 + S-LPS

Family of 3D structures of LF11 in complex with LPS.

( basic, hydrophobic,

Complex between LF11 and LPS

441 Å2 of surface area buried

polar residues)

slide8

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 282:2215–2220

2Japelj, 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

slide10

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 quencher

KSV… Stern-Volmer quenching constant

slide11

C12LF11:

-acylation enhances antimicrobial activity against G- and G+ bacteria

-acylation stabilizes secondary structure

CD spectra of LF11 and C12LF11 in

DPC micelles

Family of structures of C12LF11 in DPC1

1Japelj, B., Zorko, M., Majerle, A., Pristovšek, P.,et al.. JACS, (2007), 129: 1022-1023.

slide12

P3-55:

OCTANOYL-F WRIRIR R – NH2

1 2 3 4 5 6 7 8 9

slide14

Structure of P3-55 in DPC

Structure of P3-55 in SDS

Backbone conformation of P3-55 in DPC

slide15

Positioning and orientation of P3-55 in micelles

(NMR experiments using paramagnetic probes 5-DSA and 16-DSA)

doxyl

group

slide16

reference

5 - DSA

NOESY

TOCSY

slide18

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

DPC

DPC + P3-55

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, a, defined as a = (2I1-I2-I3)/(I1+I2+I3)

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

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

slide19

Outer membrane

Cytoplasmic membrane

Mechanism of interaction of ANEPID peptides with the membrane of Gram-negative bacteria.

slide20

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)