STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES ACTING ON BACTERIAL MEMBRANES

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STRUCTURAL PROPERTIES OF ANTIMICROBIAL PEPTIDES
ACTING ON BACTERIAL MEMBRANES
Boštjan Japelj Lek Pharmaceuticals, Drug
Discovery, Ljubljana, Slovenia
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Antibiotics miracle drugs
Bacterial resistance is becoming a major problem
in modern medicine
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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
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- 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
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LACTOFERRIN
LF11 FQWQRNIRKVR-NH2 C12LF1
1 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

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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.
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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)
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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
22152220 2Japelj, B., Pristovšek, P., Majerle,
A., Jerala, R. J Biol Chem, 2005. 280(17) p.
16955-61. 3Pristovšek, P. and J. Kidric, J Med
Chem, 1999. 42(22) 4604-13 4Pristovšek, P.,
Simcic, S., Wraber, B., Urleb, U. , J Med Chem,
2005. 48 7911-7914
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Comparison of structures LF11S-LPS, LF11SDS,
LF11DPC
LF11SDS
LF11S-LPS
LF11DPC
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N-terminal part of LF11 is protected from
fluoresscence quenching
Fluorescence quenching
LF11 FQWQRNIRKVR-NH2 1 2 3 4 5 67 8
91011
F0, FFluorescence emission intensity in the
absence and presence of the
quencher(Q) Q concentration of the
quencher KSV Stern-Volmer quenching constant
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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.
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P3-55
OCTANOYL-F W R I R I R R NH2
1 2 3 4 5 6 7 8 9
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Circular dichroism spectra
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Structure of P3-55 in DPC
Structure of P3-55 in SDS
Backbone conformation of P3-55 in DPC
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Positioning and orientation of P3-55 in
micelles (NMR experiments using paramagnetic
probes 5-DSA and 16-DSA)
doxyl group
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reference
5 - DSA
NOESY
TOCSY
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Normalized I/Iref ratios of HN-Ha cross peaks in
NOESY spectrum
P3-55 in SDS

P3-55 in DPC
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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)/(I1I2I3)
Order parameter tensor elements of DPC micelle
for the simulation of DPC micelle a) and DPC
micelle P3-55 b). SCD2/3Sxx 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, 1046380-6388
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Outer membrane
Cytoplasmic membrane
Mechanism of interaction of ANEPID peptides with
the membrane of Gram-negative bacteria.
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Acknowledgements Andreja Majerle, Primož
Pristovšek, Mateja Zorko, Roman Jerala (NIC,
Ljubljana) Miha Kotnik, Katja Kristan, Drago
Kuzman, Andrej Preželj, Jan Humljan, Petra
Iglicar, Vjekoslava Car, Uroš Urleb (Lek, Drug
Discovery, Ljubljana) co-workers from the EU
project ANEPID (Antimicrobial Endotoxin-neutralazi
ng 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)