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Structural approach towards endotoxin and microbial inhibition

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Title: Structural approach towards endotoxin and microbial inhibition


1
Structural approach towards endotoxin and
microbial inhibition Primo Pristovek, Botjan
Japelj and Roman Jerala Laboratory of
Biotechnology, National Institute of Chemistry
(NIC), Ljubljana, Slovenia
2
Lipopolysaccharide main component of the outer
membrane of Gram (-) bacteria
Recognized by the innate immune system
Source http//pathmicro.med.sc.edu/fox/
3
Lipopolysaccharide (LPS, endotoxin) innate
immune system
Source Hawkins et al., Curr. Top. Med. Chem.
2004
  • Septic shock
  • 2 million casualties annually in the world
  • No cure

4
Anti-endotoxin Peptides -natural inhibitors of
LPS polymyxin B mo-KTKKKfLKKT
1 pyrrochoricine VDKGSYLPRPTPPRPIYNRN polyph
emusin I RRWCFRVCYRG FCYRKCR-NH2
... -fragments of endotoxin-binding
proteins lalf14 GCKPTFRRLKWKYKCG 2
CAP18106-137 GLRKRLRKFRNKIKEKLKKIGQKIQGLLPKLA
CAP3720-44 NQGRHFCGGALIHARFVMTAASCFQ
LBP91-108 WKVRKSFFKLQGSFDVSV BPI82-108
NANCKISGKWKAQKRFLKMSGNFDCSI LF11 FQWQRNIRKVR
3 C12LF11 N-lauryl-LF11 4 ... 1
Pristovek Kidric, J. Med. Chem. 1999 2
Pristovek, Feher, Szilagyi, Kidric, J. Med.
Chem. 2005 3 Japelj, Pristovek, Majerle,
Jerala, J. Biol. Chem. 2006 4 Majerle, Kidric,
Jerala, J. Antimicrob. Chemother. 2003 Japelj et
al, submitted Review Jerala Porro Pristovek
Kidric, Curr. Topics Med. Chem. 2004
- physical chemistry structure - rational design
5
Structural Origin of Endotoxin Neutralization and
Antimicrobial Activity of a Lactoferrin-based
Peptide
LF11 (FQWQRNIRKVR)
free peptide
Lf11 bound to LPS
LPS (G -)
LTA (G )
Backbone RMSD 1.24 ? 0.45 Å (Japelj B.,
Pristovek P., Majerle A., Jerala R. J. Biol.
Chem. 2005)
TR-NOE Clore Gronenborn, J. Magn. Reson. 1982
LTA lipoteichoic acid ( Gram () )
6
Known Structural Data X-ray structure of LPS
bound to the FhuA protein (Ferguson et al.,
Science 282, 1998)
7
LF11 bound to - LPS - SDS micelles
LPS T-shape
SDS umbrella-shape
441 Å2 of surface area buried

8
Effect of acylation on structure / antimicrobial
activity
C12LF11 N-lauryl LF11 Acylation affects
structure and antimicrobial activity
polymyxin B
Methyl-octanoat- KTKKKfLKKT K DAB
(a,g-diaminobutyric acid ) Golden standard for
antimicrobial and anti-endotoxin activity
Poster 11 Botjan Japelj, Mateja Zorko, Andreja
Majerle, Primo Pristovek and Roman Jerala
Effects of Acylation of the Cationic Peptide
on its Structure and Antimicrobial Activity
9
Structure of a Synthetic Fragment of the
Lipopolysaccharide (LPS) Binding Protein When
Bound to LPS and Design of a Peptidic LPS
Inhibitor
LBP-14 (RVQGRWKVRASFFK) LBP86-99K95A
Pairwise RMSDs for residues 6-13 Mean global
backbone 1.71 /- 0.39 Å Mean global heavy
atom 3.82 /- 0.61 Å
free peptide LPS
Pristovek P., Simcic S., Wraber B., Urleb U.,
J. Med. Chem. 2005
10
Modeling of peptide LPS complexes
Analysis of intermolecular contacts Rational
design of peptides with enhanced LPS-binding
properties
Measurement of in vitro inhibition of LPS-induced
TNF-a production
LPS peptide
LPS
TNF-a
TNF-a
TNF-a
11
Rational Design of Peptidic Lipopolysaccharide
(LPS) Inhibitors
free peptide LPS (TRNOE)
Synthesis Measurement of biological activity
NMR structure of the bound peptide
Pristovek P., Feher C., Szilagyi L., Kidric J.
(2005) J. Med. Chem.
Pristovek P., Simcic S., Wraber B., Urleb U.
(2005) J. Med. Chem.
Pristovek P. et al., in preparation
12
Green tea catechins inhibit DNA gyrase by binding
to its ATP binding site  Helena Gradiar1, Primo
Pristovek1, Andreja Plaper2, Roman
Jerala1 1Laboratory of Biotechnology, National
Institute of Chemistry

2KRKA Pharmaceutical Company, Novo
Mesto, Slovenia
Gyrases characteristic and essential bacterial
topoisomerases, relax supercoiled DNA
1AJ6
24 kDa Gyrase B novobiocin Holdgate et al.,
Biochem. 1997
Gyrase B ADPNP D.B. Wigley
24 kDa Gyrase B coumarin Plaper et al., BBRC
2003
13
15N labeled G24 (24 kDa fragment Gyr B)
EGCG 1 0.5 EGCG 1 4
Green tea up to 30 of catechin-based
polyphenols - radical scavenging action
1H, 15N NMR assignments from Golic Grdadolnik S.
et al., in Drug discovery and design medical
aspects, IOS Press 2002
14
Morris et al., J. Comp. Chem. 1998
A gyrB EGCG
B
24 kDa Gyrase B novobiocin Holdgate et al.,
Biochem. 1997
almost equal AutoDock docking interaction
15
You need
16
Andreja Majerle Helena Gradiar Mateja
Mancek Mateja Zorko Jurka Kidric Robert
Bremak Laboratory of Biotechnology NIC,
Ljubljana
Krisztina Fehér, László Szilágyi University of
Debrecen, Hungary
Saa Simcic Branka Wraber-Herzog Institute for
Microbiology and Immunology (IMI) Faculty of
Medicine University of Ljubljana Slovenia


Andreja Plaper
Ministry of Higher Education, Science and
Technology
Uro Urleb
ANEPID - QLRT-2001-01001, Antimicrobial endotoxin
neutralizing peptides to combat infectious
diseases
Slovenian NMR Centre
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