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ANR funded project

Blanc - SIMI 7 - Chimie moléculaire, organique, de coordination, catalyse et chimie biologique (Blanc SIMI 7)
Edition 2011


PepSiFUR


Structure-function relationships of Ferric Uptake Regulators and their inhibitors: towards new peptido-siderophore antibacterial compounds

New antibacterial compounds for peptido-siderophores synthesis
The increasing number of bacterial infections resistant to the current antibiotics is an extremely worrying phenomenon, particularly in hospitals (nosocomial infections). It is therefore necessary to develop new strategies and innovative antibiotics to fight against these particularly virulent microorganisms.

From peptide aptamers to peptide inhibitors: new molecules with anti-virulence activity targeting the regulation of iron homeostasis
Iron homeostasis and virulence of pathogens are intimately linked. A key actor of this metabolism in bacteria is the Fur protein (Ferric uptake regulator), a global transcriptional regulator of the expression of the genes involved in iron homeostasis. Any perturbation of Fur activity will affect proliferation and virulence of pathogenic strains. The aim of this project was to build on the discovery of 4 peptide aptamers able to interact with Fur from E. coli and to decrease the virulence of pathogens as demonstrated in a fly infection model. The derived peptides have been checked and optimized against Fur purified from different pathogenic strains such as Francisella tularensis, Yersinia pestis or Pseudomonas aeruginosa. This allowed to i) define a new structural family of tetrameric Fur, ii) precisely delineate the peptide-protein interactions by using coupled experimental and theoretical approaches, iii) synthesize new catechol precursors for biomimetic siderophores to be used as peptide vectors targeting Fur in vivo in a Trojan Horse strategy, and iv) use these interactions to screen chemical libraries looking for synthetic interacting molecules.

Purification and characterization of new Fur proteins, peptidization, screening of chemical libraries and synthesis of catechol vectors and peptides
Besides the protein from E. coli, both the purification and the characterization of Fur from Francisella tularensis (causal agent of the tularemia), from Yersinia pestis (causal agent of the plague), and from Pseudomonas aeruginosa (causal agent of complications in cystic fibrosis patients) were performed. A series of methods were used to define and characterize their interaction with 6 - 13 aminoacid-long linear anti-Fur peptides: in silico (modeling, molecular docking), in vitro (activities, interactions, mutagenesis, structural approaches), and in vivo (two-hybrid). The most promising peptides are coupled to the last generation of biomimetic siderophores, synthetic catechol vectors, developed to promote iron incorporation in E. coli or P. aeruginosa using the so-called Trojan Horse strategy. The screening of the Prestwick chemical library for inhibition of the interaction with peptide aptamers led to select a compound X inhibiting the oligomerization of Fur from P. aeruginosa. The better knowledge of the peptide-protein interactions allowed identifying potential inhibitor molecules by virtual screening of the ZINC bank.

Results

The project has been developed for basic research, first step before development of the lead compounds identified. It is valorized by scientific articles already published, presently submitted, or still under writing. The main results have been presented in scientific meetings. We can cite the development of optimized peptide inhibitors and the analysis of their interactions with different Fur, the synthesis of molecules designed as intracellular vectors for these peptides, the unexpected discovery of a new family of tetrameric Fur and their biochemical characterization.

Outlook

As previously mentioned, no new broad-spectrum family of antibiotics has emerged for 40 years. One explanation lies in the profitability of this type of compound for large pharmaceutical companies. The placing on the market of these compounds is indeed subject to stringent rules and the operating time is short, especially given the rapid emergence of resistance. The strategy currently used by the industry is to take / buy projects in progress to seedlings (start-up) or academic laboratories phases. Our approach, targeting the balance between iron deficeincy and overload in bacteria, fits perfectly in this context. Once the proof of concept made, it should be possible to develop our discoveries and to patent the most promising molecules.

Scientific outputs and patents

Published
- Cisse, C., Mathieu, S.V., Abeih, M.B., Flanagan, L., Vitale, S., Catty, P., Boturyn, D., Michaud-Soret, I. and Crouzy, S. Inhibition of the Ferric Uptake Regulator by peptides rerived from anti-FUR peptide aptamers: coupled theoretical and experimental approaches. ACS Chem Biol, 2014, 9, 2779-2786.
- Baco E, Hoegy F, Schalk IJ, Mislin GLA. Diphenyl-benzo[1,3]dioxole-4-carboxylic acid pentafluorophenyl ester : a convenient catechol precursor in the synthesis of siderophore vectors suitable for antibiotic Trojan horse strategies. Org. Biomol. Chem. 2014, 12, 749-757.
//pubs.rsc.org/en/Content/ArticleLanding/2014/OB/c3ob41990h#!divAbstract
Submitted:
- Pérard J, Covès J, Castellan M, Solard C, Savard M, Miras R, Galop S, Signor L, Crouzy S, Michaud-Soret I, de Rosny E. Quaternary structure of Fur proteins, new subfamily of tetrameric proteins (2016) revised version submitted to Biochemistry.
- Mathieu S, Cissé C, Vitale S, Ahmadova A, Pérard J, Colas P, Miras R, Boturyn D, Covès J, Crouzy S, Michaud-Soret I. From peptide aptamers to lead inhibitors of FUR, a bacterial global transcriptional regulator of iron homeostasis and virulence (2016) submitted to ACS Chem Biol.

Partners

CNRS-TMB CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ALSACE

IBS/Métalloprotéines CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES

LCBM/BioMet CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES

ANR grant: 436 000 euros
Beginning and duration: novembre 2011 - 36 mois

Submission abstract

The overuse of antibiotics has induced the development of resistant pathogens which become an increasing threat to public health. New antibacterial compounds and new targets must be found. The Ferric Uptake Regulator (FUR) is a global regulator in most bacteria which does not exist in eukaryotes. Since a link has been well established between iron bio-availability and pathogens virulence, FUR is a potential antibacterial target. Indeed, fur mutants exhibit decreased virulence. The FUR protein uses iron as a cofactor. Once metallated, it binds to specific DNA sequences to regulate the transcription of genes involved in iron metabolism and in virulence. The aim of this project is to develop inhibitors of FUR proteins that were formerly identified by means of the peptide aptamer technology. Deciphering the structure-function relationships between FUR and these inhibitors will help to design new antibacterial drugs. Insights into the mechanism of FUR are also expected from this work.
This project involves three groups with complementary expertise and common interest in finding specific inhibitors of FUR for the design of new antibacterial drugs: Partner 1 (coordinator) - Bioinorganic biochemists and theoretical chemists from the LCBM in Grenoble, experts in metalloregulators such as FUR and experts in modeling and molecular dynamics, respectively; Partner 2 - Chemists in the group "Transport membranaire bactérien" from the laboratory "Biotechnologie et Signalisation Cellulaire" in Strasbourg, experts in chemistry of siderophore mimics and Partner 3 – Biochemists and structural biologists from the "Groupe métalloprotéines" at the "Institut de Biologie Structurale" in Grenoble.
Partner 1 has developed for several years now the concept of using FUR as an antibacterial target. This group has in hands already four antiFUR inhibitors resulting from the peptide aptamer technology. These four peptide aptamers inhibit E. coli FUR in vivo and in vitro. Moreover, infection of flies showed that the inhibitors decreased significantly the virulence of an E. coli pathogenic strain. Structural and functional studies of the interaction of these four aptamers with FUR are ongoing, involving biochemists and theoretical chemists who analyze the molecular interactions from modeling studies and metal coordination theory. Peptides as short as 13-mer coming from the anti-FUR inhibitors have already been obtained that are able to inhibit FUR and are therefore very promising.

Key achievements of this project will be:
1) To obtain such structural information on the inhibitor-target interactions that drug-design becomes possible. This is expected from X-ray structure determination, biochemical assays and modeling studies;
2) To synthesize new inhibitors derived from the lead compounds that improve their inhibition ability and allow their uptake by the bacteria. Peptido-siderophores able to deliver the inhibitor in the cytoplasm and cyclic peptides will be synthesized and assayed on FUR activity in vitro and in vivo;
3) To understand the specificity of the inhibitors and to demonstrate the efficiency of the new antiFUR inhibitors.
Many groups in the world are involved in the field of iron acquisition by bacteria with the aim of developing both basic knowledge and new therapeutic targets. However, to our knowledge, partner 1 is the only group in the international scientific community who has targeted the FUR protein and discovered antiFUR inhibitors having an effect on virulence. This gathering which associates skills in biochemistry, structural biology, chemistry and molecular dynamics, should yield significant advances in the field and is meant to be competitive.

There was no previous application for an ANR financial support for this project with these partners.

 

ANR Programme: Blanc - SIMI 7 - Chimie moléculaire, organique, de coordination, catalyse et chimie biologique (Blanc SIMI 7) 2011

Project ID: ANR-11-BS07-0007

Project coordinator:
Madame Isabelle Michaud-Soret (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES)
imichaud@nullcea.fr

 

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The project coordinator is the author of this abstract and is therefore responsible for the content of the summary. The ANR disclaims all responsibility in connection with its content.