Research topic and scientific context

Transcription

Research topic and scientific context
30 avril 2015 [ALLOCATIONS DE RECHERCHE 2015] Titredelathèse
« Reprogrammation de la bioactivation de pro‐antituberculeux : conception, développement et évaluation biologique d'inhibiteurs de régulateurs transcriptionnels. Une nouvelle approche dans le traitement de la tuberculose. » Directeur de thèse: Pr. Nicolas Willand, Inserm U1177, Faculté des Sciences Pharmaceutiques et Biologiques de Lille Co‐directeur de thèse: Dr. Alain Baulard, Inserm U1019, CIIL, Institut Pasteur de Lille Researchtopicandscientificcontext
The past decade has seen a dramatic worldwide increase in antibiotic resistant pathogenic bacteria. Despite this, many drug developers left the field, leaving an urgent, unmet need for new antibiotics, especially for treatment of infectious caused by MDR gram(‐) pathogens and Mycobacterium tuberculosis. The low number of new products in clinical development is inadequate to circumvent rapid antibiotic resistance development and novel innovative strategies are needed to avoid entering a pre‐antibiotic era. Our project is based on the peculiarity of some important antituberculous (anti‐
TB) compounds that require enzyme‐dependent bioactivation by the bacteria to acquire their activity. Resistance mechanisms to these prodrugs often rely on the appearance of mutation in the bioactivation pathways. We have recently demonstrated that small molecules can be used to reprogram the transcriptome of the bacteria in general and to wake up alternative bioactivation pathway in particular, thus reverting resistance to corresponding prodrug. The project is driven by a consortium of three academic partners that aim at accelerating the development of new, safe and highly effective combinations of drugs to restore activity of known antituberculous prodrugs and to limit the emergence of resistant strains. This objective will be addressed by combining phenotypic screens using a unique cell‐based, high‐throughput and high‐
content miniaturized screening (HT‐HCS) set up together with target‐based assays. Taking advantage of the high throughput of this platform (10.000 points / day, 384‐well plate format), we can rapidly identify a very large number of drug‐like anti‐TB molecules that will ‐ in combination with drugs used in the clinic ‐ highlight alternative bioactivation pathways of first and second‐line drugs isoniazide (INH), pyrazinamide (PZA), para‐aminosalicylic acid (PAS) or nitroimidazoles. The chemical entities that will be developed in the project will be optimized in combination with drugs to enhance the global effectiveness of the treatment, especialy against resistant bacteria. Our goal is to provide innovative, new combination treatment regimens that show in vivo efficacy and appropriate safety/pharmacokinetic profiles, which could then be progressed into preclinical regulatory studies, with industrial partners. The project input will include: 1) Platforms of screening technologies for the rapid discovery of hits a. a HT‐HCS screening platform in BSL2 and BSL3 environment (Equipex: Imaginex Biomed) optimized for high throughput screening of combinations of molecules and drugs b. a chemical library of 70,000 drug like molecules, divided into sub‐collections (Ibisa platform U1177) c. Medicinal chemistry expertise d. Impact of host‐pathogen interaction on resistance/virulence regulation e. ADME and in vivo platform 2) A validated proof of concept of our approach with the reversion of resistance to the anti‐TB drug ethionamide (ETH). We recently validated a series of small molecules that reprogram the mycobacterial pathway involved in ETH bioactivation in M. tuberculosis thus restoring full drug susceptibility in clinical ETH‐resistant strains. [ALLOCATIONSDERECHERCHE2015]
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30 avril 2015 [ALLOCATIONS DE RECHERCHE 2015] Stateoftheart
It is commonly accepted that transcription of prokaryotic operons is typically regulated by repressors and activators that contain ligand‐binding or sensor domains, connected to DNA‐binding HTH domains. In the TetR family of regulators (TFRs) found in more than 115 genera of gram‐positive, α‐, β‐, and γ‐proteobacteria, cyanobacteria and archaea, both sensors and DNA binding domains are contained in the same polypeptide. TFRs regulate the expression of proteins that are involved in the transcriptional control of a large variety of physiological processes, including multidrug efflux pumps, biosynthesis of antibiotics, response to osmotic stress and toxic chemicals, catabolic pathways, differentiation, and pathogenicity. To date, the sequences of more than 200,000 transcription factors, including 2500 TFRs are available in public databases and X‐ray structures of more than 200 TFRs have been solved. This class of protein has attracted our interest ten years ago when we studied the impact of the inhibition of EthR, a mycobacterial transcriptional factor that controls the bioactivation and thus the resistance of M. tuberculosis to ETH. Our current understanding of the bioactivation of ETH is centered on the NADPH‐specific FAD‐containing monooxygenase EthA, which transforms via oxidation process ETH into an ETH‐NAD adduct. This adduct has been shown to inhibit InhA, an NADH‐dependent enoyl‐acyl carrier protein reductase of the fatty acid biosynthesis II system required for mycolic acid biosynthesis, thus leading to cell wall deficiency and bacterial death. In 2004, we proposed a new therapeutic concept involving the inhibition of EthR by synthetic ligand to release the transcription of ethA and thereby boost the bioactivation of ETH. Based on this alternative concept, the combined efforts of our consortium, led to the discovery, design and development of a first generation of small molecules boosting ETH in a TB‐infected mice model. By combining our EthR target‐based assays and a phenotypic assay based on High‐Content Screening involving macrophages infected with fluorescent mycobacteria, we discovered a new generation of small molecules able to boost the activity of ETH without interacting with EthR. We therefore hypothesized that these new molecules trigger a new EthR‐independent route of ETH bioactivation. This led to the identification of a new ETH bioactivation pathway. The high potential of this discovery for the clinic was demonstrated by the full restoration of ETH activity on MDR and ETH‐resistant strains in the presence of second generation boosters (ETH‐F2B) compared to ETH‐F1B. Proof of concept was recently obtained in mice were ETH‐F2B was able to restore the activity of ETH against an ETH‐resistant Mtb strain. Like ETH, a large panel of antituberculosis drugs of first and second‐line treatments are pro‐
antibiotics (or prodrugs) and therefore require enzymatic bioactivation to exhibit their antibacterial activity. Their transformation is catalyzed by drug‐specific mycobacterial enzymes. For example, pyrazinamide (PZA) is activated by the pyrazinamidase PncA and isoniazid (INH) by the catalase peroxidase KatG. Bioactivation of the new compounds PA824 (presently in Phase II) and Deltyba (delamanid, compationate use in EU) involve the glucose‐6‐phosphate dehydrogenase (G6PD), and Rv3547 catalytic enzyme. Therefore the resistance to these drugs occurs mainly via mutation in the bioactivation genes, and we propose to screen those particular strains with our libraries of drug like compounds in combination to known TB drugs to identify and optimize molecules that will restore activity against MDR/XDR strains by targeting new regulated bioactivation pathways, following the successful concept described for ETH. [ALLOCATIONSDERECHERCHE2015]
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30 avril 2015 [ALLOCATIONS DE RECHERCHE 2015] Outcomesoftheproject
1. The discovery of a new bioactivation pathway for at least one of four important antitubercular prodrugs (INH, PZA, OPC and PAS) facing major resistance obstacles in the clinic 2. Hit to lead optimization of at least one family of drug‐like molecules awakening a new prodrug bioactivation pathway 3. in vivo proof of concept of our approach Partnersdescription:
Thesis co‐director: Dr. Alain Baulard, CIIL, INSERM U1019 /CNRS UMR 8204, Institut Pasteur de Lille Collaborator: Dr. Priscille Brodin, CIIL, INSERM U1019 /CNRS UMR 8204, Institut Pasteur de Lille Collaborator: Dr Roland Brosch, équipe Pathogénomique Mycobacterienne Intégrée, Institut Pasteur Paris, France Alain Baulard and Priscille Brodin are specialists in M. tuberculosis functional and chemical genomics and have developed innovative tools to study combinations of molecules using automated confocal fluorescence microscopy. Using this approach, more than 200,000 compounds have already been screened. Both will participate in the development of screening assays, in the validation of combinations using secondary biochemical assays and in the validation of the targets. Roland Brosch will bring to the consortium its expertise in Mtb genomics. He will perform transcriptomic analysis and knock‐out of genes to validate the target engagement of identified hits. Programandworkschedule:
During the first nine months of 2015, we will perform the screening of resistant M. Tb strains with our libraries of drug like compounds in combination to known TB drugs. Hits that will restore activity against those strains will be further optimized and their mode of action will be studied. Aim of the thesis: the PhD student will provide medicinal chemistry to transform identified hits into a lead for in vivo testing and subsequently into a preclinical candidate. The role of the PhD student will be to perform a rational pharmacomodulation of the hits identified during the phenotypic screenings using medicinal chemistry. In a second phase of the thesis, the candidate will select an optimized analogue to study the mode of action of this molecule and validate its final target. Finally, the optimization of the pharmacokinetic properties of the lead compound will allow performing in vivo study for the proof of concept. Steps
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Step 1: Following screenings and validation of hits, two chemical series will be selected for further optimization. Several criteria will be considered: chemical accessibility, originality, ease to introduce chemical diversity. Step 2: Synthesis of focused libraries of analogues and study structure‐activity relationships will be performed. A lead compound will be selected for step 3 to allow studying its mode of action. Step 3: Validation of the mode of action of the lead using transcriptomic. Step 4: The study of the pharmacokinetic properties of the lead will allow identifying weaknesses prior optimization. Step 5: A candidate will be selected to perform in vivo studies. [ALLOCATIONSDERECHERCHE2015]
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30 avril 2015 [ALLOCATIONS DE RECHERCHE 2015] ListofPublications:
1. Villemagne, B.; Flipo, M.; Blondiaux, N.; Crauste, C.; Malaquin, S.; Leroux, F.; Piveteau, C.; Villeret, V.; Brodin, P.; Villoutreix, B.; Sperandio, O.; Soror, S.; Wohlkönig, A.; Wintjens, R.; Deprez, B.; Baulard, A. R.; Willand, N. Ligand Efficiency Driven Design of New Inhibitors of Mycobacterium tuberculosis Transcriptional Repressor EthR Using Fragment Growing, Merging, and Linking Approaches. J. Med. Chem. 2014, 57, 4876. 2. Tatum N.J., Villemagne B., Willand N., Deprez B., Liebeschuetz J.W., Baulard A.R. , Pohl, E. Structural and docking studies of potent ethionamide boosters. Acta Crystallographica Section C: Crystal Structure Communications, 2013, 69 (11), pp. 1243‐1250. 3. Carette, X., Blondiaux, N., Willery, E., Hoos, S., Lecat‐Guillet, N., Lens, Z., Wohlkönig, A., Wintjens, R., Soror, S. H., Frénois, F., Dirié, B., Villeret, V., England, P., Lippens, G., Deprez, B., Locht, C., Willand, N. and Baulard, A. R., Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands. Nucleic Acids Research 2012, 40, 3018‐3030. 4. Flipo, M., Desroses, M., Lecat‐Guillet, N., Villemagne, B., Blondiaux, N., Leroux, F., Piveteau, C., Mathys, V., Flament, M.‐P., Siepmann, J., Villeret, V., Wohlkönig, A., Wintjens, R., Soror, S. H., Christophe, T., Jeon, H. K., Locht, C., Brodin, P., Déprez, B., Baulard, A. R. and Willand, N., Ethionamide Boosters. 2. Combining Bioisosteric Replacement and Structure‐Based Drug Design To Solve Pharmacokinetic Issues in a Series of Potent 1,2,4‐Oxadiazole EthR Inhibitors. Journal of Medicinal Chemistry 2012, 55, 68‐83. 5. Flipo, M., Willand, N., Lecat‐Guillet, N., Hounsou, C., Desroses, M., Leroux, F., Lens, Z., Villeret, V., Wohlkönig, A., Wintjens, R., Christophe, T., Kyoung Jeon, H., Locht, C., Brodin, P., Baulard, A. R. and Déprez, B., Discovery of Novel N‐Phenylphenoxyacetamide Derivatives as EthR Inhibitors and Ethionamide Boosters by Combining High‐Throughput Screening and Synthesis. Journal of Medicinal Chemistry 2012, 55, 6391‐6402. 6. Villemagne, B., Crauste, C., Flipo, M., Baulard, A. R., Déprez, B. and Willand, N., Tuberculosis: The drug development pipeline at a glance. European Journal of Medicinal Chemistry 2012, 51, 1‐16. 7. Flipo, M., Desroses, M., Lecat‐Guillet, N., Dirié, B., Carette, X., Leroux, F., Piveteau, C., Demirkaya, F., Lens, Z., Rucktooa, P., Villeret, V., Christophe, T., Jeon, H. K., Locht, C., Brodin, P., Déprez, B., Baulard, A. R. and Willand, N., Ethionamide Boosters: Synthesis, Biological Activity, and Structure−Ac vity Rela onships of a Series of 1,2,4‐Oxadiazole EthR Inhibitors. Journal of Medicinal Chemistry 2011, 54, 2994‐3010. 8. Willand, N., Desroses, M., Toto, P., Dirié, B., Lens, Z., Villeret, V., Rucktooa, P., Locht, C., Baulard, A. and Deprez, B., Exploring Drug Target Flexibility Using in Situ Click Chemistry: Application to a Mycobacterial Transcriptional Regulator. ACS Chemical Biology, 2010, 5, 1007‐1013. 9. Willand, N., Dirié, B., Carette, X., Bifani, P., Singhal, A., Desroses, M., Leroux, F., Willery, E., Mathys, V., Déprez‐Poulain, R., Delcroix, G., Frénois, F., Aumercier, M., Locht, C., Villeret, V., Déprez, B. and Baulard, A. R., Synthetic EthR inhibitors boost antituberculous activity of Ethionamide. Nature Medicine, 2009, 15, 537 ‐ 544. [ALLOCATIONSDERECHERCHE2015]
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