Tuberculosis Drug Boost

Team:

Team Leader: Nicolas Willand PhD - Pr
Marion Flipo PhD - Assistant Pr.
Marion Prieri - PhD student
Maryline Bourotte - Postdoc
Ingrid Ciliberti - Researcher
Nicolas Probst - Postdoc

Mission: Improving the current regimen of TB using new innovative approaches

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is still a leading cause of death. Co-infection with the human immunodeficiency virus contributes substantially to the morbidity and mortality from TB, and to the emergence of multidrug resistance. Multidrug-Resistant Tuberculosis (MDR-TB) must be treated for 2 to 4 years with second-line drugs. These drugs are less effective and are often associated with serious side effects, which reduce patient’s compliance and thus lead to high rates of recurrence and mortality.

Several antibiotics used to treat tuberculosis must be bioactivated by the bacteria to become highly toxic for the pathogen. A new therapeutic concept emerged in 2000 from the observation that thioamides (Ethionamide (ETH) and Prothionamide) activation is processed by a monooxygenase called EthA and is under the control of a transcriptional repressor called EthR.

Approach: Boosting activity of known antibiotics

Our strategy to boost Ethionamide activity lies on the discovery and optimization of drug-like inhibitors of EthR. This inhibitor could be then used in association with lower doses of Ethionamide in a regimen of higher tolerance and improved efficacy.

Latest results:

We combined our efforts with the research group of Alain Baulard from Pasteur Institute of Lille (Center for Infection and Immunity of Lille). This led to the design and development of the 1,2,4-oxadiazole drug-like series of molecule as inhibitors of EthR and boosters of ethionamide activity in vivo (Nature Medicine, 2009).

We demonstrated that the sensitivity of M. tuberculosis to ETH can be substantially increased in vitro and in vivo using specific EthR inhibitors. Critically, our boosters were able to triple the activity of ETH in a TB-infected mice model. Our last generation of ETH-boosters given orally at 20 mg/kg/day was shown to boost ETH 4 times in an intravenously infected TB mice model (data not yet published). We are now working on the identification of a preclinical candidate in collaboration with Bioversys (http://www.bioversys.com/index.php/pipeline-research/tuberculosis)

Tuberculosis remains a leading cause of death

Tuberculosis (TB) is a common infectious disease that is caused by Mycobacterium tuberculosis (Mtb) and accounts for more than 1.3 million deaths and 8.6 million new cases each year worldwide (http://www.who.int/tb/publications/global_report/fr/). Whilst TB prevalence and associated mortality are in decline, the increasing number of multi- (MDR), extensively (XDR) and totally drug-resistant TB cases still forces the discovery of new therapeutic alternatives.[1]

The scientific community has recently agreed that the key to improving TB therapy relies on shortening the duration of treatment and increasing the efficacy of the treatment against MDR and XDR strains.[2]

Thionamides: candidates for boosting. Ethionamide (ETH) and Prothionamide (PTH) are the most frequently used drugs for the treatment of drug-resistant tuberculosis and their therapeutic use has been reinforced by the results of a recent meta-analysis.[3] Consequently, as the number of MDR and XDR cases is growing worldwide, the importance of ETH is steadily increasing. Moreover ETH and PTH are the only second-line drugs with potential bactericidal activity.[4] However, ETH has an unfavorable therapeutic index, and gastrointestinal intolerance is its Achilles’ heel. Gradual dose increases to the highest tolerable dose is therefore recommended.

Bioactivation of ETH by the mycobacterial monooxygenase EthA produces a NAD-adduct which in turn inhibits the final target, InhA, an essential enzyme involved in cell-wall synthesis.[5] The expression of ethA, is tightly controlled by the transcriptional repressor EthR. As such, genetically engineered ethR BCG knockouts revealed a bacteria twenty-five times more sensitive to ETH, demonstrating that the mycobacteria controls its sensitivity to ETH.[6]

Boosting strategy: Proof of concept. A new therapeutic concept emerged from this observation: recently, we designed and developed the first drug-like molecules able to inhibit EthR. The results published in Nature Medicine [7] and in Journal of Medicinal Chemistry [8-10] demonstrated that the sensitivity of M. tuberculosis to ETH can be substantially increased in vitro and in vivo using specific EthR inhibitors. Critically, our booster compounds were able to triple the activity of ETH in a TB-infected mice model. Our lead booster (BDM41906) formulated in beta-cyclodextrine and administrated orally at 20 mg/kg/day was shown to boost ETH 4 times in an intravenously infected TB mice model (data not yet published).

Discovery of backup series:

Using HTS. A screening of our chemical library of drug-like molecules (14640 compounds) has been performed using reporter gene assay in Mycobacterium smegmatis. From this screening a new chemical family of EthR inhibitors bearing an N-phenylphenoxyacetamide motif was identified. The X-ray structure of the most potent compound crystallized with EthR inspired the synthesis of a 960-member focused library. These compounds were tested in vitro using a rapid thermal shift assay on EthR to accelerate the optimization. The best compounds were synthesized on a larger scale and confirmed as potent ethionamide boosters on M. tuberculosis-infected macrophages. Finally, the cocrystallization of the best optimized analogue with EthR revealed an unexpected reorientation of the ligand in the binding pocket. [10]

Using a fragment-based approach. As a complementary way to identify potent inhibitors of EthR we have developed Fragment-based approaches. We combined, SPR assay, X-ray crystallography, in silico design and medicinal chemistry for the rapid discovery and optimization of new chemotypes of EthR inhibitors.

Using in situ click-chemistry. In situ click chemistry has been successfully applied to probe the ligand binding domain of EthR. Specific protein-templated ligands were generated in situ from one azide and six clusters of 10 acetylenic fragments. Comparative X-ray structures of EthR complexed with either clicked ligand BDM14950 or its azide precursor showed ligand dependent conformational impacts on the protein architecture. This approach revealed two mobile phenylalanine residues that control the access to a previously hidden hydrophobic pocket that can be further exploited for the development of structurally diverse EthR inhibitors. [11]

Work in progress. We are currently working on the development of a preclinical candidate and identifying new chemotypes, which display different risk profiles.


[1] Villemagne, B., et al. (2012) European Journal of Medicinal Chemistry. 51, 1-16; [2] Ginsberg, A. M. et al. (2007), Nat Med. 13, 290-294; [3] Ahuja, S. D., et al. (2012) PLoS Med. 9, e1001300; [4] Dooley, K. E., et al. (2012) Clinical Infectious Diseases. 55, 572-581; [5] Wang, F., et al. (2007) J Exp Med. 204, 73-78; [6] Baulard, A. R., et al. (2000) J Biol Chem. 275, 28326-28331; [7] Willand, N., et al. (2009) Nat Med. 15, 537-544; [8] Flipo, M., et al. (2011) Journal of Medicinal Chemistry. 54, 2994-3010; [9] Flipo, M., et al. (2012) Journal of Medicinal Chemistry. 55, 68-83; [10] Flipo, M., et al. (2012) Journal of Medicinal Chemistry. 55, 6391-6402; [11] Willand, N., ACS Chemical Biology, 2010, 5, 1007–1013.

Publications on TB Drug Boost strategies:

  • Prevet, H., Moune, M., Tanina, A., Kemmer, C., Herledan, A., Frita, R., Wohlkönig, A., Bourotte, M., Villemagne, B., Leroux, F., Gitzinger, M., Baulard, A. R., Déprez, B., Wintjens, R., Willand, N., & Flipo, M. A fragment-based approach towards the discovery of N-substituted tropinones as inhibitors of Mycobacterium tuberculosis transcriptional regulator EthR2. Eur. J. Med. Chem.,2019, 167: 426-438.10.1016/j.ejmech.2019.02.023
  • Machelart, A., Salzano, G., Li, X., Demars, A., Debrie, A.-S., Menendez-Miranda, M., Pancani, E., Jouny, S., Hoffmann, E., Deboosere, N., Belhaouane, I., Rouanet, C., Simar, S., Talahari, S., Giannini, V., Villemagne, B., Flipo, M., Brosch, R., Nesslany, F., Deprez, B., Muraille, E., Locht, C., Baulard, A. R., Willand, N., Majlessi, L., Gref, R., & Brodin, P. Intrinsic Antibacterial Activity of Nanoparticles Made of β-Cyclodextrins Potentiates Their Effect as Drug Nanocarriers against Tuberculosis. ACS Nano,2019, 13(4): 3992-4007.10.1021/acsnano.8b0790
  • Pastor, A., Machelart, A., Li, X., Willand, N., Baulard, A., Brodin, P., Gref, R., & Desmaële, D. A novel codrug made of the combination of ethionamide and its potentiating booster: synthesis, self-assembly into nanoparticles and antimycobacterial evaluation. Org. Biomol. Chem.,2019, 17(20): 5129-5137.10.1039/C9OB00680J
  • Tanina, A., Wohlkönig, A., Soror, S. H., Flipo, M., Villemagne, B., Prevet, H., Déprez, B., Moune, M., Perée, H., Meyer, F., Baulard, A. R., Willand, N., & Wintjens, R. A comprehensive analysis of the protein-ligand interactions in crystal structures of Mycobacterium tuberculosis EthR. Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics, 2019, 1867(3): 248-258. 10.1016/j.bbapap.2018.12.003
  • Prieri, M., Frita, R., Probst, N., Sournia-Saquet, A., Bourotte, M., Déprez, B., Baulard, A. R., & Willand, N. Efficient analoging around ethionamide to explore thioamides bioactivation pathways triggered by boosters in Mycobacterium tuberculosis. Eur. J. Med. Chem.,2018, 159: 35-46.10.1016/j.ejmech.2018.09.038
  • Costa-Gouveia, J., Pancani , E., Jouny, S., Machelart, A., Delorme, V., Salzano, G., Iantomasi, R., Piveteau, C., Queval, C., Song, O.-R., Flipo, M., Deprez, B., Saint-André, J.-P., Hureaux, J., Majlessi, L., Willand, N., Baulard, A., Brodin, P., & Gref, R. Combination therapy for tuberculosis treatment: pulmonary administration of ethionamide and booster co-loaded nanoparticles. Scientific Reports,2017, 7: Article number: 5390 10.1038/s41598-017-05453-3
  • Blondiaux, N., Moune, M., Desroses, M., Frita, R., Flipo, M., Mathys, V., Soetaert, K., Kiass, M., Delorme, V., Djaout, K., Trebosc, V., Kemmer, C., Wintjens, R., Wohlkönig, A., Antoine, R., Huot, L., Hot, D., Coscolla, M., Feldmann, J., Gagneux, S., Locht, C., Brodin, P., Gitzinger, M., Déprez, B., Willand, N., & Baulard, A. R. Reversion of antibiotic resistance in Mycobacterium tuberculosis by spiroisoxazoline SMARt-420. Science,2017, 355(6330): 1206-1211. doi:10.1126/science.aag1006
  • Wohlkönig, A., Remaut, H., Moune, M., Tanina, A., Meyer, F., Desroses, M., Steyaert, J., Willand, N., Baulard, A. R., & Wintjens, R. Structural analysis of the interaction between spiroisoxazoline SMARt-420 and the Mycobacterium tuberculosis repressor EthR2. Biochemical and Biophysical Research Communications,2017, 487(2): 403-408.doi:10.1016/j.bbrc.2017.04.074
  • Villemagne, B., Flipo, M., Blondiaux, N., Crauste, C., Malaquin, S., Leroux, F., Piveteau, C., Villeret, V., Brodin, P., Villoutreix, B. O., Sperandio, O., Soror, S. H., Wohlkonig, 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. Journal of Medicinal Chemistry,2014 57(11): 4876-4888.10.1021/jm500422b
  • Crauste, C., Willand, N., Villemagne, B., Flipo, M., Willery, E., Carette, X., Moune Dimala, M., Drucbert, A. S., Danze, P. M., Deprez, B., & Baulard, A. Unconventional Surface Plasmon Resonance signals reveal quantitative inhibition of transcriptional repressor EthR by synthetic ligands. Analytical Biochemistry,2014, 452: 54-66. 10.1016/j.ab.2014.02.011
  • 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,2013, 69(11): 1243-1250.doi:10.1107/S0108270113028126
  • Flipo, M., Willand, N., Lecat-Guillet, N., Hounsou, C., Desroses, M., Leroux, F., Lens, Z., Villeret, V., Wohlkönig, A., Wintjens, R., Christophe, T., Jeon, H. K., Locht, C., Brodin, P., Baulard, A., & Deprez, B. Discovery of novel N-phenyl-phenoxyacetamide derivatives as EthR inhibitors and ethionamide boosters by combining High-Throughput Screening and Synthesis. Journal of Medicinal Chemistry,2012, 55(14): 6391–6402. doi:10.1021/jm300377g
  • Villemagne, B., Crauste, C., Flipo, M., Baulard, A. R., Deprez, B., & Willand, N. Tuberculosis: The drug development pipeline at a glance. European Journal of Medicinal Chemistry,2012, 51: 1–16. doi: 10.1016/j.ejmech.2012.02.033
  • Carette, X., Blondiaux, N., Willery, E., Hoos, S., Lecat-Guillet, N., Lens, Z., Wohlkonig, A., Wintjens, R., Soror, S., Frenois, F., Dirié, B., Villeret, V., England, P., Lippens, G., Deprez, B., Locht, C., Willand, N., & Baulard, A. Structural activation of the transcriptional repressor EthR from M.tuberculosis by single amino-acid change mimicking natural and synthetic ligands. Nucleic Acids Res,2012, 40(7): 3018-3030. doi: 10.1093/nar/gkr1113

  • 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., Deprez, B., Baulard, A. R., & 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(1): 68-83.doi: 10.1021/jm200825u
  • Flipo, M., Desroses, M., Lecat-Guillet, N., Dirie, B., Carette, X., Leroux, F., Piveteau, C., Demirkaya, F., Lens, Z., Rucktooa, P., Villeret, V., Christophe, T., Jeon, H. K., Locht, C., Brodin, P., Deprez, B., Baulard, A. R., & Willand, N. Ethionamide Boosters: Synthesis, Biological Activity, and Structure-Activity Relationships of a Series of 1,2,4-Oxadiazole EthR Inhibitors. Journal of Medicinal Chemistry,2011, 54(8): 2994-3010. doi:10.1021/jm200076a
  • Nicolas Willand, M. D., Patrick Toto, Bertrand Diri, Zo Lens, Vincent Villeret, Prakash Rucktooa, Camille Locht, Alain Baulard, and Benoit Deprez. Exploring Drug Target Flexibility Using in Situ Click Chemistry: Application to a Mycobacterial Transcriptional Regulator. ACS Chemical Biology,2010, 5(11): 1007–1013. doi: 10.1021/cb100177g
  • Willand, N. ; Dirie, B.; Carette, X.; Bifani, P.; Singhal, A.; Desroses, M.; Leroux, F.; Willery, E.; Mathys, V.; Deprez-Poulain, R.; Delcroix, G.; Frenois, F.; Aumercier, M.; Locht, C.; Villeret, V.; Deprez, B.; Baulard, A. R., Synthetic EthR inhibitors boost antituberculous activity of ethionamide. Nat Med 2009, 15, (5), 537. doi:10.1038/nm.1950
  • Frenois, F.; Baulard, A. R.; Villeret, V., Insights into mechanisms of induction and ligands recognition in the transcriptional repressor EthR from Mycobacterium tuberculosis. Tuberculosis (Edinb) 2006, 86, (2), 110-4.
  • Frenois, F.; Engohang-Ndong, J.; Locht, C.; Baulard, A. R.; Villeret, V., Structure of EthR in a ligand bound conformation reveals therapeutic perspectives against tuberculosis. Mol Cell 2004, 16, (2), 301-7.
  • (WO/2008/003861) Compounds having a potentiating effect on the activity of ethionamide and uses therof.

Awards:

  • Innovation Prize 20016 : Bioversys and TBBoost Team laureates of the Innovation Prize
  • AGETIP Thesis Prize 2013 to Baptiste Villemagne.
  • SCT Prix d'Encouragement à la Recherche 2011- SCT/Servier (Mar 2012) to Nicolas Willand, Paris, France.
  • MedChem Poster Prize (Nov. 2011) to Baptiste Villemagne, B. Annual One-Day Meeting on Medicinal Chemistry of SRC & KVCV: Emerging Targets and Treatments: Opportunities and Challenges for Drug Design, Ghent, Belgium.
  • SCT Jeunes Chercheurs Poster Prize (Feb. 2011) to Baptiste Villemagne. Design of EthR inhibitors as potent Ethionamide boosters using fragment based drug design (FBDD) approaches, Paris, France.
  • Prix de la vocation en Chimie Thérapeutique (dotation Servier) 2011 to Baptiste Villemagne. Ethionamide Boosters as a New Strategy to Fight Tuberculosis
  • Oséo Emergence 2009 : This project has been awarded by the French government in 2009 and received a grant dedicated to the creation of a private company that will ensure the commercial development of the project.
  • Best Oral Communication PRIM Scientific day (Mar 2010) Baptiste Villemagne "Validation of a Fragment-Based inhibitors - Growing strategy"
  • NOVARTIS Grant 2010 to Bouchra Hajjaj « for master degree internships in schools of the Federation Gay Lussac ».
  • SCT Jeunes Chercheurs Poster Prize (Feb. 2011) to Baptiste Villemagne. Design of EthR inhibitors as potent Ethionamide boosters using fragment based drug design (FBDD) approaches.

National and International collaborations:

  • Alain Baulard, INSERM U1019, CNRS UMR8204, Univ. Lille Nord de France, Institut Pasteur de Lille, Lille, France.
  • Priscille Brodin, INSERM U1019, CNRS UMR8204, Univ. Lille Nord de France, Institut Pasteur de Lille, Lille, France.
  • Vanessa Mathys, Tuberculosis and Mycobacteria, Communicable and Infectious Diseases, Scientific Institute of Public Health (WIV-ISP), Brussel, Belgium.
  • Juergen Siepmann, INSERM U1008, School of Pharmacy, Univ. Lille Nord de France, Lille, France.
  • Olivier Sperandio, INSERM UMR-S 973/MTi, Université Paris Diderot, Paris, France.
  • Vincent Villeret, Institut de Recherche Interdisciplinaire - IRI - USR3078 CNRS - Univ Lille Nord de France, Villeneuve d'Ascq, France.
  • René Wintjens, Structural Biology Unit, Institute of Molecular Biology and Medicine, Université Libre de Bruxelles, Gosselies, Belgium.
  • Alexandre Wohlkonig, Structural Biology Brussels and Molecular and Cellular Interactions, VIB, Brussel, Belgium.
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