Team Leader: Nicolas Willand PhD - Pr
Marion Flipo PhD - Assistant Pr.
Marion Prieri - PhD student
Maryline Bourotte - Postdoc
Ingrid Ciliberti - Researcher
Nicolas Probst - Postdoc
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.
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.
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 (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.
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.
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. 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. 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. 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.
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  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).
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. 
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. 
Work in progress. We are currently working on the development of a preclinical candidate and identifying new chemotypes, which display different risk profiles.
 Villemagne, B., et al. (2012) European Journal of Medicinal Chemistry. 51, 1-16;  Ginsberg, A. M. et al. (2007), Nat Med. 13, 290-294;  Ahuja, S. D., et al. (2012) PLoS Med. 9, e1001300;  Dooley, K. E., et al. (2012) Clinical Infectious Diseases. 55, 572-581;  Wang, F., et al. (2007) J Exp Med. 204, 73-78;  Baulard, A. R., et al. (2000) J Biol Chem. 275, 28326-28331;  Willand, N., et al. (2009) Nat Med. 15, 537-544;  Flipo, M., et al. (2011) Journal of Medicinal Chemistry. 54, 2994-3010;  Flipo, M., et al. (2012) Journal of Medicinal Chemistry. 55, 68-83;  Flipo, M., et al. (2012) Journal of Medicinal Chemistry. 55, 6391-6402;  Willand, N., ACS Chemical Biology, 2010, 5, 1007–1013.