Structural analysis of transcription factors involved in Mycobacterium tuberculosis mycolic acid biosynthesis

Tuberculosis (TB) remains the leading cause of death due to a single infectious agent with more than 1.5 million people killed each year. In 2018, the World Health Organization (WHO) estimated that one third of the world’s population was infected with Mycobacterium tuberculosis (Mtb), the pathogen responsible for the disease.In 2000, EthR, a mycobacterial transcriptional repressor, was identified as a key modulator of ethionamide (ETH) bioactivation. ETH is one of the main second-line drugs used to treat drug-resistant strains and it is a prodrug that is activated in Mtb by the mono-oxygenase EthA and then inhibits InhA, an enzyme involved in the mycolic acid biosynthesis. In 2009, it was demonstrated that co-administration of ETH with the drug-like inhibitors of EthR was able to boost ETH activity by a factor three in a mouse-model of TB-infection, thus validating EthR protein as a target for a new therapeutic strategy. The first part of this thesis deals with the validation and deep characterization of the solved EthR-ligand structures based on all analysis of how each ligand bind to the EthR. In this section, based on the study of both co-crystal structures and the physicochemical properties of the ligands, we have rationalized the information currently available and understood the interaction of all EthR inhibitors in order to lead to more effective inhibitor design.More recently, another mycobaterial repressor, denoted EthR2, was identified as a putative target that appears to be functionally comparable to EthR (then the locus has been termed EthA2/EthR2, due to its similarity to the EthA/EthR locus). Furthermore, a spiroisoxazoline family of small-molecules, generically denoted as SMARt, has been identified as effective ligand of EthR2. However, according to the data present in the literature, this spiroisoxazoline family can also bind to the former EthR. In order to investigate this proposition, I have solved these small molecules in complex with EthR and compared their binding interactions to the EthR2 protein as well. The opportunity for the design small-molecules is capable of targeting both repressors, thereby opening the way to a dual-target approach.Finally, the third part of this thesis is devoted to the mycobacterial transcriptional factor MabR (Rv2242). Several studies identified this protein as a regulatory transcription factor of the fatty acid synthase II operon, which is mainly responsible for the mycolic acid biosynthesis in Mtb. I therefore purified to homogeneity and characterized the MabR protein as well as I determined the crystal structure of its C-terminal part. Finally, the functional role of MabR is largely discussed, and the way on how to interfere with its DNA binding ability is commented with respect to our results.Doctorat en Sciences biomédicales et pharmaceutiques (Pharmacie)info:eu-repo/semantics/nonPublishe

Structural analysis of the interaction between spiroisoxazoline SMARt-420 and the Mycobacterium tuberculosis repressor EthR2.

Inhibition of transcriptional regulators of bacterial pathogens with the aim of reprogramming their metabolism to modify their antibiotic susceptibility constitutes a promising therapeutic strategy. One example is the bio-activation of the anti-tubercular pro-drug ethionamide, which activity could be enhanced by inhibiting the transcriptional repressor EthR. Recently, we discovered that inhibition of a second transcriptional repressor, EthR2, leads to the awakening of a new ethionamide bio-activation pathway. The x-ray structure of EthR2 was solved at 2.3 Å resolution in complex with a compound called SMARt-420 (Small Molecule Aborting Resistance). Detailed comparison and structural analysis revealed interesting insights for the upcoming structure-based design of EthR2 inhibitors as an alternative to revert ethionamide resistance in Mycobacterium tuberculosis.SCOPUS: ar.jSCOPUS: ar.jinfo:eu-repo/semantics/publishe

A comprehensive analysis of the protein-ligand interactions in crystal structures of Mycobacterium tuberculosis EthR

International audienceThe Mycobacterium tuberculosis EthR is a member of the TetR family of repressors, controlling the expression of EthA, a mono-oxygenase responsible for the bioactivation of the prodrug ethionamide. This protein was established as a promising therapeutic target against tuberculosis, allowing, when inhibited by a drug-like molecule, to boost the action of ethionamide. Dozens of EthR crystal structures have been solved in complex with ligands. Herein, we disclose EthR structures in complex with 18 different small molecules and then performed in-depth analysis on the complete set of EthR structures that provides insights on EthR-ligand interactions. The 81 molecules solved in complex with EthR show a large diversity of chemical structures that were split up into several chemical clusters. Two of the most striking common points of EthR-ligand interactions are the quasi-omnipresence of a hydrogen bond bridging compounds with Asn 179 and the high occurrence of - interactions involving Phe 110. A systematic analysis of the proteinligand contacts identified eight hot spot residues that defined the basic structural features governing the binding mode of small molecules to EthR. Implications for the design of new potent inhibitors are discussed

A fragment-based approach towards the discovery of N-substituted tropinones as inhibitors of Mycobacterium tuberculosis transcriptional regulator EthR2

Tuberculosis (TB) caused by the pathogen Mycobacterium tuberculosis, represents one of the most challenging threat to public health worldwide, and with the increasing resistance to approved TB drugs, it is needed to develop new strategies to address this issue. Ethionamide is one of the most widely used drugs for the treatment of multidrug-resistant TB. It is a prodrug that requires activation by mycobacterial monooxygenases to inhibit the enoyl-ACP reductase InhA, which is involved in mycolic acid biosynthesis. Very recently, we identified that inhibition of a transcriptional repressor, termed EthR2, derepresses a new bioactivation pathway that results in the boosting of ethionamide activation. Herein, we describe the identification of potent EthR2 inhibitors using fragment-based screening and structure-based optimization. A target-based screening of a fragment library using thermal shift assay followed by X-ray crystallography identified 5 hits. Rapid optimization of the tropinone chemical series led to compounds with improved in vitro potency.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Discovery of the first Mycobacterium tuberculosis MabA (FabG1) inhibitors through a fragment-based screening

International audienceMycobacterium tuberculosis (M.tb), the etiologic agent of tuberculosis, remains the leading cause of death from a single infectious agent worldwide. The emergence of drug-resistant M.tb strains stresses the need for drugs acting on new targets. Mycolic acids are very long chain fatty acids playing an essential role in the architecture and permeability of the mycobacterial cell wall. Their biosynthesis involves two fatty acid synthase (FAS) systems. Among the four enzymes (MabA, HadAB/BC, InhA and KasA/B) of the FAS-II cycle, MabA (FabG1) remains the only one for which specific inhibitors have not been reported yet. The development of a new LC-MS/MS based enzymatic assay allowed the screening of a 1280 fragment-library and led to the discovery of the first small molecules that inhibit MabA activity. A fragment from the anthranilic acid series was optimized into more potent inhibitors and their binding to MabA was confirmed by 19 F ligand-observed NMR experiments

The small-molecule SMARt751 reverses Mycobacterium tuberculosis resistance to ethionamide in acute and chronic mouse models of tuberculosis.

The sensitivity of Mycobacterium tuberculosis, the pathogen that causes tuberculosis (TB), to antibiotic prodrugs is dependent on the efficacy of the activation process that transforms the prodrugs into their active antibacterial moieties. Various oxidases of M. tuberculosis have the potential to activate the prodrug ethionamide. Here, we used medicinal chemistry coupled with a phenotypic assay to select the N-acylated 4-phenylpiperidine compound series. The lead compound, SMARt751, interacted with the transcriptional regulator VirS of M. tuberculosis, which regulates the mymA operon encoding a monooxygenase that activates ethionamide. SMARt751 boosted the efficacy of ethionamide in vitro and in mouse models of acute and chronic TB. SMARt751 also restored full efficacy of ethionamide in mice infected with M. tuberculosis strains carrying mutations in the ethA gene, which cause ethionamide resistance in the clinic. SMARt751 was shown to be safe in tests conducted in vitro and in vivo. A model extrapolating animal pharmacokinetic and pharmacodynamic parameters to humans predicted that as little as 25 mg of SMARt751 daily would allow a fourfold reduction in the dose of ethionamide administered while retaining the same efficacy and reducing side effects.info:eu-repo/semantics/publishe