Structural activation of the transcriptional repressor EthR from Mycobacterium tuberculosis by single amino acid change mimicking natural and synthetic ligands

Ethionamide is an antituberculous drug for the treatment of multidrug-resistant Mycobacterium tuberculosis. This antibiotic requires activation by the monooxygenase EthA to exert its activity. Production of EthA is controlled by the transcriptional repressor EthR, a member of the TetR family. The sensitivity of M. tuberculosis to ethionamide can be artificially enhanced using synthetic ligands of EthR that allosterically inactivate its DNA-binding activity. Comparison of several structures of EthR co-crystallized with various ligands suggested that the structural reorganization of EthR resulting in its inactivation is controlled by a limited portion of the ligand-binding-pocket. In silico simulation predicted that mutation G106W may mimic ligands. X-ray crystallography of variant G106W indeed revealed a protein structurally similar to ligand-bound EthR. Surface plasmon resonance experiments established that this variant is unable to bind DNA, while thermal shift studies demonstrated that mutation G106W stabilizes EthR as strongly as ligands. Proton NMR of the methyl regions showed a lesser contribution of exchange broadening upon ligand binding, and the same quenched dynamics was observed in apo-variant G106W. Altogether, we here show that the area surrounding Gly106 constitutes the molecular switch involved in the conformational reorganization of EthR. These results also shed light on the mechanistic of ligand-induced allosterism controlling the DNA binding properties of TetR family repressors

Exploring drug target flexibility using in situ click chemistry: Application to a mycobacterial transcriptional regulator

In situ click chemistry has been successfully applied to probe the ligand binding domain of EthR, a mycobacterial transcriptional regulator known to control the sensitivity of Mycobacterium tuberculosis to several antibiotics. 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. This report shows that protein-directed in situ chemistry allows medicinal chemists to explore the conformational space of a ligand-binding pocket and is thus a valuable tool to guide drug design in the complex path of hit-to-lead processes. © 2010 American Chemical Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Ethionamide boosters: Synthesis, biological activity, and structure-activity relationships of a series of 1,2,4-oxadiazole EthR inhibitors

We report in this article an extensive structure-activity relationships (SAR) study with 58 thiophen-2-yl-1,2,4-oxadiazoles as inhibitors of EthR, a transcriptional regulator controling ethionamide bioactivation in Mycobacterium tuberculosis. We explored the replacement of two key fragments of the starting lead BDM31343. We investigated the potency of all analogues to boost subactive doses of ethionamide on a phenotypic assay involving M. tuberculosis infected macrophages and then ascertained the mode of action of the most active compounds using a functional target-based surface plasmon resonance assay. This process revealed that introduction of 4,4,4-trifluorobutyryl chain instead of cyanoacetyl group was crucial for intracellular activity. Replacement of 1,4-piperidyl by (R)-1,3-pyrrolidyl scaffold did not enhance activity but led to improved pharmacokinetic properties. Furthermore, the crystal structures of ligand-EthR complexes were consistent with the observed SAR. In conclusion, we identified EthR inhibitors that boost antibacterial activity of ethionamide with nanomolar potency while improving solubility and metabolic stability. © 2011 American Chemical Society.SCOPUS: ar.jinfo:eu-repo/semantics/publishe