Crystal structure of the Mycobacterium tuberculosis VirS regulator reveals its interaction with the lead compound SMARt751.

Ethionamide (ETO) is a prodrug that is primarily used as a second-line agent in the treatment of tuberculosis. Among the bacterial ETO activators, the monooxygenase MymA has been recently identified, and its expression is regulated by the mycobacterial regulator VirS. The discovery of VirS ligands that can enhance mymA expression and thereby increase the antimycobacterial efficacy of ETO, has led to the development of a novel therapeutic strategy against tuberculosis. This strategy involves the selection of preclinical candidates, including SMARt751. We report the first crystal structure of the AraC-like regulator VirS, in complex with SMARt751, refined at 1.69 Å resolution. Crystals were obtained via an in situ proteolysis method in the requisite presence of SMARt751. The elucidated structure corresponds to the ligand-binding domain of VirS, adopting an α/ß fold with structural similarities to H-NOX domains. Within the VirS structure, SMARt751 is situated in a completely enclosed hydrophobic cavity, where it forms hydrogen bonds with Asn11 and Asn149 as well as van der Waals contacts with various hydrophobic amino acids. Comprehensive structural comparisons within the AraC family of transcriptional regulators are conducted and analyzed to figure out the effects of the SMARt751 binding on the regulatory activity of VirS.

Discovery of dual-active ethionamide boosters inhibiting the Mycobacterium tuberculosis ESX-1 secretion system.

Drug-resistant Mycobacterium tuberculosis (Mtb) remains a major public health concern requiring complementary approaches to standard anti-tuberculous regimens. Anti-virulence molecules or compounds that enhance the activity of antimicrobial prodrugs are promising alternatives to conventional antibiotics. Exploiting host cell-based drug discovery, we identified an oxadiazole compound (S3) that blocks the ESX-1 secretion system, a major virulence factor of Mtb. S3-treated mycobacteria showed impaired intracellular growth and a reduced ability to lyse macrophages. RNA sequencing experiments of drug-exposed bacteria revealed strong upregulation of a distinct set of genes including ethA, encoding a monooxygenase activating the anti-tuberculous prodrug ethionamide. Accordingly, we found a strong ethionamide boosting effect in S3-treated Mtb. Extensive structure-activity relationship experiments revealed that anti-virulence and ethionamide-boosting activity can be uncoupled by chemical modification of the primary hit molecule. To conclude, this series of dual-active oxadiazole compounds targets Mtb via two distinct mechanisms of action.

Both Nitro Groups Are Essential for High Antitubercular Activity of 3,5-Dinitrobenzylsulfanyl Tetrazoles and 1,3,4-Oxadiazoles through the Deazaflavin-Dependent Nitroreductase Activation Pathway.

3,5-Dinitrobenzylsulfanyl tetrazoles and 1,3,4-oxadiazoles, previously identified as having high in vitro activities against both replicating and nonreplicating mycobacteria and favorable cytotoxicity and genotoxicity profiles were investigated. First we demonstrated that these compounds act in a deazaflavin-dependent nitroreduction pathway and thus require a nitro group for their activity. Second, we confirmed the necessity of both nitro groups for antimycobacterial activity through extensive structure-activity relationship studies using 32 structural types of analogues, each in a five-membered series. Only the analogues with shifted nitro groups, namely, 2,5-dinitrobenzylsulfanyl oxadiazoles and tetrazoles, maintained high antimycobacterial activity but in this case mainly as a result of DprE1 inhibition. However, these analogues also showed increased toxicity to the mammalian cell line. Thus, both nitro groups in 3,5-dinitrobenzylsulfanyl-containing antimycobacterial agents remain essential for their high efficacy, and further efforts should be directed at finding ways to address the possible toxicity and solubility issues, for example, by targeted delivery.