Mycobacterial Epoxide Hydrolase EphD Is Inhibited by Urea and Thiourea Derivatives.

The genome of the human intracellular pathogen Mycobacterium tuberculosis encodes an unusually large number of epoxide hydrolases, which are thought to be involved in lipid metabolism and detoxification reactions needed to endure the hostile environment of host macrophages. These enzymes therefore represent suitable targets for compounds such as urea derivatives, which are known inhibitors of soluble epoxide hydrolases. In this work, we studied in vitro the effect of the thiourea drug isoxyl on six epoxide hydrolases of M. tuberculosis using a fatty acid substrate. We show that one of the proteins inhibited by isoxyl is EphD, an enzyme involved in the metabolism of mycolic acids, key components of the mycobacterial cell wall. By analyzing mycolic acid profiles, we demonstrate the inhibition of EphD epoxide hydrolase activity by isoxyl and two other urea-based inhibitors, thiacetazone and AU1235, inside the mycobacterial cell.

Mycobacterial Targets for Thiourea Derivatives: Opportunities for Virtual Screening in Tuberculosis Drug Discovery.

Tuberculosis (TB) remains a primary global health concern, necessitating the discovery and development of new anti-TB drugs, mainly to combat drug-resistant strains. In this context, thiourea derivatives have emerged as promising candidates in TB drug discovery due to their diverse chemical structures and pharmacological properties. This review aimed to explore this potential, identifying and exploring molecular targets for thiourea derivatives in Mycobacterium tuberculosis (Mtb) and the potential application of virtual screening techniques in drug discovery. We have compiled a comprehensive list of possible molecular targets of thiourea derivatives in Mtb. The enzymes are primarily involved in the biosynthesis of various cell wall components, including mycolic acids, peptidoglycans, and arabinans, or targets in the branched-chain amino acid biosynthesis (BCAA) pathway and detoxification mechanisms. We discuss the potential of these targets as critical constituents for the design of novel anti-TB drugs. Besides, we highlight the opportunities that virtual screening methodologies present in identifying potential thiourea derivatives that can interact with these molecular targets. The presented findings contribute to the ongoing efforts in TB drug discovery and lay the foundation for further research in designing and developing more effective treatments against this devastating disease.

Mechanisms of Resistance Associated with the Inhibition of the Dehydration Step of Type II Fatty Acid Synthase in Mycobacterium tuberculosis.

Isoxyl (ISO) and thiacetazone (TAC) are two antitubercular prodrugs that abolish mycolic acid biosynthesis and kill Mycobacterium tuberculosis (Mtb) through the inhibition of the essential type II fatty acid synthase (FAS-II) dehydratase HadAB. While mutations preventing ISO and TAC either from being converted to their active form or from covalently modifying their target are the most frequent spontaneous mutations associated with high-level resistance to both drugs, the molecular mechanisms underlying the high-level ISO and TAC resistance of Mtb strains harboring missense mutations in the second, nonessential, FAS-II dehydratase HadBC have remained unexplained. Using a combination of genetic, biochemical, and biophysical approaches and molecular dynamics simulation, we here show that all four reported resistance mutations in the HadC subunit of HadBC alter the stability and/or specific activity of the enzyme, allowing it in two cases (HadBCV85I and HadBCK157R) to compensate for a deficiency in HadAB in whole Mtb bacilli. The analysis of the mycolic acid profiles of Mtb strains expressing the mutated forms of HadC further points to alterations in the activity of the mycolic acid biosynthetic complex and suggests an additional contributing resistance mechanism whereby HadC mutations may reduce the accessibility of HadAB to ISO and TAC. Collectively, our results highlight the importance of developing optimized inhibitors of the dehydration step of FAS-II capable of inhibiting both dehydratases simultaneously, a goal that may be achievable given the structural resemblance of the two enzymes and their reliance on the same catalytic subunit HadB.

Covalent modification of the Mycobacterium tuberculosis FAS-II dehydratase by Isoxyl and Thiacetazone.

Isoxyl and Thiacetazone are two antitubercular prodrugs formerly used in the clinical treatment of tuberculosis. Although both prodrugs have recently been shown to kill Mycobacterium tuberculosis through the inhibition of the dehydration step of the type II fatty acid synthase pathway, their detailed mechanism of inhibition, the precise number of enzymes involved in their activation and the nature of their activated forms remained unknown. We here demonstrate that both Isoxyl and Thiacetazone specifically and covalently react with a cysteine residue (Cys61) of the HadA subunit of the dehydratase thereby inhibiting HadAB activity. Our results unveil for the first time the nature of the active forms of Isoxyl and Thiacetazone and explain the basis for the structure-activity relationship of and resistance to these thiourea prodrugs. Our results further indicate that the flavin-containing monooxygenase EthA is most likely the only enzyme required for the activation of ISO and TAC in mycobacteria.