Residues of helix ɑ2 are critical for catalytic efficiency of mycobacterial alkylhydroperoxide reductase subunit C

The ability of Mycobacteria to overcome oxidative stress is of paramount importance for its survival within the host. One of the key enzymes that are involved in protecting the bacterium from reactive oxygen species is the catalase-peroxidase (KatG). However, in strains resistant to the antibiotic isoniazid, KatG is rendered ineffective, which is associated with an increased expression of alkylhydroperoxide reductase subunit C (AhpC). Mycobacterial AhpC possesses a unique helical displacement when compared to its bacterial counterparts. Here, via mutagenesis studies, we demonstrate the importance of this helix for redox modulation of the catalytic activity of AhpC. Along with structural insights from crystallographic data, the impact of critical residues on the structure and flexibility of the helix and on AhpC oligomerization is described.Ministry of Education (MOE)Nanyang Technological UniversityThis study was supported by the AcademicResearch Fund (AcRF) Tier 1 ID889 Ministry of Edu-cation, Singapore, to GG, SMSC is grateful to receivean NTU Research Scholarship at Nanyang Technolog-ical University, Singapore

Targeting the menaquinol binding loop of mycobacterial cytochrome bd oxidase

Mycobacteria have shown enormous resilience to survive and persist by remodeling and altering metabolic requirements. Under stringent conditions or exposure to drugs, mycobacteria have adapted to rescue themselves by shutting down their major metabolic activity and elevate certain survival factor levels and efflux pathways to survive and evade the effects of drug treatments. A fundamental feature in this adaptation is the ability of mycobacteria to vary the enzyme composition of the electron transport chain (ETC), which generates the proton motive force for the synthesis of adenosine triphosphate via oxidative phosphorylation. Mycobacteria harbor dehydrogenases to fuel the ETC, and two terminal respiratory oxidases, an aa3-type cytochrome c oxidase (cyt-bcc-aa3) and a bacterial specific cytochrome bd-type menaquinol oxidase (cyt-bd). In this study, we employed homology modeling and structure-based virtual screening studies to target mycobacteria-specific residues anchoring the b558 menaquinol binding region of Mycobacterium tuberculosis cyt-bd oxidase to obtain a focused library. Furthermore, ATP synthesis inhibition assays were carried out. One of the ligands MQL-H2 inhibited both NADH2- and succinate-driven ATP synthesis inhibition of Mycobacterium smegmatis inside-out vesicles in micromolar potency. Similarly, MQL-H2 also inhibited NADH2-driven ATP synthesis in inside-out vesicles of the cytochrome-bcc oxidase deficient M. smegmatis strain. Since neither varying the electron donor substrates nor deletion of the cyt-bcc oxidase, a major source of protons, hindered the inhibitory effects of the MQL-H2, reflecting that MQL-H2 targets the terminal oxidase cytochrome bd oxidase, which was consistent with molecular docking studies.NRF (Natl Research Foundation, S’pore)Accepted versio

Antituberculosis Activity of the Antimalaria Cytochrome bcc Oxidase Inhibitor SCR0911

The ability to respire and generate adenosine triphosphate (ATP) is essential for the physiology, persistence, and pathogenicity of Mycobacterium tuberculosis, which causes tuberculosis. By employing a lead repurposing strategy, the malarial cytochrome bc1 inhibitor SCR0911 was tested against mycobacteria. Docking studies were carried out to reveal potential binding and to understand the binding interactions with the target, cytochrome bcc. Whole-cell-based and in vitro assays demonstrated the potency of SCR0911 by inhibiting cell growth and ATP synthesis in both the fast- and slow-growing M. smegmatis and M. bovis bacillus Calmette-Guérin, respectively. The variety of biochemical assays and the use of a cytochrome bcc deficient mutant strain validated the cytochrome bcc oxidase as the direct target of the drug. The data demonstrate the broad-spectrum activity of SCR0911 and open the door for structure-activity relationship studies to improve the potency of new mycobacteria specific SCR0911 analogues.</p

Antituberculosis activity of the antimalaria cytochrome bcc oxidase inhibitor SCR0911

The ability to respire and generate adenosine triphosphate (ATP) is essential for the physiology, persistence, and pathogenicity of Mycobacterium tuberculosis, which causes tuberculosis. By employing a lead repurposing strategy, the malarial cytochrome bc1 inhibitor SCR0911 was tested against mycobacteria. Docking studies were carried out to reveal potential binding and to understand the binding interactions with the target, cytochrome bcc. Whole-cell-based and in vitro assays demonstrated the potency of SCR0911 by inhibiting cell growth and ATP synthesis in both the fast- and slow-growing M. smegmatis and M. bovis bacillus Calmette–Guérin, respectively. The variety of biochemical assays and the use of a cytochrome bcc deficient mutant strain validated the cytochrome bcc oxidase as the direct target of the drug. The data demonstrate the broad-spectrum activity of SCR0911 and open the door for structure–activity relationship studies to improve the potency of new mycobacteria specific SCR0911 analogues.NRF (Natl Research Foundation, S’pore)Accepted versio

Dual inhibition of the terminal oxidases eradicates antibiotic‐tolerant Mycobacterium tuberculosis

Abstract The approval of bedaquiline has placed energy metabolism in the limelight as an attractive target space for tuberculosis antibiotic development. While bedaquiline inhibits the mycobacterial F1F0 ATP synthase, small molecules targeting other components of the oxidative phosphorylation pathway have been identified. Of particular interest is Telacebec (Q203), a phase 2 drug candidate inhibitor of the cytochrome bcc:aa3 terminal oxidase. A functional redundancy between the cytochrome bcc:aa3 and the cytochrome bd oxidase protects M. tuberculosis from Q203‐induced death, highlighting the attractiveness of the bd‐type terminal oxidase for drug development. Here, we employed a facile whole‐cell screen approach to identify the cytochrome bd inhibitor ND‐011992. Although ND‐011992 is ineffective on its own, it inhibits respiration and ATP homeostasis in combination with Q203. The drug combination was bactericidal against replicating and antibiotic‐tolerant, non‐replicating mycobacteria, and increased efficacy relative to that of a single drug in a mouse model. These findings suggest that a cytochrome bd oxidase inhibitor will add value to a drug combination targeting oxidative phosphorylation for tuberculosis treatment