Expanding the squaramide library as mycobacterial ATP synthase inhibitors: Innovative synthetic pathway and biological evaluation

Mycobacterial ATP synthase is a validated therapeutic target for combating drug-resistant tuberculosis. Inhibition of this enzyme has been featured as an efficient strategy for the development of new antimycobacterial agents against drug-resistant pathogens. In this study, we synthesised and explored two distinct series of squaric acid analogues designed to inhibit mycobacterial ATP synthase. Among the extensive array of compounds investigated, members of the phenyl-substituted sub-library emerged as primary hits. To gain deeper insights into their mechanisms of action, we conducted advanced biological studies, focusing on the compounds displaying a direct binding of a nitrogen heteroatom to the phenyl ring, resulting in the highest potency. Our investigations into spontaneous mutants led to the validation of a single point mutation within the atpB gene (Rv1304), responsible for encoding the ATP synthase subunit a. This genetic alteration sheds light on the molecular basis of resistance to squaramides. Furthermore, we explored the possibility of synergy between squaramides and the reference drug clofazimine using a checkerboard assay, highlighting the promising avenue for enhancing the effectiveness of existing treatments through combined therapeutic approaches. This study contributes to the expansion of investigating squaramides as promising drug candidates in the ongoing battle against drug-resistant tuberculosis.

Automated summary

This study aims to develop new antimycobacterial agents against drug-resistant tuberculosis by inhibiting mycobacterial ATP synthase. Squaric acid analogues were synthesized and explored, with phenyl-substituted compounds showing the best inhibition effect. Deeper insights into their mechanisms of action were gained, and a single point mutation in the atpB gene responsible for ATP synthase subunit a was validated as the molecular basis of resistance to squaramides. The study also highlights the potential synergy between squaramides and clofazimine, providing a promising avenue for enhancing the effectiveness of existing treatments.