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.

Department of Organic Chemistry Faculty of Science Palacký University 17 listopadu 12 77146 Olomouc Czech Republic

Department of Physical Chemistry Faculty of Science Palacký University 17 listopadu 12 771 46 Olomouc Czech Republic

Laboratory of Microbiology Parasitology and Hygiene S7 Faculty of Pharmaceutical Biomedical and Veterinary Sciences University of Antwerp Wilrijk Belgium

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