Identification and validation of l-asparaginase as a potential metabolic target against Mycobacterium tuberculosis

Multidrug-resistant Mycobacterium tuberculosis (Mtb) has emerged as a major health challenge, necessitating the search for new molecular targets. A secretory amidohydrolase, l-asparaginase of Mtb (MtA), originally implicated in nitrogen assimilation and neutralization of acidic microenvironment inside human alveolar macrophages, has been proposed as a crucial metabolic enzyme. To investigate whether this enzyme could serve as a potential drug target, it was studied for structural details and active site-specific inhibitors were tested on cultured Mycobacterial strain. The structural details of MtA obtained through comparative modeling and molecular dynamics simulations provided insights about the orchestration of an alternate reaction mechanism at the active site. This was contrary to the critical Tyr flipping mechanism reported in other asparaginases. We report the novel finding of Tyr to Val replacement in catalytic triad I along with the structural reorganization of a β-hairpin loop upon substrate binding in MtA active site. Further, 5 MtA-specific, active-site-based inhibitors were obtained by following a rigorous differential screening protocol. When tested on Mycobacterium culture, 3 of these, M3 (ZINC 4740895), M26 (ZINC 33535), and doxorubicin showed promising results with inhibitory concentrations (IC ₅₀ ) of 431, 100, and 56 µM, respectively. Based on our findings and considering stark differences with human asparaginase, we project MtA as a promising molecular target against which the selected inhibitors may be used to counteract Mtb infection effectively.