Structural insights into multitargeting Mycobacterium tuberculosis Pkn kinases

Tuberculosis remains one of the leading major global health challenges, driven by the emergence of multidrug-resistant and extensively drug-resistant bacterial strains. Resistant strains complicate treatment, which often requires prolonged use of toxic second- and third-line drugs. Protein phosphorylation plays critical roles in Mycobacterium tuberculosis, with serine/threonine kinases PknA, PknB, and PknG being essential for survival, virulence, and persistence. In this study, we screened an in-house kinase inhibitor library to identify compounds targeting these kinases. Four structurally diverse hits from the screening inhibiting all three kinases in vitro were selected for further analysis. Hits were evaluated for their ability to inhibit M. tuberculosis growth and characterized structurally using X-ray crystallography, molecular docking, and isothermal titration calorimetry. Our findings provide a structural framework for the development of multitargeting kinase inhibitors, offering a potential strategy to combat drug-resistant M. tuberculosis.IMPORTANCEDrug-resistant tuberculosis is a growing global health threat that is increasingly difficult to treat with existing antibiotics, necessitating the discovery of new therapeutic strategies. This study focuses on protein kinases, key regulatory enzymes that help Mycobacterium tuberculosis survive, cause disease, and persist in the host. By identifying small molecules that can simultaneously block multiple essential kinases, this work introduces a promising multitarget approach to combat tuberculosis. Using structural and biophysical methods, we reveal how these compounds interact with their targets, providing a clear blueprint for improving their effectiveness. These insights advance the rational design of next-generation antitubercular drugs and open new avenues for tackling multidrug- and extensively drug-resistant tuberculosis.