Structural and mechanistic study of a novel inhibitor of M. tuberculosis cytochrome bc1:aa3

Drug-resistant tuberculosis (TB) continues to challenge treatment options, necessitating the exploration of new compounds of novel targets. The mycobacterial respiratory complex cytochrome bc1:aa3 has emerged as a promising target, exemplified by the success of first-in-class inhibitor Q203 in phase 2 clinical trial. However, to fully exploit the potential of this target and to identify the best-in-class inhibitor more compounds need evaluation. Here, we introduce JNJ-2901, a novel inhibitor of the M. tuberculosis cytochrome bc1:aa3, demonstrating activity against multidrug-resistant M. tuberculosis clinical strains at sub-nanomolar concentration and 4-log reduction in bacterial burden in mouse model of TB infection. Inhibitory studies on purified enzyme validate the nanomolar inhibitions observed in mycobacterial cells. Additionally, cryo-EM structure analysis of cytochrome bc1:aa3 bound to JNJ-2901 reveals the binding pocket at the menaquinol oxidation site (Qp), akin to other substate analogue inhibitors like Q203 and TB47. Validation of binding site is further achieved by generating and isolating the JNJ-2901 resistant mutations in M. tuberculosis, followed by purification and resistance analysis of the resistant cytochrome bc1:aa3 complex. Our comprehensive work lay the foundation for further clinical validations of JNJ-2901.