Elucidation of essentiality of lumazine synthase (RibH) for Mycobacterium tuberculosis survival and discovery of potent inhibitors for enhanced antimycobacterial therapy

Abstract Tuberculosis (TB) remains a pressing global health crisis, necessitating urgent interventions to combat drug resistance and improve treatment efficacy. In this study, we elucidate the essential role of lumazine synthase (RibH) for the survival of Mycobacterium tuberculosis (M. tb). Through CRISPRi-based gene knockdown in M. tb, we confirm that deficiency of RibH – the penultimate enzyme in riboflavin biosynthesis cannot be compensated by exogenous supply of Riboflavin, FAD, or FMN. Further, by high-throughput molecular docking-based virtual screening of ~600,000 compounds and in vitro whole cell growth assays, we identify 3 RibH inhibitors that exhibit potent antimycobacterial activity. These compounds also effectively eradicate intracellular M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister mycobacteria. Furthermore, they synergistically enhance the bactericidal effect of Isoniazid and Rifampicin. These compounds exhibit high affinity binding to lumazine synthase in vitro and display favorable absorption, distribution, metabolism and excretion (ADME) properties, toxicity profiles, with a high selective index in mammalian cells. By providing mechanistic evidence for the essentiality of RibH in M. tb survival and identification of potent inhibitors that bind to RibH, this study contributes to the development of superior TB treatment strategies and advances the global fight against this devastating disease.