Elucidation of the essentiality of lumazine synthase (RibH) forMycobacterium tuberculosissurvival and discovery of potent inhibitors for enhanced antimycobacterial therapy

Tuberculosis (TB) continues to be a global health crisis, necessitating urgent interventions to address drug resistance and improve treatment efficacy. In this study, we validate the indispensable role of lumazine synthase (RibH), a vital enzyme in the riboflavin biosynthetic pathway, in the survival of Mycobacterium tuberculosis (M. tb) using a CRISPRi-based conditional gene knockdown strategy. We show that genetic and functional ablation of RibH in M. tb cannot be compensated by exogenous supply of Riboflavin, or co-factors Flavin Adenine Dinucleotide (FAD) or Flavin Mononucleotide (FMN). Capitalizing on the essentiality of RibH, we employ a high-throughput molecular docking approach to screen ∼600,000 compounds and identify inhibitors of RibH. Through in vitro screening of 55 shortlisted compounds, we discover 3 inhibitors that exhibit potent antimycobacterial activity. These compounds effectively also eradicate intracellular M. tb during macrophage infection and prevent the resuscitation of the nutrient-starved persister bacteria. Moreover, these 3 compounds synergistically enhance the bactericidal effect of first-line anti-TB drugs, Isoniazid and Rifampicin. Corroborating with the in silico predicted high docking scores along with favorable ADME and toxicity profiles, all 3 compounds demonstrate exceptional binding affinity towards purified lumazine synthase enzyme in vitro , and display an acceptable safety profile in mammalian cells, with a high selective index. By providing mechanistic evidence for the essentiality of RibH in M. tb survival, and discovering potent RibH inhibitors with outstanding antimycobacterial activity, our study contributes to the development of superior TB treatment strategies and advances the global fight against this devastating disease.