TBI-166 inhibits DlaT and LpdC, exhibiting synergistic effects with Bedaquiline and Pyrazinamide against Mycobacterium tuberculosis

Tuberculosis (TB), particularly drug-resistant tuberculosis (DR-TB), remains a critical global challenge and underscores the urgent need for novel drugs and innovative combination regimens with distinct mechanisms of action. Here, we characterize an all-oral three-drug regimen comprising TBI-166, bedaquiline(BDQ), and pyrazinamide(PZA), which displays strong synergistic antimicrobial activity in vitro against both replicating and non-replicating Mycobacterium tuberculosis (MTB) and has previously shown superior bactericidal and sterilizing efficacy to standard HRZ and BPaL regimens in murine TB models(1). Time-kill studies demonstrate that the triple regimen outperforms dual-drug combinations, accelerating bacterial clearance across multiple physiological states. Mechanistic investigations revealed that the TBI-166-BDQ-PZA combination induces a comprehensive collapse of energy and redox homeostasis, marked by profound ATP depletion, robust accumulation of reactive oxygen species (ROS), and marked disruption of the intracellular NAD(H) pool. TBI-166, a novel riminophenazine analogue of clofazimine (CFZ) currently in phase II clinical trials, emerged as a key contributor to this metabolic stress. Metabolomic profiling and {superscript 1}3C-based flux analysis show that TBI-166 slows glycolysis and the tricarboxylic acid (TCA) cycle while enhancing flux through the pentose phosphate and nicotinate pathways, thereby lowering the NADH/NAD ratio and diminishing MTB metabolic flexibility under environmental stress. In parallel, TBI-166 downregulates the dormancy regulator DosR and its regulon, further compromising adaptation to non-replicating states. Multi-omics analyses, together with biochemical and biophysical assays, identify the pyruvate dehydrogenase complex (PDHc) components DlaT and LpdC as direct molecular targets of TBI-166, with drug binding leading to potent inhibition of their enzymatic activities. Collectively, these findings define the mechanism of action of TBI-166 and provide a molecular rationale for its inclusion in potent, all-oral, short-course regimens. More broadly, they highlight the therapeutic potential of metabolically targeted combinations that destabilize energy metabolism, redox balance, and metabolic adaptability to improve DR-TB treatment outcomes.