REBASTINIB, OLVEREMBATINIB, AND NAS-181 INHIBIT MYCOBACTERIUM ABSCESSUS BY DISRUPTING MYCOTHIOL HOMEOSTASIS

Nontuberculous mycobacteria (NTM), particularly Mycobacterium abscessus , pose a major clinical challenge due to intrinsic drug resistance and limited therapeutic options. In this study, we evaluated three repurposed kinase inhibitors, such as rebastinib, olverembatinib, and NAS-181for their antimycobacterial potential against M. abscessus . All three compounds displayed measurable in vitro activity, with MIC values of 22 μM, 40 μM, and 9 μM, respectively. Whole-genome sequencing of resistant mutants revealed nonsynonymous mutations in key enzymes of the mycothiol biosynthesis pathway, including D87A and W227G substitutions in MshC ( MAB_2116 ) and a Q51T substitution in MshA ( MAB_4057c ), implicating disruption of redox homeostasis as a likely mechanism of action. Consistent with this, biochemical analyses demonstrated a concentration-dependent decrease in intracellular mycothiol (MSH) and its precursor GlcN-Ins upon treatment with each compound. Cytotoxicity profiling indicated tolerable activity thresholds (rebastinib, 30 μM; olverembatinib, 60 μM; NAS-181, 14 μM), enabling further macrophage infection assays. All three compounds significantly reduced intracellular M. abscessus burden in THP-1 macrophages without affecting host cell viability. In a mouse pulmonary infection model, treatment with rebastinib, olverembatinib, or NAS-181 (5 mg/kg) resulted in maintenance of body weight, decreased lung CFU counts, and reduced spleen enlargement compared to vehicle controls, with efficacy comparable to amikacin. Collectively, these findings identify rebastinib, olverembatinib, and NAS-181 as promising repurposed drug candidates that target mycothiol biosynthesis, providing a foundation for the development of novel therapeutic strategies against drug-resistant M. abscessus .