Pharmacological targeting of the mycothiol cysteine ligase MshC in Mycobacteriumabscessus

The prevalence of pulmonary diseases caused by Mycobacterium abscessus is on the rise and surpasses tuberculosis in many countries. However, intrinsic resistance of this pathogen to antibiotics profoundly limits our therapeutic arsenal, and new chemotherapeutic agents and targets are urgently needed. Mycothiol (MSH), the major thiol in mycobacteria, plays an important role in resistance to oxidative stress. Previous work in Mycobacterium smegmatis and Mycobacterium tuberculosis indicates that the ATP-dependent cysteine ligase MshC catalyzes the penultimate step in MSH biogenesis. Here, we investigated whether MshC represents a potent drug target in M. abscessus. CRISPRi was used to demonstrate that mshC is essential in vitro and during infection in zebrafish larvae. Subsequently, in silico drug screening identified three potential MshC-binding molecules, namely rebastinib, olverembatinib and SB-224289, significantly inhibiting M. abscessus growth and MSH production. Spontaneous missense mutations in MshC generated cross-resistance to all three compounds. Mapping the mutations conferring resistance on a MshC three-dimensional homology model defined the enzyme catalytic site as the potential drug binding cavity. Additionally, overexpression of mshC in a susceptible M. abscessus strain increased resistance levels to rebastinib, olverembatinib and SB-224289, but not to rifabutin. Conversely, reduced mshC expression levels in the CRISPRi mutant increased susceptibility to the inhibitors. Systematic assessment of olverembatinib and SB-224289 in combination with 18 additional drugs revealed distinct synergistic and antagonistic interactions. These results emphasize a yet unexploited chemical structure class against M. abscessus and highlight the potential of targeting the MSH pathway for future translational developments against M. abscessus lung diseases.