Tianyu Liu,Yong Nian,Ying Liu,Simin Xu,Chujie Lu,Wei Yang,Wei Chen,Wei Lin
The resistance of nontuberculous mycobacteria (NTM) to conventional anti-tuberculosis drugs and its growing infection rate year by year urgently require new treatment strategies. Structure-based virtual screening, which can greatly improve efficiency and reduce costs in the early stage of drug development, is an indispensable part of modern drug discovery. In this study, the crystal structure of the mycothiol disulfide reductase from Mycobacterium abscessus ( Mab Mtr) was determined. Through virtual screening, compound AK-968/11492032 was identified as a promising candidate capable of fitting well into the potential MSSM-binding pocket of Mab Mtr. It was discovered that AK-968/11492032 and its derivatives ( Y6B and Y6C ) could produce antimicrobial effects on the Mycobacterial type strain Mycobacterium smegmatis . Moreover, microscale thermophoresis analysis was employed to evaluate the high binding affinity of the compounds to Mab Mtr. Furthermore, the key residues (S14, I47, H451) of Mab Mtr involved in the interaction with AK-968/11492032 were predicted and confirmed through molecular docking and mutational analysis, Mab Mtr was verified as the target for it to exert antibacterial effects through in vitro enzyme activity and in vivo gene knockout, complementation, and overexpression. These findings provide a potential development target to develop effective and specific anti-NTM drugs.
The conversion of sterols to steroid synthons by engineered mycobacteria comprises one of the basic ways for the production of steroid medications in the pharmaceutical industry. Here, we revealed that high amounts of reactive oxygen species (ROS) generate during the conversion process of sterols, which impairs the cell viability of mycobacterial cells and thus hinders the conversion of sterols to steroid synthons. Accordingly, the endogenous antioxidants for detoxifying ROS in mycobacteria, ROS scavenging enzymes and low molecular weight thiols, were examined. The results revealed that three antioxidants, catalase (CAT), mycothiol (MSH), and ergothioneine (EGT), demonstrated efficacy toward neutralizing the excessive ROS produced during sterol metabolism . CAT overexpression or MSH or EGT augmentation enhanced the conversion of phytosterols to 22-hydroxy-23,24-bisnorchol-4-ene-3-one (4-HBC) by 18.9%, 23.8%, and 32.1%, respectively, and also enhanced the cell viability, indicating the benefits of these antioxidants in reducing ROS-induced stress. Further combinatorial augmentation of CAT, MSH, and EGT demonstrated enhanced effects toward intracellular ROS scavenging, resulting in 54.2% greater cell viability and 47.5% enhancement in 4-HBC production. These findings indicated that the excessive ROS induces cell stress, in turn limiting the conversion of sterols, whereas neutralization of the excessive ROS by combined control of CAT, MSH, and EGT serves as an effective strategy to boost the conversion productivity of sterols to steroid synthons.
危险品化学品经营许可证（带存储）