A novel natural compound from Cleistocalyx operculatus enhances the anti-mycobacterial response by inhibiting ferroptosis.

Tuberculosis is the leading cause of death among chronic infectious diseases caused by a single pathogen. Macrophages serve as the first line of immune defense, and pulmonary epithelial cells are essential for lung structural integrity and function. However, these cells are highly susceptible to death following Mycobacterium tuberculosis (Mtb) infection. Preserving cell viability has been shown to significantly enhance host anti-infective capacity. This study aimed to screen an in-house natural product library derived from Lingnan-region herbal medicines in China for cell-protective activity in H37Rv-infected cells and investigate the underlying mechanisms. Cell viability in macrophages and epithelial cells was assessed using CCK-8 and LDH assays. Pathways involved in cell death inhibition were explored via RNA-seq. Natural product target proteins were identified using DARTS and LC-MS. Gene and protein expression were validated by qRT-PCR and western blotting. Intracellular H37Rv viability was determined by CFU counting. A novel natural compound, cleistoperlone J (CJ), was identified as a cell viability-protective agent. CJ inhibited ferroptosis by modulating cellular antioxidant balance. Glucose-6-phosphate dehydrogenase (G6PD), a key enzyme in glutathione metabolism, acted as a critical mediator of CJ. By binding to G6PD, CJ enhanced its level and activity via inhibition of ubiquitination-mediated degradation, thereby suppressing ferroptosis in Mtb-infected cells. Notably, both CJ and the G6PD activator AG1 potentiated the antibacterial activity of isoniazid in Mtb-infected macrophages. These findings suggest that CJ holds promise as an adjunctive therapeutic agent for tuberculosis chemotherapy by inhibiting ferroptosis in Mtb-infected cells.