Nargenicin

Nargenicin A1, an antibacterial polyketide macrolide produced by Nocardia sp. CS682, was enhanced by increasing the pool of precursors using different sources. Furthermore, by using engineered strain Nocardia sp. ACC18 and supplementation of glucose and glycerol, enhancement was ~7.1 fold in comparison to Nocardia sp. CS682 without supplementation of any precursors. The overproduced compound was validated by mass spectrometry and nuclear magnetic resonance analyses. The novel glycosylated derivatives of purified nargenicin A1 were generated by efficient one-pot reaction systems in which the syntheses of uridine diphosphate (UDP)-α-D-glucose and UDP-α-D-2-deoxyglucose were modified and combined with glycosyltransferase (GT) from Bacillus licheniformis. Nargenicin A1 11-O-β- D-glucopyranoside, nargenicin A1 18-O-β-D-glucopyranoside, nargenicin A111 18-O-β-D- diglucopyranoside, and nargenicin 11-O-β-D-2-deoxyglucopyranoside were generated. Nargenicin A1 11-O-β-D-glucopyranoside was structurally elucidated by ultra-high performance liquid chromatography-photodiode array (UPLC-PDA) conjugated with high-resolution quantitative time-of-flight-electrospray ionization mass spectroscopy (HR-QTOF ESI-MS/MS), supported by one- and two-dimensional nuclear magnetic resonance studies, whereas other nargenicin A1 glycosides were characterized by UPLC-PDA and HR-QTOF ESI-MS/MS analyses. The overall conversion studies indicated that the one-pot synthesis system is a highly efficient strategy for production of glycosylated derivatives of compounds like macrolides as well. Furthermore, assessment of solubility indicated that there was enhanced solubility in the case of glycoside, although a substantial increase in activity was not observed.

Natural products provide a rich source of potential antimicrobials for treating infectious diseases for which drug resistance has emerged. Foremost among these diseases is tuberculosis. Assessment of the antimycobacterial activity of nargenicin, a natural product that targets the replicative DNA polymerase ofStaphylococcus aureus, revealed that it is a bactericidal genotoxin that induces a DNA damage response inMycobacterium tuberculosis(Mtb) and inhibits growth by blocking the replicative DNA polymerase, DnaE1. Cryo-electron microscopy revealed that binding of nargenicin toMtbDnaE1 requires the DNA substrate such that nargenicin is wedged between the terminal base pair and the polymerase and occupies the position of both the incoming nucleotide and templating base. Comparative analysis across three bacterial species suggests that the activity of nargenicin is partly attributable to the DNA binding affinity of the replicative polymerase. This work has laid the foundation for target-led drug discovery efforts focused onMtbDnaE1.

Tacrolimus is widely used as an immunosuppressant to reduce the risk of rejection after organ transplantation, but its cytotoxicity is problematic. Nargenicin A1 is an antibiotic extracted fromNocardia argentinensisand is known to have antioxidant activity, though its mode of action is unknown. The present study was undertaken to evaluate the protective effects of nargenicin A1 on DNA damage and apoptosis induced by tacrolimus in hirame natural embryo (HINAE) cells. We found that reduced HINAE cell survival by tacrolimus was due to the induction of DNA damage and apoptosis, both of which were prevented by co-treating nargenicin A1 or N-acetyl-l-cysteine, a reactive oxygen species (ROS) scavenger, with tacrolimus. In addition, apoptosis induction by tacrolimus was accompanied by increases in ROS generation and decreases in adenosine triphosphate (ATP) levels caused by mitochondrial dysfunction, and these changes were significantly attenuated in the presence of nargenicin A1, which further indicated tacrolimus-induced apoptosis involved an oxidative stress-associated mechanism. Furthermore, nargenicin A1 suppressed tacrolimus-induced B-cell lymphoma-2 (Bcl-2) down-regulation, Bax up-regulation, and caspase-3 activation. Collectively, these results demonstrate that nargenicin A1 protects HINAE cells against tacrolimus-induced DNA damage and apoptosis, at least in part, by scavenging ROS and thus suppressing the mitochondrial-dependent apoptotic pathway.