Discovery of anti-MRSA carpatamides' congeners by heterologous expression along with their mechanism investigation targeting FabI and biofilm formation.

Methicillin-resistant Staphylococcus aureus (MRSA) remains a significant clinical challenge, necessitating the discovery of novel anti-MRSA agents. Previous bioinformatic analysis identified a candidate biosynthetic gene cluster (BGC) of ctd for carpatamides in Streptomyces parvus 1268. Through heterologous expression of ctd and subsequent fermentation and isolation, we have identified five novel carpatamide derivatives of carpatamides N - R (1-5), and a known compound of daryamide A (6). The structures and absolute configurations of compounds 1-6 were determined by ESI-HRMS, NMR, and ECD calculations. Compound 1 exhibited significant antitumor activity against non-small cell lung cancer cell line A549 with an IC50 value of 7.43 μM. Meanwhile, the antibacterial bioactivity results showed that carpatamides N - O (1-2) displayed excellent antibacterial bioassay against Gram-positive bacteria, including MRSA with MIC values of 0.5-2.0 μg/mL, outperforming vancomycin. Further mechanism investigation through molecular dynamics (MD) simulations and biofilm-related experiments suggests that compounds 1 and 2 may exert their anti-MRSA activity by inhibiting the function of NADPH-dependent enoyl-acyl carrier protein reductase (FabI) and the formation of biofilms of MRSA.