Eeyarestatin 24 impairs SecYEG‐dependent protein trafficking and inhibits growth of clinically relevant pathogens

Eeyarestatin 1 (ES1) is an inhibitor of endoplasmic reticulum (ER) associated protein degradation, Sec61‐dependent Ca2+ homeostasis and protein translocation into the ER. Recently, evidence was presented showing that a smaller analog of ES1, ES24, targets the Sec61‐translocon, and captures it in an open conformation that is translocation‐incompetent. We now show that ES24 impairs protein secretion and membrane protein insertion in Escherichia coli via the homologous SecYEG‐translocon. Transcriptomic analysis suggested that ES24 has a complex mode of action, probably involving multiple targets. Interestingly, ES24 shows antibacterial activity toward clinically relevant strains. Furthermore, the antibacterial activity of ES24 is equivalent to or better than that of nitrofurantoin, a known antibiotic that, although structurally similar to ES24, does not interfere with SecYEG‐dependent protein trafficking. Like nitrofurantoin, we find that ES24 requires activation by the NfsA and NfsB nitroreductases, suggesting that the formation of highly reactive nitroso intermediates is essential for target inactivation in vivo. ES24, but not nitrofurantoin, inhibits SecYEG‐dependent protein transport in bacteria and exhibits antibacterial activity toward various clinically relevant strains. In addition, ES24 affects many other processes important to Escherichia coli, hinting at a multifaceted mode of action of ES24.