An Unnatural Enantiomer of Trichodermamide F Inhibits Type III Secretion System-Mediated Hemolysis

Trichodermamides D-F were isolated in 2017, along with known trichodermamides A-C, from the mangrove-derived endophytic fungus Penicillium janthinellum. Although the pronounced bioactivities of trichodermamides A-C, including cytotoxic and antimicrobial activities, have been reported, the bioactivities of trichodermamides D-F have not yet been determined. The absence of reported lethal activity prompted us to focus on nonlethal biological targets and to conduct enzymatic and phenotypic screening aimed at identifying antivirulence activity. On the other hand, we recently developed a scalable, chiral-pool-based asymmetric synthesis of the trichodermamides A-F. Using this synthetic platform, 11 natural and unnatural enantiomers of trichodermamides D-F, along with selected synthetic intermediates, were evaluated to elucidate structure-activity relationships. This broad screening effort identified that the unnatural enantiomer of trichodermamide F exhibited selective inhibitory activity against the bacterial type III secretion system (T3SS), with an IC₅₀ of 76.7 μM, while showing no antibacterial activity. This study revealed the first trichodermamide derivative that targets the nonlethal T3SS, highlighting the importance of considering unnatural enantiomers and demonstrating the untapped antivirulence potential of the trichodermamide scaffold.