Isocyanides inhibit bacterial pathogens by covalent targeting of essential metabolic enzymes.

Isonitrile natural products, also known as isocyanides, demonstrate potent antimicrobial activities, yet our understanding of their molecular targets remains limited. Here, we focus on the so far neglected group of monoisonitriles to gain further insights into their antimicrobial mode of action (MoA). Screening a focused monoisonitrile library revealed a potent growth inhibitor with a different MoA compared to previously described isonitrile antibiotics. Chemical proteomics competitive cysteine reactivity profiling, uncovered covalent modifications of two essential metabolic enzymes involved in the fatty acid biosynthetic process (FabF) and the hexosamine pathway (GlmS) at their active site cysteines. In-depth studies with the recombinant enzymes demonstrated concentration-dependent labeling, covalent binding to the catalytic site and corresponding functional inhibition by the isocyanide. Thermal proteome profiling and full proteome studies of compound-treated further highlighted the destabilization and dysregulation of proteins related to the targeted pathways. Cytotoxicity and the inhibition of cytochrome P450 enzymes require optimization of the hit molecule prior to therapeutic application. The here described novel, covalent isocyanide MoA highlights the versatility of the functional group, making it a useful tool and out-of-the-box starting point for the development of innovative antibiotics.