Inhibition of surfactin biosynthesis in Bacillus subtilis using cell-permeable adenylation domain inhibitors.

Abstract

Several natural bacterial virulence factors are biosynthesized by nonribosomal peptide synthetases (NRPSs). Therefore, NRPSs producing such natural products have emerged as attractive antibiotic targets. We have previously reported N-(aminoacyl)sulfamoyladenosine (aminoacyl-AMS) derivatives with chemical modifications at 2'-OH group of the adenosine skeleton that inhibit NRPS amino acid adenylation (A) domains in recombinant enzyme systems, cellular lysates, and bacterial cells. The introduction of a couple of functionalities at the 2'-OH group preserve the binding affinity toward NRPS amino acid A-domains and improve cell permeability of the AMS scaffold. However, the effects of these compounds on secondary metabolism have not yet been explored. In this study, we validated an affinity-based protein profiling (AfBPP) probe, Leu-AMS-BPyne, for in-cell applications, including imaging of NRPS activities in bacteria. Next, we synthesized L-Leu-AMS derivatives incorporating methyl, benzyl, and cyanomethyl functionalities at the 2'-OH group and investigated inhibitory activity toward intracellular surfactin-NRPSs in the surfactin-producer Bacillus subtilisATCC 21332 using the AfBPP probe. Finally, we demonstrated the attenuation of surfactin production using a Leu-AMS-BPyne probe and L-Leu-AMS derivatives in B. subtilis. These results indicate that chemical modifications at the 2'-OH group provide a way to develop cell-permeable and functional NRPS A-domain inhibitors.