Inhibition of Surfactin Biosynthesis in Bacillus Subtilis Using Cell-Permeable Adenylation Domain Inhibitors

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