Design and synthesis of inhibitors of nonribosomal peptide synthetase adenylation domains

Bacteria synthesize and utilize nonribosomal peptides (NRPs) for a variety of functions including iron acquisition, biofilm formation, and commensalism. Small-molecule probes that inhibit NRP biosynthesis are valuable tools for studying these processes and may have therapeutic applications. The NRP amino acid adenylation domains facilitate the initial steps of NRP biosynthesis, and thus are attractive targets for inhibitor design. However, inhibitor development to date has been hampered by cross-inhibition of the mechanistically related aminoacyl-tRNA synthetases (aaRSs). We noted that the tightly bound aminoacyl-AMP intermediate, produced in both amino acid adenylation domains and the aaRSs, adopts a cisoid or transoid conformation, respectively. To selectively inhibit amino acid adenylation domains, we designed macrocyclic AMP-analogs that are locked in the cisoid conformation. The macrocycles contain a two- or three-carbon linker between the beta carbon of the amino acid moiety and C8 of the adenine ring and a sulfamate in place of the phosphate group. Herein, we demonstrate that these compounds are potent inhibitors of the cysteine adenylation domain activity of yersiniabactin synthetase HMWP2 and, unlike the corresponding linear aminoacyl-AMP analogs, do not inhibit protein translation in vitro. The second major project was devoted to improving the cellular activity of the lead antibiotic compound, salicyl-AMS. Towards this end we identified several conformationally constrained saliycl-AMS analogs that potently inhibit YbtE. We developed an assay to monitor salicyl-AMS stability and cellular accumulation and showed that this compound was stable under conditions similar to the cellular assay. Finally, we developed an improved large-scale synthesis of salicyl-AMS that enables future studies in more advanced models of infection. The work described herein involves the discovery of novel inhibitors, which selectively target the amino acid adenylation domains, and strategies and methods to advance salicyl-AMS as a therapeutic.