Structure–activity relationship studies of c-di-AMP synthase inhibitor, bromophenol-thiohydantoin

C-di-AMP, a bacterial second messenger, regulates various processes in Gram-positive bacteria and mycobacteria. Small molecule inhibitors of c-di-AMP metabolic enzymes could affect bacterial growth and viability. A medium throughput screening identified bromophenol-thiohydantoin (BTH) as the first inhibitor of c-di-AMP synthase, DisA. Herein, we performed SAR studies of bromophenol-thiohydantoin to identify the salient features on BTH that are important for DisA inhibition. Seemingly minor substitution changes (e.g., aromatic bromo to chloro substitutions) resulted in dramatic changes in ligand potency. Bromophenol TH is specific for c-di-AMP synthase and did not inhibit RocR (c-di-GMP PDE), YybT (c-di-AMP PDE) or WspR (c-di-GMP synthase).