Structure-guided inhibitor design targeting CntL provides the first chemical validation of the staphylopine metallophore system in bacterial metal acquisition.

To survive in the metal-scarce environment within the host, pathogens synthesize various small molecular metallophores to facilitate the acquisition of transition metals. The cobalt and nickel transporter (Cnt) system synthesizes and transports staphylopine, a nicotianamine-like metallophore, and serves as a primary transition metal uptake system in Gram-positive bacteria including the human pathogen Staphylococcus aureus. In this study, we report the design of the first inhibitor of the Cnt system by targeting the key aminobutanoyltransferase CntL which is involved in the biosynthesis of staphylopine. Through structure-guided fragment linking and optimization, a class of acceptor-adenosine dual-site inhibitors against S. aureus CntL (Sa CntL) were designed and synthesized. The most potent inhibitor, compound 9 , demonstrated a Δ T m value of 9.4 °C, a K d value of 0.021 ± 0.004 μM, and an IC 50 value of 0.06 μM against Sa CntL. The detailed mechanism by which compound 9 inhibits Sa CntL has been elucidated through a high-resolution co-crystal structure. Treatment with compound 9 resulted in a moderate downregulation of intracellular concentrations of iron, nickel, and cobalt ions in the S. aureus cells cultured in the metal-scarce medium, providing the first chemical validation of the important role of Cnt system in bacterial metal acquisition. Our findings pave the way for the development of CntL-based antibacterial agents in future. [Display omitted] • Structure-guided fragment linking led to the first inhibitors of the Cnt system. • The most potent compound 9 demonstrated nanomolar activity against S. aureus CntL. • Dual-site inhibitory mechanism of 9 to CntL was elucidated by cocrystal structure. • Treatment with 9 downregulated transition metal levels in S. aureus cells. [ABSTRACT FROM AUTHOR]