Abstract
Staphylococcus aureus (S. aureus) is a major global health concern, causing various infections and presenting challenges due to antibiotic resistance. In particular, methicillin-resistant S. aureus, vancomycin-intermediate S. aureus (VISA), and vancomycin-resistant S. aureus pose significant obstacles in treating S. aureus infections. Therefore, the critical need for novel drugs to counter these resistant forms is pressing. Two-component systems (TCSs), integral to bacterial regulation, offer promising targets for disruption. In this study, a comprehensive approach, involving pharmacophore-based inhibitor screening, along with biochemical and biophysical analyses were conducted to identify, characterize, and validate potential inhibitors targeting the response regulator VraRC of S. aureus. The constructed pharmacophore model, Phar-VRPR-N3, demonstrated effectiveness in identifying a potent inhibitor, TST1N-224 (IC(50) = 60.2 ± 4.0 μM), against the formation of the VraRC-DNA complex. Notably, TST1N-224 exhibited strong binding to VraRC (KD = 23.4 ± 1.2 μM) using a fast-on-fast-off binding mechanism. Additionally, NMR-based molecular modeling revealed that TST1N-224 predominantly interacts with the α9- and α10-helixes of the DNA-binding domain of VraR, where the interactive and functionally essential residues (N165, K180, S184, and R195) act as hotspots for structure-based inhibitor optimization. Furthermore, TST1N-224 evidently enhanced the susceptibility of VISA to both vancomycin and methicillin. Importantly, TST1N-224 distinguished by 1,2,5,6-tetrathiocane with the 3 and 8 positions modified with ethanesulfonates holds significant potential as a lead compound for the development of new antimicrobial agents.