Combatting Antibiotic-Resistant : Discovery of TST1N-224, a Potent Inhibitor Targeting Response Regulator VraRC, through Pharmacophore-Based Screening and Molecular Characterizations.

() is a major global health concern, causing various infections and presenting challenges due to antibiotic resistance. In particular, methicillin-resistant , vancomycin-intermediate (VISA), and vancomycin-resistant pose significant obstacles in treating 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 . The constructed pharmacophore model, , demonstrated effectiveness in identifying a potent inhibitor, (IC = 60.2 ± 4.0 μM), against the formation of the VraRC-DNA complex. Notably, 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 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, evidently enhanced the susceptibility of VISA to both vancomycin and methicillin. Importantly, 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.