Computer-Aided Discovery of a β-Lactam-Independent Deep-Pocket PBP2a Binding Scaffold for Combating MRSA via Pharmacophore-Guided Scaffold Hopping and Bioisosteric Replacement

Methicillin-resistant Staphylococcus aureus (MRSA) remains a major driver of antimicrobial resistance due to expression of penicillin-binding protein 2a (PBP2a), a transpeptidase whose conformational regulation limits the efficacy of most β-lactam antibiotics. Structural studies have shown that PBP2a activity is modulated through a distal regulatory pocket that controls catalytic-site accessibility, yet exploitation of this mechanism for inhibitor design remains limited. The present study applied a pharmacophore-guided computer-aided drug design (CADD) strategy to identify β-lactam–independent scaffolds capable of engaging this regulatory region. A literature-guided workflow integrating similarity screening, pharmacophore modeling, scaffold hopping, and bioisosteric replacement was implemented. Ceftaroline was selected as a reference ligand based on clinical relevance and structural similarity analysis. Docking validation revealed limited interaction of ceftaroline with key regulatory residues within the PBP2a deep pocket, particularly Asp516, Tyr519, and Gln521, residues implicated in allosteric signal propagation and conformational control of the enzyme. Residue-level interaction analysis was therefore used to guide rational scaffold redesign. Three novel analogues were generated through scaffold hopping and targeted bioisosteric modification and evaluated using molecular docking with PyRx followed by interaction analysis in Discovery Studio. Among the designed compounds, Analogue 2 demonstrated the most favorable predicted binding affinity and interaction stability, establishing directional hydrogen bonding with Asp516 and Gln521 and improved interaction density within the regulatory pocket. These interactions were not observed for the β-lactam reference ligand. Pharmacophore validation confirmed alignment between similarity-derived candidates and the redesigned scaffolds, supporting the robustness of the computational design framework. Collectively, these findings demonstrate that rational scaffold redesign can overcome structural limitations associated with β-lactam antibiotics and identify chemically distinct scaffolds capable of engaging the PBP2a regulatory pocket. This study proposes a reproducible computational strategy for discovering non-β-lactam PBP2a modulators and highlights the role of CADD-driven medicinal chemistry in accelerating antimicrobial discovery.