| Summary: | Penicillin-binding proteins (PBPs) catalyze the final stages for peptidoglycan cell-wall bio-synthesis. Mutations in the PBP2a subunit can attenuate <i>β</i>-lactam antibiotic activity, resulting in unimpeded cell-wall formation and methicillin-resistant <i>Staphylococcus aureus</i> (MRSA). A double mutation in PBP2a (i.e., N146K and E150K) is resistant to <i>β</i>-lactam inhibitors; however, (<i>E</i>)-3-(2-(4-cyanostyryl)-4-oxoquinazolin-3(4<i>H</i>)-yl) benzoic acid (QNZ), a heterocyclic antibiotic devoid of a <i>β</i>-lactam ring, interacts non-covalently with PBP2a allosteric site and inhibits PBP enzymatic activity. In the search for novel inhibitors that target this PBP2a allosteric site in acidic medium, an <i>in silico</i> screening was performed. Chemical databases including eMolecules, ChEMBL, and ChEBI were virtually screened for candidate inhibitors with a physicochemical similarity to QNZ. PBP2a binding affinities from the screening were calculated based on molecular docking with co-crystallized ligand QNZ serving as a reference. Molecular minimization calculations were performed for inhibitors with docking scores lower than QNZ (calc. −8.3 kcal/mol) followed by combined MD simulations and MM-GBSA binding energy calculations. Compounds eMol26313223 and eMol26314565 exhibited promising inhibitor activities based on binding affinities (Δ<i>G</i><sub>binding</sub>) that were twice that of QNZ (−38.5, −34.5, and −15.4 kcal/mol, respectively). Structural and energetic analyses over a 50 ns MD simulation revealed high stability for the inhibitors when complexed with the double mutated PBP2a. The pharmacokinetic properties of the two inhibitors were predicted using an <i>in silico</i> ADMET analysis. Calculated binding affinities hold promise for eMol26313223 and eMol26314565 as allosteric inhibitors of PBP2a in acidic medium and establish that further <i>in vitro</i> and <i>in vivo</i> inhibition experimentation is warranted.
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