Structure-Informed Prioritization of Phytochemical Antistreptococcal Candidates: Integrated Molecular Docking and In Vitro Proof-of-Concept Validation Against Streptococcus agalactiae and Streptococcus pyogenes

Abstract

Localized phytochemical formulations may provide complementary strategies for controlling mucosal colonization by Streptococcus agalactiae and Streptococcus pyogenes, but computational prioritization requires orthogonal biological validation. This study integrated molecular docking against the redox-sensing transcriptional repressor Rex of S. agalactiae and a protein tyrosine phosphatase target of S. pyogenes (SP-PTP) with in vitro broth microdilution, biofilm, target-bridging, and epithelial-tolerance experiments. Isoflavone showed the most favorable natural-compound interaction with Rex (ΔG = −8.3 kcal/mol), whereas secoisolariciresinol diglucoside (SDG) led the natural-ligand ranking for SP-PTP (ΔG = −5.2 kcal/mol). The complete formulation produced MIC50 values of 0.031% and 0.063% v/v against S. agalactiae and S. pyogenes, respectively; inhibited biofilm formation by 89.2% and 80.1%; and preserved >92% epithelial viability at 1× MIC. Among the three natural candidates with complete matched broth data, target-normalized docking and composite MIC50/MIC90/MBC50 ranks were perfectly concordant in both species; pooled species-stratified Spearman analysis yielded ρ = 1.000 (exact p = 0.0556; n = 6). In contrast, the extended panel including antibacterial comparators showed negligible concordance (ρ = 0.081; p = 0.7826; n = 14). Principal component analysis assigned 74.8% of variance to an in vitro potency axis and 24.6% to a largely orthogonal docking axis. Thus, the experiments confirmed the target-specific ordering of the natural candidates but did not support extrapolation of docking rank across mechanistically heterogeneous antibacterial classes. Intracellular target inhibition, genetic causality, and component synergy remain unestablished.