Reactivity Over Recognition: Covalent Screening of Natural Products against Falcipain-2 and Falcipain-3

We present a reproducible, reactivity-weighted covalent-screening workflow that separates molecular recognition, intrinsic electrophile reactivity, and matched host-protease covalent capture, and we validate it before applying it. Screening 1,012 South African Natural Compounds Database entries against falcipain-2 and falcipain-3, a warhead/β-accessibility cascade retained 185 compounds, and a consensus of recognition docking, uniform-mode covalent docking, MM-GBSA, DFT reactivity descriptors, and predicted developability prioritized SANC00867. A retrospective benchmark (256 falcipain-2 actives, 5,016 propertymatched decoys) shows recognition docking alone does not enrich covalent falcipain-2 actives (ROC-AUC 0.53), and that the covalent signal is carried by warhead reactivity, not recognition: adding warhead reactivity significantly improves retrieval over docking on both propertymatched and stringent warhead-matched decoy benchmarks (ROC-AUC 0.62 vs 0.56, DeLong p = 0.015 in the stringent test), while docking alone remains near-random throughout; covalent pose recovery is validated against a crystallographic vinyl-sulfone complex, and the dual-target ranking is robust to food-vacuole (pH 5.5) preparation (Spearman ρ = 0.77 and 0.97 for FP-2 and FP-3). Across capped para-substituted cinnamate models, global electrophilicity and the site-local β-carbon Fukui index diverge under strong electron withdrawal (4-NO2 raises global ω yet collapses the β-carbon f+ through LUMO localization), so warhead tuning is governed by local, not global, electrophilicity. A matched host-cathepsin covalent counter-screen shows SANC00867’s apparent selectivity does not survive receptor-normalized analysis, reported as a cross-target scoring result rather than a selectivity claim. The workflow thus delivers a validated, transparent prioritization framework and a chemically interpretable design rule, with all inputs, scripts, and settings deposited for reuse.