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Evidence that bottom-up methods uncover hidden interactions: A photoaffinity probe derived from an HHAT inhibitor, activated by UV (probe 11, half-life 32 s), crosslinked five residues—Pro212, Val213, His215 (reentrant loop 2) and Glu399, Val402—identifying the first small-molecule binding site in a mammalian MBOAT; 40-fold excess of the parent inhibitor IMP-1575 (Ki 38 nM, competitive with palmitoyl-CoA) competed crosslinking to background, confirming site specificity HHAT photoaffinity binding-site mapping. Similarly, truncation analysis plus MD of a PFOS-binding aptamer revealed fluorocarbon-chain epitopes and a π–sulfur interaction invisible to conventional assays, enabling a GNP-qPCR sensor with 5.8 pM LOD (0–100 pM linear, R² = 0.99) PFOS aptamer qPCR with MD epitope mapping. In ligand discovery, MD with mixed solvents (20% isopropanol or acetamide in water) outperformed GRID at mapping water displaceability in Hsp90 and HIV-1 protease sites MDmix mixed-solvent mapping.

Caveat—artifact control is essential: the validation-cross framework stresses that binding must be shown on both ligand and protein, with integrity checks across orthogonal methods, because screening assays generate false positives Validation cross framework. Bottom-up findings therefore require orthogonal confirmation (e.g., gene silencing validated insect gut receptors such as ST6 and the enolase-binding protein Insect gut receptor discovery review).

Analysis plan: (1) Run bottom-up discovery—photoaffinity labeling with LC-MS/MS residue mapping, aptamer truncation + MD (GROMACS/AutoDock), mixed-solvent MDmix; (2) score candidates by ΔG and crosslink competition; (3) validate orthogonally (mutagenesis, SPR/ITC, in vivo knockdown); (4) quantify uncertainty (95% CI on IC50/KD; coverage limits, e.g., 68% HHAT coverage).