Leveraging Cryptic Ligand Envelopes through Enhanced Molecular Simulations

Protein-bound ligands can adopt a range of different conformations, collectively defining a ligand envelope that has proven to be crucial for the design of potent and selective drugs. Yet, the cryptic nature of this ligand envelope makes it difficult to visualize, characterize, and ultimately exploit for drug design. Using enhanced molecular dynamics simulations, here, we provide a general framework to reconstruct the cryptic ligand envelope that is dynamically accessible by protein-bound small molecules in solution. We apply this approach to quantify hidden conformational heterogeneity in structurally complex ligands including the marine natural product plitidepsin. The computed conformational heterogeneity expands the small-molecule footprint beyond that typically observed in experiments, also revealing key thermodynamic and kinetic properties of single ligand-target interactions. The model agrees quantitatively with solution NMR, X-ray crystallography, and biochemical measurements, showcasing a versatile strategy to integrate receptor-bound ligand conformational ensembles in molecular design.