Structural excavation of cryptic sites in proteins using glycol decoys: implications in drug-design

Cryptic binding sites are visible in ligand-bound protein structures but are occluded in unbound structures. Targeting these sites for therapy provides an attractive option for proteins not tractable by classical binding sites. However, owing to their hidden nature, it is difficult to identify cryptic sites. Consequently, approaches for discovery are being actively pursued. Here, we show that ethylene glycol and propylene glycol act as finders of cryptic sites in proteins. Initial serendipitous observation made through crystallography experiments on a protein (RBSX-W6A) revealed the ability of ethylene glycol to identify a cryptic site in RBSX-W6A. The same cryptic site was found upon repeating the crystallography experiments with propylene glycol, supporting the cryptic-site finding potential of small glycols. Explicit-solvent molecular dynamics (MD) simulations of RBSX-W6A with exposed-state of the cryptic site revealed closure of the site in absence of glycol molecule reiterating what previous simulation studies have shown that cryptic sites are unstable without ligands and prefer to stay in closed state in their absence. Also, no change in the cryptic site was observed in explicit-solvent MD simulations of RBSX-W6A with occluded-state of the cryptic site, showing that water molecules are not able to open the cryptic site. The combined crystal structure and simulation results highlight the role of small glycols in identifying the cryptic site of RBSX-W6A. Further, through protein dataset construction and analysis, we demonstrate the generality of the cryptic-site finding potential of these glycols. Our analyses show that ethylene glycol molecules identify cryptic sites in pharmaceutically important proteins (G-actin, Myosin II, Bcl-xL, Glutamate receptor 2) for which the same cryptic sites become apparent upon binding of biologically relevant ligands. Our study suggests potential application of small glycols in experimental and computational approaches to identify cryptic sites in undruggable and/or difficult targets, making these proteins amenable to drug-design strategies and expanding the druggable proteome.