How does a small molecule bind at a cryptic binding site?

Abstract

Protein-protein interactions (PPIs) are ubiquitous biomolecular processes that are central to virtually all aspects of cellular function. Identifying small molecules that modulate specific disease-related PPIs is a strategy with enormous promise for drug discovery. The design of drugs to disrupt PPIs is challenging, however, because many potential drug-binding sites at PPI interfaces are “cryptic”: When unoccupied by a ligand, cryptic sites are often flat and featureless, and thus not readily recognizable in crystal structures, with the geometric and chemical characteristics of typical small-molecule binding sites only emerging upon ligand binding. The rational design of small molecules to inhibit specific PPIs would benefit from a better understanding of how such molecules bind at PPI interfaces. To this end, we have conducted unbiased, all-atom MD simulations of the binding of four small-molecule inhibitors (SP4206 and three SP4206 analogs) to interleukin 2 (IL2)—which performs its function by forming a PPI with its receptor—without incorporating any prior structural information about the ligands’ binding. In multiple binding events, a small molecule settled into a stable binding pose at the PPI interface of IL2, resulting in a protein–small-molecule binding site and pose virtually identical to that observed in an existing crystal structure of the IL2-SP4206 complex. Binding of the small molecule stabilized the IL2 binding groove, which when the small molecule was not bound emerged only transiently and incompletely. Moreover, free energy perturbation (FEP) calculations successfully distinguished between the native and non-native IL2–small-molecule binding poses found in the simulations, suggesting that binding simulations in combination with FEP may provide an effective tool for identifying cryptic binding sites and determining the binding poses of small molecules designed to disrupt PPI interfaces by binding to such sites. Author summary Small-molecule drugs typically function by binding to and modulating the biological activity of their protein targets. Drug-binding sites resemble pockets or grooves on the surface of the target protein, and are generally present even when the drug is not bound. In the case of “cryptic” binding sites, however, the pocket or groove only takes shape during the drug-binding process, prior to which the geometric features of a typical binding site are absent. Cryptic sites commonly occur at protein-protein interfaces, for example, so targeting such sites could facilitate the design of drugs capable of modulating specific protein-protein interactions—an approach with great therapeutic potential. In practice, targeting cryptic sites is typically difficult, in part because much less is known about how small molecules bind to cryptic sites than to conventional sites. In the work reported here, we used molecular dynamics simulations to study the process of a drug binding at a cryptic binding site, and showed that simulations are capable of predicting the location and geometry of a drug binding. The improved understanding of how small molecules bind at cryptic sites afforded by approaches like the one presented here could aid the rational design of small molecules that target such sites.