Many proteins in their unbound structures lack surface pockets appropriately sized for drug binding. Hence, a variety of experimental and computational tools have been developed for the identification of cryptic sites that are not evident in the unbound protein but form upon ligand binding, and can provide tractable drug target sites. The goal of this review is to discuss the definition, detection, and druggability of such sites, and their potential value for drug discovery. Novel methods based on molecular dynamics simulations are particularly promising and yield a large number of transient pockets, but it has been shown that only a minority of such sites are generally capable of binding ligands with substantial affinity. Based on recent studies, current methodology can be improved by combining molecular dynamics with fragment docking and machine learning approaches.

Development of small molecule inhibitors of protein−protein interactions (PPIs) is hampered by our poor understanding of the druggability of PPI target sites. Here, we describe the combined application of alanine-scanning mutagenesis, fragment screening, and FTMap computational hot spot mapping to evaluate the energetics and druggability of the highly charged PPI interface between Kelch-like ECH-associated protein 1 (KEAP1) and nuclear factor erythroid 2 like 2 (Nrf2), an important drug target. FTMap identifies four binding energy hot spots at the active site. Only two of these are exploited by Nrf2, which alanine scanning of both proteins shows to bind primarily through E79 and E82 interacting with KEAP1 residues S363, R380, R415, R483, and S508. We identify fragment hits and obtain X-ray complex structures for three fragments via crystal soaking using a new crystal form of KEAP1. Combining these results provides a comprehensive and quantitative picture of the origins of binding energy at the interface. Our findings additionally reveal non-native interactions that might be exploited in the design of uncharged synthetic ligands to occupy the same site on KEAP1 that has evolved to bind the highly charged DEETGE binding loop of Nrf2. These include π-stacking with KEAP1 Y525 and interactions at an FTMapidentified hot spot deep in the binding site. Finally, we discuss how the complementary information provided by alaninescanning mutagenesis, fragment screening, and computational hot spot mapping can be integrated to more comprehensively evaluate PPI druggability.

Beyond rule-of-five (bRo5) compounds are increasingly used in drug discovery. Here we analyze 37 target proteins that have bRo5 drugs or clinical candidates. Targets can benefit from bRo5 drugs if they have “complex” hot spot structure with four or more hots spots, including some strong ones. Complex I targets show positive correlation between binding affinity and molecular weight. These targets are conventionally druggable, but reaching additional hot spots enables improved pharmaceutical properties. Complex II targets, mostly protein kinases, also have strong hot spots but show no correlation between affinity and ligand molecular weight, and the primary motivation for creating larger drugs is to increase selectivity. Each target considered as complex III has some specific reason for requiring bRo5 drugs. Finally, targets with “simple” hot spot structure, i.e., three or fewer weak hot spots, must use larger compounds that interact with surfaces beyond the hot spot region to achieve acceptable affinity.

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