Abstract
Allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites, make it possible to target proteins considered “undruggable” and to develop new therapies that circumvent existing resistance. Despite growing interest in allosteric drug discovery, rational design is limited by a lack of sufficient structural information about alternative binding sites in proteins. Previously, we used Markov State Models (MSMs) to identify such “cryptic pockets,” and here we describe a method for identifying compounds that bind in these cryptic pockets and modulate enzyme activity. Experimental tests validate our approach by revealing both an inhibitor and two activators of TEM β-lactamase (TEM). To identify hits, a library of compounds is first virtually screened against either the crystal structure of a known cryptic pocket or an ensemble of structures containing the same cryptic pocket that is extracted from an MSM. Hit compounds are then screened experimentally and characterized kinetically in individual assays. We identify three hits, one inhibitor and two activators, demonstrating that screening for binding to allosteric sites can result in both positive and negative modulation. The hit compounds have modest effects on TEM activity, but all have higher affinities than previously identified inhibitors, which bind the same cryptic pocket but were found, by chance, via a computational screen targeting the active site. Site-directed mutagenesis of key contact residues predicted by the docking models is used to confirm that the compounds bind in the cryptic pocket as intended. Because hit compounds are identified from docking against both the crystal structure and structures from the MSM, this platform should prove suitable for many proteins, particularly targets whose crystal structures lack obvious druggable pockets, and for identifying both inhibitory and activating small-molecule modulators.
Highlights
Rational drug design based on a single protein structure captured, for instance, by x-ray crystallography typically focuses on molecules that bind to and sterically block a key functional site
Druggable pockets that appear in these alternate structures, called cryptic pockets, present the opportunity to design allosteric drugs, which bind to proteins in regions other than their main ligand-binding or active sites and are known to have distinct benefits over drugs targeting active sites [3]
12,695 compounds we screened in silico, we tested the 40 compounds with the highest docking scores for their ability to modulate TEM activity in a high throughput plate-based assay, and identified 5 hit compounds
Summary
Rational drug design based on a single protein structure captured, for instance, by x-ray crystallography typically focuses on molecules that bind to and sterically block a key functional site. This approach is inapplicable to proteins that lack obvious druggable pockets or scenarios where activation, rather than inhibition, is desired. While there are examples of high-throughput experimental screens that have serendipitously identified compounds that bind cryptic pockets [7] and screens designed for finding allosteric modulators [8], our goal is to develop a structure-based approach to rationally target cryptic pockets in proteins for drug design
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