Abstract
Protein–protein interactions (PPIs) are central to biological mechanisms, and can serve as compelling targets for drug discovery. Yet, the discovery of small molecule inhibitors of PPIs remains challenging given the large and typically shallow topography of the interacting protein surfaces. Here, we describe a general approach to the discovery of orthosteric PPI inhibitors that mimic specific secondary protein structures. Initially, hot residues at protein–protein interfaces are identified in silico or from experimental data, and incorporated into secondary structure-based queries. Virtual libraries of small molecules are then shape-matched against the queries, and promising ligands docked to target proteins. The approach is exemplified experimentally using two unrelated PPIs that are mediated by an α-helix (p53/hDM2) and a β-strand (GKAP/SHANK1-PDZ). In each case, selective PPI inhibitors are discovered with low μM activity as determined by a combination of fluorescence anisotropy and 1H–15N HSQC experiments. In addition, hit expansion yields a series of PPI inhibitors with defined structure–activity relationships. It is envisaged that the generality of the approach will enable discovery of inhibitors of a wide range of unrelated secondary structure-mediated PPIs.
Highlights
We demonstrate the power of the approach using two unrelated exemplar PPI targets: (i) p53/hDM2, an a-helixmediated PPI37 that is a clinically relevant oncology target;[38] and (ii) GKAP/SHANK1-PDZ,[39] a b-strand-mediated PPI which
A computational work ow that involved FastROCS matching to secondary structure queries identi ed candidate inhibitors
Experimental screening of subsets of these candidates identi ed multiple genuine inhibitors demonstrating the approach to be valid for improving hit rate
Summary
The discovery of small-molecule modulators of protein–protein interactions (PPIs) is a central challenge in both chemical biology and medicinal chemistry.[1,2,3,4,5,6] For a number of PPI targets, potent PPI inhibitors and stabilizers have been successfully discovered, for example by optimisation of high-throughput screening hits, fragment-based discovery approaches or virtual methods targeting p53/hDM2,7–10 the BCL-2 family[11,12,13,14,15] and other interactions.[16,17,18,19,20,21] the paucity of small-molecule PPI inhibitors that have progressed as clinical candidates,[3]within the context of an enormous protein–protein interactome,[22] provides continued motivation for development of novel and general small-molecule discovery approaches. PPIs are known to involve shallow, relatively large interfaces of varied topography, the identi cation of hotspots23,24 – i.e., amino-acid residues that contribute signi cantly to binding – can provide focus for ligand design efforts. Edge Article plays a key role at the synaptic junction[40] and is representative of PDZ-mediated interactions, which remain challenging for small-molecule inhibitor discovery.[41,42] For both targets, we used this approach to virtually screen $4 million compounds for which physical samples were available in AstraZeneca's compound collection and validated the approach experimentally using uorescence anisotropy and 1H–15N HSQC biophysical screens.
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