Mimicking Intermolecular Interactions of Tight Protein–Protein Complexes for Small‐Molecule Antagonists

Abstract

Tight protein-protein interactions (Kd < 100 nM) that occur over a large binding interface (> 1,000 Å2) are highly challenging to disrupt with small molecules. Historically, the design of small molecules to inhibit protein-protein interactions has focused on mimicking the position of interface protein ligand side chains. Here, we explore mimicry of the pairwise intermolecular interactions of the native protein ligand with residues of the protein receptor to enrich commercial libraries for small-molecule inhibitors of tight protein-protein interactions. We use the high-affinity interaction (Kd = 1 nM) between the urokinase receptor (uPAR) and its ligand urokinase (uPA) to test our methods. We introduce three methods for rank-ordering small molecules docked to uPAR: (i) a new fingerprint approach that represents uPA’s pairwise interaction energies with uPAR residues; (ii) a pharmacophore approach to identify small molecules that mimic the position of uPA interface residues; and (iii) a combined fingerprint and pharmacophore approach. Our work led to small molecules with novel chemotypes that inhibited a tight uPAR•uPA protein-protein interaction with single-digit micromolar IC50s. We also report the extensive work that identified several of the hits as either lacking stability, thiol reactive, or redox active. This work suggests that mimicking the binding profile of the native ligand and the position of interface residues can be an effective strategy to enrich commercial libraries for small-molecule inhibitors of tight protein-protein interactions.

Chemical Space Overlap with Critical Protein–Protein Interface Residues in Commercial and Specialized Small‐Molecule Libraries

“…Glide was used to score the Vina-dockedb inding modes in place using the Gli-deHTVS scoring function. [59,60] Ligand pharmacophore:Apreviously described [13] pharmacophore-based approach was adapted and used to identify how docking compounds overlapped with and mimicked known hot spots of the protein ligand in each of the protein-protein complexes.Aset of pharmacophore hypotheses was constructed corresponding to the physicochemical properties of the protein ligand side chain using the Phase package in Schrçdinger. [61,62] Phase has six built-in types of pharmacophore features:1 )hydrogen-bond acceptor( A);2 )hydrogen-bond donor( D);3 )hydrophobe (H);4 )negative ionizable (N);5 )positive ionizable (P);a nd 6) aromaticr ing (R).…”

“…There is growing interest in applying computational methods for the discovery of small‐molecule PPI inhibitors, particularly for tight secondary and tertiary interactions. We have successfully used virtual screening to identify inhibitors of the tight tertiary interaction between the urokinase receptor uPAR and its protein ligand uPA with compounds ranging from sub‐micromolar to micromolar affinity . In one case, we screened multiple structures that were sampled from explicit‐solvent molecular‐dynamics simulations and identified a sub‐micromolar affinity compound .…”

“…We have successfully used virtual screeningt oi dentify inhibitors of the tight tertiary interaction between the urokinase receptor uPAR and its protein ligand uPAw ith compounds ranging from submicromolar to micromolar affinity. [11][12][13] In one case, we screened multiple structures that were sampled from explicitsolventm olecular-dynamics simulations and identified as ubmicromolar affinity compound. [11] The predicted structure of the compound was independently confirmed by X-ray crystallography.…”

“…[14] More recently,w ei ntroducedafingerprint method that uses the native protein ligand as ag uide to identify small molecules that mimic the interaction of the protein ligand. [13] There is growing interest in the use of structure-based virtual screening to identify small molecules that inhibit challenging protein-protein interactions (PPIs). In this study,w ei nvestigated how effectively chemical library members docked at the PPI interface mimic the position of criticals ide-chain residues known as "hot spots".…”

Unconventional Secondary Structure Mimics: Ladder‐Rungs

“…Assays used to monitor the effects of small molecules on uPA⋅uPAR tend to test for parameters such as cell migration and invasion to reflect influence on metastatic spread; the work of Meroueh and co‐workers on testing small molecules discovered by virtual screening on the same target was a good model for our studies …”