Agonists and Antagonists of Protease-Activated Receptor 2 Discovered within a DNA-Encoded Chemical Library Using Mutational Stabilization of the Target

Abstract

<jats:p> The discovery of ligands via affinity-mediated selection of DNA-encoded chemical libraries is driven by the quality and concentration of the protein target. G-protein-coupled receptors (GPCRs) and other membrane-bound targets can be difficult to isolate in their functional state and at high concentrations, and therefore have been challenging for affinity-mediated selection. Here, we report a successful selection campaign against protease-activated receptor 2 (PAR2). Using a thermo-stabilized mutant of PAR2, we conducted affinity selection using our &gt;100-billion-compound DNA-encoded library. We observed a number of putative ligands enriched upon selection, and subsequent cellular profiling revealed these ligands to comprise both agonists and antagonists. The agonist series shared structural similarity with known agonists. The antagonists were shown to bind in a novel allosteric binding site on the PAR2 protein. This report serves to demonstrate that cell-free affinity selection against GPCRs can be achieved with mutant stabilized protein targets. </jats:p>

Novel Nucleic Acid Binding Small Molecules Discovered Using DNA-Encoded Chemistry

“…Individual DNA-encoded chemical libraries were synthesized using a DNA-recorded split and pool procedure in which DNA tagging and building block installation events occurred during each split. We have described this general methodology before [3,4,5,6,7,8,27,28]. The synthetic schemes defining each library (A and B) are shown in Figure 1.…”

“…Altering the target concentration, the inclusion of selectivity targets, and the addition of known binders to compete with library compounds are individual variables that can be explored in parallel in a single-selection campaign. We have previously reported the result of multiple parallel selection conditions that successfully identified novel small-molecule inhibitors of therapeutic protein targets, including BTK [3], PAR2 [4], sEH [5], InhA [6], Mcl1 [7], and ATAD2 [8]: Other groups have reported successes with similar platforms [1].…”

Encoded Library Technologies as Integrated Lead Finding Platforms for Drug Discovery

“…Two recent reports on protease-activated receptor (PAR2) exemplify this well. Screening of DELs against a thermo-stabilized mutant of the receptor identified two ligand families binding to distinct sites on the target [67]. The first family, originating from a capped dipeptide library, showed considerable similarity to known agonists and indeed exhibited the same effect in cellular assays.…”

“…This may also contribute to the observation that a larger number of DEL literature reports describe the identification of enzyme inhibitors binding to well defined pockets [8]. Nonetheless, the number of examples successfully identifying binders to challenging targets like PPIs [63,65,66,88,89] and MoAs such as agonists, positive allosteric modulators and antagonists for G-protein coupled receptors (GPCRs) [67,68,90,91,92] grows at an increasing rate. Key contributors to the recent success against GPCRs were advances in production of membrane proteins [93] and stabilization of specific conformations [94,95].…”

Functionality‐Independent DNA Encoding of Complex Natural Products

“…Novel compounds exhibiting nanomolar potency have been selected through this strategy . Recently, several ligands for GPCRs were also selected from a DEL . The recent announcement of the DELopen platform by Wuxi AppTec (DELopen.org) provides academic research institutions open access to its 80 billion member DEL, underlining another unique feature of DELs—the power of sharing.…”