Structural insight into allosteric modulation of protease-activated receptor 2

Abstract

Protease-activated receptors (PARs) are a family of G-protein-coupled receptors (GPCRs) that are irreversibly activated by proteolytic cleavage of the N terminus, which unmasks a tethered peptide ligand that binds and activates the transmembrane receptor domain, eliciting a cellular cascade in response to inflammatory signals and other stimuli. PARs are implicated in a wide range of diseases, such as cancer and inflammation. PARs have been the subject of major pharmaceutical research efforts but the discovery of small-molecule antagonists that effectively bind them has proved challenging. The only marketed drug targeting a PAR is vorapaxar, a selective antagonist of PAR1 used to prevent thrombosis. The structure of PAR1 in complex with vorapaxar has been reported previously. Despite sequence homology across the PAR isoforms, discovery of PAR2 antagonists has been less successful, although GB88 has been described as a weak antagonist. Here we report crystal structures of PAR2 in complex with two distinct antagonists and a blocking antibody. The antagonist AZ8838 binds in a fully occluded pocket near the extracellular surface. Functional and binding studies reveal that AZ8838 exhibits slow binding kinetics, which is an attractive feature for a PAR2 antagonist competing against a tethered ligand. Antagonist AZ3451 binds to a remote allosteric site outside the helical bundle. We propose that antagonist binding prevents structural rearrangements required for receptor activation and signalling. We also show that a blocking antibody antigen-binding fragment binds to the extracellular surface of PAR2, preventing access of the tethered ligand to the peptide-binding site. These structures provide a basis for the development of selective PAR2 antagonists for a range of therapeutic uses.

The Symbiotic Relationship Between Drug Discovery and Organic Chemistry

“…[106] In 2018, the preparation of a4 0-trillion member DEL-the largest collection of synthetic molecules in the world, so far-was announced by Nuevolution. Recent successs tories involving the use of aD EL include the discoveryo fareceptor interacting protein kinase 1( RIPK1) inhibitor, [107] ap rotease-activated receptor 2( PAR2) allosteric ligand, [108] or an egative allosteric modulator of the b 2 -adrenergic receptor (b 2 AR; Figure 21). [109] Despite these prominente xamples, DEL technologys tillh as some challenges that need to be addressed.…”

Discovery of new GPCR ligands to illuminate new biology

“…3). A spectacular result of recent crystallography, and one case of a large DNA-encoded library screening 30 , is that two other allosteric sites on GPCRs are targetable by small molecules: the intracellular region where G protein binds 31,32 and an intramembrane region on the outer surface of the helical bundle 33,34 . These sites appear conserved among family A GPCRs, suggesting that they may be broadly targeted.…”

Muscarinic receptor regulates extracellular signal regulated kinase by two modes of arrestin binding

“…For example, the crystal structure of the seven transmembrane domains of M 1 R was fully resolved (41), but the C tail and third intracellular loop of the receptor were not due to flexibility and truncation for insertion of a reference protein (T4 lysozyme), respectively. Therefore, in this study, we used I-TASSER modeling to complete the structure of M 1 R. The PAR1 and a new PAR2 structure are also available, but the C terminus tail and ICL3 regions are not included (55,56). With I-TASSER, the c-score indicates the accuracy of the predictions and 90% accuracy of the protein fold is expected for a c-score > −1.5.…”