Identification of an antithrombotic allosteric modulator that acts through helix 8 of PAR1

Abstract

G protein-coupled receptors (GPCRs) can assume multiple conformations and possess multiple binding sites. Whereas endogenous agonists acting at the orthosteric binding site stabilize the active receptor conformation, small molecules that act at nonorthosteric sites can stabilize alternative conformations. The large majority of these allosteric modulators associate with extracellular loops of GPCRs. The role of intracellular domains in mediating allosteric modulation is largely unknown. In screening a small-molecule library for inhibitors of platelet activation, we identified a family of compounds that modified PAR1-mediated granule secretion. The most potent inhibitory compound, termed JF5, also demonstrated noncompetitive inhibition of the α 2A -adrenergic receptor. Aggregation studies using a battery of platelet GPCR agonists demonstrated that sensitivity to JF5 was limited to GPCRs that possessed a constrained eighth helix, as defined by a C-terminal palmitoylation site and interactions with TM7 and the i1 loop. Inhibition by JF5 was overcome in a PAR1 mutant in which the eighth helix was deleted, confirming a role for helix 8 in JF5 activity. Evaluation of downstream signaling showed that JF5 was selective with regard to G protein coupling, blocking signaling mediated by G αq but not G α12 . The compound inhibited thrombus formation in vivo following vascular injury with an IC 50 of ∼1 mg/kg. These results indicate a role for helix 8 in conferring sensitivity to small molecules, and show that this sensitivity can be exploited to control platelet activation during thrombus formation.thrombosis | chemical genetics | thrombin receptor | platelet signaling

Parmodulins inhibit thrombus formation without inducing endothelial injury caused by vorapaxar

“…22 Binding studies were performed in triplicate in 1.5 mL Eppendorf tubes at a final assay volume of 200 mL. Bovine serum albumin (0.1%) was included in the incubation buffer and filter plates were presoaked in 0.1% polyethyleneimine to reduce binding of [ H]haTRAP (25 nM) was mixed with the indicated concentration of compounds or vehicle in binding buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 , 1 mM ethylene glycol tetraacetic acid (EGTA), 0.1% bovine serum albumin).…”

“…22 Experiments using epinephrine were performed in the presence of 50 U/mL hirudin to prevent thrombin formation. Parmodulins were used at the lowest concentration, resulting in .90% inhibition of the aggregation of washed platelets in response to 5 mM SFLLRN.…”

“…22,[25][26][27] We compared the activity of these compounds with that of orthosteric antagonists of PAR1. Orthosteric antagonists included vorapaxar, atopaxar, SCH79797, and BMS-200261 ( Figure 1B).…”

“…The PAR4 cytoplasmic tail was used because none of these compounds inhibits platelet activation mediated through PAR4. 22,[25][26][27] Expression levels of receptors were quantified using an alkaline phosphatase assay (supplemental Figure 1). All compounds inhibited [Ca 21 ] i flux stimulated by the PAR1-specific agonist SFLLRN when tested in COS-7 cells overexpressing PAR1 ( Figure 1C).…”

The Therapeutic Potential of Allosteric Ligands for Free Fatty Acid Sensitive GPCRs

“…Although the publication of these structures has opened the door to increased structure-based drug development through homology models [50,51], this is much more challenging for allosteric ligands, as a suitable allosteric site must first be identified. Further complicating this issue, there are now examples of allosteric ligands that interact with both extracellular loop 2 (ECL2) [52,53], and the intracellular carboxyl terminal tail [54,55] of GPCRs, two regions that are poorly conserved and therefore difficult to predict through homology models. Considering these factors, at present it is clear that structure-based design approaches are often not the best suited to identifying novel allosteric leads.…”