6u7c | pdb_00006u7c
Human GRK2 in complex with Gbetagamma subunits and CCG258747
Structural highlights
FunctionARBK1_HUMAN Specifically phosphorylates the agonist-occupied form of the beta-adrenergic and closely related receptors, probably inducing a desensitization of them. Key regulator of LPAR1 signaling. Competes with RALA for binding to LPAR1 thus affecting the signaling properties of the receptor. Desensitizes LPAR1 and LPAR2 in a phosphorylation-independent manner.[1] Publication Abstract from PubMedG protein-coupled receptor (GPCR) kinases (GRKs) play a key role in terminating signals initiated by agonist-bound GPCRs. However, chronic stimulation of GPCRs, such as that which occurs in heart failure, leads to the over-expression of GRKs and maladaptive down regulation of GPCRs on the cell surface. We previously reported the discovery of potent and selective families of GRK inhibitors based on either the paroxetine or GSK180736A scaffold. A new inhibitor, CCG258747, based on paroxetine, demonstrates increased potency against the GRK2 subfamily and favorable pharmacokinetic parameters in mice. CCG258747 and the closely related compound CCG258208 also showed high selectivity for the GRK2 subfamily in a kinome panel of 104 kinases. We developed a cell-based assay to screen the ability of CCG258747 and ten other inhibitors with different GRK subfamily selectivities and with either the paroxetine or GSK180736A scaffold to block internalization of the micro-opioid receptor (MOR). CCG258747 showed the best efficacy in blocking MOR internalization among the compounds tested. Further we show that compounds based on paroxetine had much better cell permeability than those based on GSK180736A, explaining why GSK180736A-based inhibitors, although being potent in vitro, do not always show efficacy in cell-based assays. This study validates the paroxetine scaffold as the most effective for GRK inhibition in living cells, confirms that GRK2 predominantly drives internalization of MOR in the cell-lines we tested, and underscores the utility of high-resolution cell-based assays for assessment of compound efficacy. SIGNIFICANCE STATEMENT: G protein-coupled receptor kinases are attractive targets for developing therapeutics for heart failure because cardiac-selective knockouts of GRK2 and GRK5 in mice have shown improved heart function following injury. We have synthesized a new GRK2-subfamily selective inhibitor, CCG258747, that has nanomolar potency against GRK2 and excellent selectivity over other kinases. A live-cell receptor internalization assay was developed to test the ability of this and other GRK2 inhibitors to impart efficacy on a GRK-dependent process in cells. Our data indicates that CCG258747 blocked the internalization of the mu-opioid receptor most efficaciously because it has the best ability to cross cell membranes. This assay will aid in selecting inhibitors to pursue in future mouse studies. A New Paroxetine-Based GRK2 Inhibitor Reduces Internalization of the mu-Opioid Receptor.,Bouley RA, Weinberg ZY, Waldschmidt HV, Yen YC, Larsen SD, Puthenveedu MA, Tesmer JJG Mol Pharmacol. 2020 Mar 31. pii: mol.119.118661. doi: 10.1124/mol.119.118661. PMID:32234810[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. See AlsoReferences
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