Molecular Mechanism of Selectivity among G Protein-Coupled Receptor Kinase 2 Inhibitors

Abstract

G protein-coupled receptors (GPCRs) are key regulators of cell physiology and control processes ranging from glucose homeostasis to contractility of the heart. A major mechanism for the desensitization of activated GPCRs is their phosphorylation by GPCR kinases (GRKs). Overexpression of GRK2 is strongly linked to heart failure, and GRK2 has long been considered a pharmaceutical target for the treatment of cardiovascular disease. Several lead compounds developed by Takeda Pharmaceuticals show high selectivity for GRK2 and therapeutic potential for the treatment of heart failure. To understand how these drugs achieve their selectivity, we determined crystal structures of the bovine GRK2-G␤␥ complex in the presence of two of these inhibitors. Comparison with the apoGRK2-G␤␥ structure demonstrates that the compounds bind in the kinase active site in a manner similar to that of the AGC kinase inhibitor balanol. Both balanol and the Takeda compounds induce a slight closure of the kinase domain, the degree of which correlates with the potencies of the inhibitors. Based on our crystal structures and homology modeling, we identified five amino acids surrounding the inhibitor binding site that we hypothesized could contribute to inhibitor selectivity. However, our results indicate that these residues are not major determinants of selectivity among GRK subfamilies. Rather, selectivity is achieved by the stabilization of a unique inactive conformation of the GRK2 kinase domain.

Raf Kinase Inhibitor Protein (RKIP) Dimer Formation Controls Its Target Switch from Raf1 to G Protein-coupled Receptor Kinase (GRK) 2

“…RKIP-mediated GRK2 inhibition increases cAMP signaling and enhances contractility of isolated neonatal cardiomyocytes (4). Despite significant efforts to develop small molecule GRK2 inhibitors, a specific and applicable GRK2 inhibitor is still not available (20,27). The phosphomimetic and the dimeric mutants of RKIP, RKIP SK153/7EE , and RKIP…”

“…As controls, we used either mock-transfected cells or cells transfected with N-terminal RKIP fragments, which did not contain the loop sequence (supplemental Fig. S4, A (RKIP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] ) and C (RKIP )). In accordance with our previous results, prevention of RKIP dimerization by loop 127-150 was accompanied by inhibition of PKC-induced RKIP-GRK2 assembly (Fig.…”

Endothelin-converting Enzyme 1 and β-Arrestins Exert Spatiotemporal Control of Substance P-induced Inflammatory Signals

“…To investigate the contributions of GRK2 and ␤ARRs to NK 1 R resensitization, NCM-NK 1 R cells were preincubated with vehicle or 1 nM SP because GRKs and ␤ARRs preferentially mediate desensitization to submaximal agonist concentrations. The GRK2 inhibitor CMPD103A (26) increased the magnitude and duration of the initial response to SP (80 Ϯ 11% increase in maximal response compared with vehicle, p Ͻ 0.05, Fig. 9C) and inhibited resensitization of SPinduced Ca 2ϩ signaling (20 Ϯ 1.8% of control resensitization, 120-min recovery; p Ͻ 0.0001; Fig.…”

Atypical Actions of G Protein-Coupled Receptor Kinases

“…Unfortunately, information on the affi nities of these compounds for GRK3, GRK4, and GRK6 is not available. Crystal structure of GRK2 and CMPD103A complex revealed that CMPD103A binds GRK2 at the kinase active site (Thal et al, 2011). As GRK2 plays an important role in progression of cardiovascular diseases such as heart failure and myocardial infarction (Rockman et al, 2002;Brinks et al, 2010), a selective GRK2 inhibitor should be useful for treatment of these diseases.…”

“…Takeda compounds CMPD101 and CMPD103A inhibited GRKs, with a selectivity order of GRK2>GRK5>GRK1 (Thal et al, 2011) (Fig. 2).…”