G protein-coupled receptors (GPCRs) are central to many physiological processes. Regulation of this superfamily of receptors is controlled by GPCR kinases (GRKs), some of which have been implicated in heart failure. GSK180736A, developed as a Rho-associated coiled-coil kinase 1 (ROCK1) inhibitor, was identified as an inhibitor of GRK2, and co-crystallized in the active site. Guided by its binding pose overlaid with the binding pose of a known potent GRK2 inhibitor, Takeda103A, a library of hybrid inhibitors was developed. This campaign produced several compounds possessing high potency and selectivity for GRK2 over other GRK subfamilies, PKA, and ROCK1. The most selective compound, 12n (CCG-224406), had an IC50 for GRK2 of 130 nM, greater than 700-fold selectivity over other GRK subfamilies, and no detectable inhibition of ROCK1. Four of the new inhibitors were crystallized with GRK2 to give molecular insights into the binding and kinase selectivity of this class of inhibitors.
Selective inhibitors of individual subfamilies of G protein-coupled receptor kinases (GRKs) would serve as useful chemical probes as well as leads for therapeutic applications ranging from heart failure to Parkinson’s disease. To identify such inhibitors, differential scanning fluorimetry was used to screen a collection of known protein kinase inhibitors that could increase the melting points of the two most ubiquitously expressed GRKs: GRK2 and GRK5. Enzymatic assays on 14 of the most stabilizing hits revealed that three exhibit nanomolar potency of inhibition for individual GRKs, some of which exhibiting orders of magnitude selectivity. Most of the identified compounds can be clustered into two chemical classes: indazole/dihydropyrimidine-containing compounds that are selective for GRK2 and pyrrolopyrimidine-containing compounds that potently inhibit GRK1 and GRK5 but with more modest selectivity. The two most potent inhibitors representing each class, GSK180736A and GSK2163632A, were cocrystallized with GRK2 and GRK1, and their atomic structures were determined to 2.6 and 1.85 Å spacings, respectively. GSK180736A, developed as a Rho-associated, coiled-coil-containing protein kinase inhibitor, binds to GRK2 in a manner analogous to that of paroxetine, whereas GSK2163632A, developed as an insulin-like growth factor 1 receptor inhibitor, occupies a novel region of the GRK active site cleft that could likely be exploited to achieve more selectivity. However, neither compound inhibits GRKs more potently than their initial targets. This data provides the foundation for future efforts to rationally design even more potent and selective GRK inhibitors.
Inhibition of G protein‐coupled receptor kinase 2 (GRK2) and GRK5 has emerged as potential therapeutic routes for the treatment of heart failure and cardiac hypertrophy. However, there are no commercially available small molecule therapeutics selective for either of these kinases. High‐throughput screening campaigns with representative GRKs from each subfamily (GRK1, GRK2, and GRK5) have identified first generation small molecule inhibitors with low‐micromolar potency and orders of magnitude selectivity between GRKs. The most potent of these scaffolds have additionally been shown to mediate increased contractility in isolated cardiomyocytes as well as whole animals, thus demonstrating their potential to be developed into viable therapeutics. Crystallographic analysis of several GRK‐scaffold complexes reveal molecular mechanisms of selectivity among GRKs and AGC kinases. Based on these structures, iterative rounds of rational design have resulted in compounds with nanomolar potency, high subfamily selectivity, which has provided new insights into structure activity relationships. These compounds form the basis of a novel ‘GRK inhibitor toolkit’ that can be used to elucidate GRK roles in cellular signaling and spur development of novel therapeutics. Grant Funding Source: NIH grants [HL071818 & HL086865] to J.J.G.T. and the American Heart Association [N014938] K.T.H.
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