The Open Question of How GPCRs Interact with GPCR Kinases (GRKs).

G protein-coupled receptors (GPCRs), which regulate a vast number of eukaryotic processes, are desensitized by various mechanisms but, most importantly, by the GPCR kinases (GRKs). Ever since GRKs were first identified, investigators have sought to determine which structural features of GRKs are used to select for the agonist-bound states of GPCRs and how this binding event in turn enhances GRK catalytic activity. Despite a wealth of molecular information from high-resolution crystal structures of GRKs, the mechanisms driving activation have remained elusive, in part because the GRK N-terminus and active site tether region, previously proposed to serve as a receptor docking site and to be key to kinase domain closure, are often disordered or adopt inconsistent conformations. However, two recent studies have implicated other regions of GRKs as being involved in direct interactions with active GPCRs. Atomic resolution structures of GPCR-GRK complexes would help refine these models but are, so far, lacking. Here, we assess three distinct models for how GRKs recognize activated GPCRs, discuss limitations in the approaches used to generate them, and then experimentally test a hypothetical GPCR interaction site in GRK2 suggested by the two newest models.

Structure-Based Design of Selective, Covalent G Protein-Coupled Receptor Kinase 5 Inhibitors.

The ability of G protein-coupled receptor (GPCR) kinases (GRKs) to regulate desensitization of GPCRs has made GRK2 and GRK5 attractive targets for treating heart failure and other diseases such as cancer. Although advances have been made toward developing inhibitors that are selective for GRK2, there have been far fewer reports of GRK5 selective compounds. Herein, we describe the development of GRK5 subfamily selective inhibitors, 5 and 16d that covalently interact with a nonconserved cysteine (Cys474) unique to this subfamily. Compounds 5 and 16d feature a highly amenable pyrrolopyrimidine scaffold that affords high nanomolar to low micromolar activity that can be easily modified with Michael acceptors with various reactivities and geometries. Our work thereby establishes a new pathway toward further development of subfamily selective GRK inhibitors and establishes Cys474 as a new and useful covalent handle in GRK5 drug discovery.

Structural Determinants Influencing the Potency and Selectivity of Indazole-Paroxetine Hybrid G Protein-Coupled Receptor Kinase 2 Inhibitors.

G protein-coupled receptor kinases (GRKs) phosphorylate activated receptors to promote arrestin binding, decoupling from heterotrimeric G proteins, and internalization. GRK2 and GRK5 are overexpressed in the failing heart and thus have become therapeutic targets. Previously, we discovered two classes of GRK2-selective inhibitors, one stemming from GSK180736A, a Rho-associated coiled-coil containing kinase 1 (ROCK1) inhibitor, the other from paroxetine, a selective serotonin-reuptake inhibitor. These two classes of compounds bind to the GRK2 active site in a similar configuration but contain different hinge-binding "warheads": indazole and benzodioxole, respectively. We surmised from our prior studies that an indazole would be the stronger hinge binder and would impart increased potency when substituted for benzodioxole in paroxetine derivatives. To test this hypothesis, we synthesized a series of hybrid compounds that allowed us to compare the effects of inhibitors that differ only in the identity of the warhead. The indazole-paroxetine analogs were indeed more potent than their respective benzodioxole derivatives but lost selectivity. To investigate how these two warheads dictate selectivity, we determined the crystal structures of three of the indazole hybrid compounds (CCG224061, CCG257284, and CCG258748) in complex with GRK2-Gßγ Comparison of these structures with those of analogous benzodioxole-containing complexes confirmed that the indazole-paroxetine hybrids form stronger interactions with the hinge of the kinase but also stabilize a distinct conformation of the kinase domain of GRK2 compared with previous complexes with paroxetine analogs. This conformation is analogous to one that can be assumed by GRK5, at least partially explaining the loss in selectivity.

Navigating the conformational landscape of G protein-coupled receptor kinases during allosteric activation.

G protein-coupled receptors (GPCRs) are essential for transferring extracellular signals into carefully choreographed intracellular responses controlling diverse aspects of cell physiology. The duration of GPCR-mediated signaling is primarily regulated via GPCR kinase (GRK)-mediated phosphorylation of activated receptors. Although many GRK structures have been reported, the mechanisms underlying GRK activation are not well-understood, in part because it is unknown how these structures map to the conformational landscape available to this enzyme family. Unlike most other AGC kinases, GRKs rely on their interaction with GPCRs for activation and not phosphorylation. Here, we used principal component analysis of available GRK and protein kinase A crystal structures to identify their dominant domain motions and to provide a framework that helps evaluate how close each GRK structure is to being a catalytically competent state. Our results indicated that disruption of an interface formed between the large lobe of the kinase domain and the regulator of G protein signaling homology domain (RHD) is highly correlated with establishment of the active conformation. By introducing point mutations in the GRK5 RHD-kinase domain interface, we show with both in silico and in vitro experiments that perturbation of this interface leads to higher phosphorylation activity. Navigation of the conformational landscape defined by this bioinformatics-based study is likely common to all GPCR-activated GRKs.