G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitors: Current

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G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitors: Current Trends and Future Perspectives Manuela Guccione,† Roberta Ettari,*,† Sabrina Taliani,‡ Federico Da Settimo,‡ Maria Zappalà,† and Silvana Grasso† †

Dipartimento di Scienze Chimiche, Biologiche, Farmaceutiche ed Ambientali, Università degli Studi di Messina, Viale Annunziata, 98168 Messina, Italy ‡ Dipartimento di Farmacia, Università di Pisa, Via Bonanno Pisano 6, 56126 Pisa, Italy ABSTRACT: G-protein-coupled receptor kinase 2 (GRK2) is a G-protein-coupled receptor kinase that is ubiquitously expressed in many tissues and regulates various intracellular mechanisms. The up- or down-regulation of GRK2 correlates with several pathological disorders. GRK2 plays an important role in the maintenance of heart structure and function; thus, this kinase is involved in many cardiovascular diseases. GRK2 up-regulation can worsen cardiac ischemia; furthermore, increased kinase levels occur during the early stages of heart failure and in hypertensive subjects. GRK2 up-regulation can lead to changes in the insulin signaling cascade, which can translate to insulin resistance. Increased GRK2 levels also correlate with the degree of cognitive impairment that is typically observed in Alzheimer’s disease. This article reviews the most potent and selective GRK2 inhibitors that have been developed. We focus on their mechanism of action, inhibition profile, and structure−activity relationships to provide insight into the further development of GRK2 inhibitors as drug candidates.

1. INTRODUCTION Protein kinases are a ubiquitous group of enzymes that catalyze the phosphorylation of specific substrates by transferring the γphosphate group from ATP (or GTP) to serine, threonine, or tyrosine residues in the cytoplasmic tails and loops of the target. This phosphorylation process causes conformational changes that affect protein functions.1,2 As important components of signal transduction, protein kinases play a key role in a variety of cellular mechanisms, including apoptosis, cell proliferation, gene expression, glycogen metabolism, immune response, neurotransmission, and oncogenesis. An alteration of phosphorylation processes is directly involved in many diseases, such as cancer, diabetes, autoimmune disorders, inflammation, neurodegeneration, and cardiovascular disorders. Thus, kinases are widely studied at the structural, biochemical, and cellular levels.3,4 AGC kinases are one of the seven major groups of human protein kinase superfamilies and mediate various cellular functions. Mutations and/or dysregulation of AGC kinases contributes to the pathogenesis of many human diseases, including cancer and diabetes. The AGC kinase family consists of 60 members, including GPCR kinases (GRKs), which are a family of seven serine/threonine kinases.5,6 The physiological importance of GRKs is prominent in several human pathologies. The pathological conditions characterized by GPCR malfunction occur because of a genetic alteration and/ or changes in the GRK expression levels. Currently, mice © 2016 American Chemical Society

lacking specific GRK isoforms are useful tools for studying the importance of individual GRKs within an organism and to understand the involvement of each protein in receptor signaling pathways and vital processes.7 The GRK family consists of seven isoforms (GRK1−GRK7) that are subdivided into three main groups based on their sequence homology: the visual GRK subfamily (GRK1 and GRK7); β-adrenergic receptor kinase (β-ARK) subfamily (GRK2 and GRK3); and GRK4 subfamily (GRK4, GRK5, and GRK6).8,9 All GRK isoforms are multidomain proteins that consist of an amino-terminal region unique to the GRK family, a regulator of G protein signaling (RGS) homology (RH) domain, which regulates GPCR signaling by phosphorylationindependent mechanisms, a serine/threonine protein kinase domain (KD), and a carboxyl-terminal domain.5,10,11 Differences in the expression, structure, and function of GRKs have been observed. GRK1, GRK4, and GRK7 are expressed in limited tissues: GRK1 and GRK7 are expressed in the retina,12 whereas GRK4 is expressed at its highest levels in the testis13,14 and to a lesser extent in the brain,13 kidney,15 and uterus myometrium.16 In contrast, GRK2, GRK3, GRK5, and GRK6 are ubiquitously expressed in mammalian tissues.17 GRKs specifically recognize and phosphorylate agonistactivated GPCRs on serine and threonine residues.5,18 GRKReceived: December 15, 2015 Published: June 30, 2016 9277

DOI: 10.1021/acs.jmedchem.5b01939 J. Med. Chem. 2016, 59, 9277−9294

Journal of Medicinal Chemistry

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Figure 1. GPCR agonist activation and GRK2-mediated receptor desensitization. Agonist stimulation produces a conformational change in the GPCR, which promotes the binding of G protein to the intracellular binding site on the receptor. The activated Gα and Gβγ subunits are responsible for the activation of specific effectors, which produce different second messengers that can generate a wide range of cellular responses. The desensitization process of GPCRs is triggered by the interaction of GRK2 with the Gβγ subunits. This interaction promotes the recruitment of the kinase to the plasma membrane and the phosphorylation of the agonist-activated receptor. An increase in receptor affinity toward β-arrestin proteins causes them to bind, which results in Gα subunit uncoupling and the arrest of signal propagation. The internalization process proceeds through clathrin-mediated receptor endocytosis, leading to receptor degradation or dephosphorylation and recycling to the plasma membrane.

phosphorylation of PDGFRβ led to desensitization and a reduction in receptor activation.23 In contrast, the phosphorylation of EGFR did not influence its activity.24 In mammalian cell cultures, GRK2 phosphorylated smoothened (Smo), a cell surface receptor with the same seventransmembrane domain structure of GPCRs.5,25 The GRK2mediated phosphorylation of Smo induced its ciliary accumulation and promoted its transition to an active open conformation via binding with β-arrestin 2.26 Smo is involved in a signaling pathway that regulates embryonic development and adult tissue homeostasis. The importance of Smo has been confirmed by the onset of several disorders, including cancer, due to its malfunction.26 GRK2 can bind and phosphorylate many cytoskeletal proteins. Tubulin phosphorylation leads to increases in the number of Gβγ subunits and phospholipids.27 Ezrin is activated by the phosphorylation of its Thr567 residue.28 Radixin is activated via the phosphorylation of its Thr564 residue.29 Vascular polypeptide endothelin 1 (ET-1) stimulates GRK2 activity, which produces ET-1-mediated phosphorylation and degradation of the insulin receptor substrate.30 GRK2 is responsible for the phosphorylation of additional substrates, such as unidentified mitochondrial proteins with a molecular weight between 30−60 kDa.31 Transcription factors also undergo GRK2 phosphorylation after a previous activation by membrane receptors. The GRK2 phosphorylation of Smad proteins prevented the nuclear

mediated receptor phosphorylation is one of the wellcharacterized mechanisms for GPCRs desensitization.17,19 GPCRs normally detect a variety of extracellular signals at the plasma membrane, thus modulating many biological processes. Receptor desensitization (Figure 1) is an important mechanism to maintain homeostasis and prevent various diseases. Desensitization occurs via a two-step mechanism: (i) a GPCR interaction with a GRK protein that results in a phosphorylation reaction and (ii) arrestin binding to the phosphorylated receptors and receptor−G protein uncoupling.17,20 As a result of β-arrestin binding, phosphorylated receptors are targeted for clathrin-mediated endocytosis and internalized via coated pits. In the endosome, receptors can undergo different processes: (i) dephosphorylation and arrestin dissociation, resensitization, and recycling to the cell surface; (ii) lysosomal proteolysis; and (iii) activation of additional intracellular signaling pathways.21,22 GRKs are mainly involved in agonist-activated GPCR phosphorylation and desensitization; however, many other types of signaling proteins are substrates for GRKs. GRKs control a variety of pathways that regulate the cell cycle, from growth to death, and other vital processes, such as cell development and immune response.5 Several studies have investigated the phosphorylation of nonGPCR substrates by GRK2.5 GRK2 can phosphorylate serine residues on two tyrosine receptor kinases: platelet-derived growth factor receptor-β (PDGFRβ) and EGFR.23 GRK2 9278



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translocation of transforming growth factor-β family components and their gene expression.32 Considering the wide variety of nonreceptor substrates that interact with GRK2, the mechanisms of these interactions may be substrate-specific.5 GRKs regulate several signaling proteins in a manner unique to the aforementioned phosphorylation-dependent receptor desensitization. This alternative mechanism is related to the activity of the RH domain located within the N-terminal region of GRKs.11,33 The RH domain shows a homology with RGS proteins, which are multifunctional signaling proteins that directly bind to activated Gα subunits. RGS proteins block Gα signaling by preventing or limiting the binding of GTP to Gα. This process leads to Gα deactivation and termination of downstream signals.34 While the RH domain is responsible for the interaction with the Gα subunit, isoforms 2 and 3 of the GRK family possess the pleckstrin homology (PH) domain, which can interact with Gβγ subunits.5 These two isoforms are directly involved in the desensitization of β-adrenergic receptors (β-ARs)35 and muscarinic receptors.36 Agonist-activated β-ARs allow the membrane-anchored Gβγ protein to bind to GRKs, thus stimulating GRK translocation from the cytoplasm to the plasma membrane.37 This relocation is necessary for kinase/ receptor interactions and phosphorylation processes. Previous studies have also demonstrated that charged phospholipids can differentially regulate GRK2 activity.38 Specifically, the ability of several membrane lipids, such as phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PIP2), to phosphorylate human m2 mAChR was examined. PS caused a 2- to 3-fold enhancement of GRK2-mediated receptor phosphorylation, whereas PIP2 strongly inhibited GRK2 activity.38 PS and PIP2 were further tested to determine if they share the same binding site on GRK2. The results showed that PS and PIP2 acted in a concentration-dependent manner; furthermore, PS prevented PIP2/GRK2 interactions and vice versa, thus suggesting a common binding site for these two membrane lipids.38 PS and PIP2 also acted in a concentration-dependent manner after the addition of Gβγ. Gβγ increased the effects of PS on GRK2mediated receptor phosphorylation and reduced the inhibition of GRK2 by PIP2.38 These results suggest that phospholipids and Gβγ bind to different binding sites located within the PH domain; therefore, GRK2 could be considered a lipiddependent protein kinase that can be both up- and downregulated by phospholipids.38

Studies in GRK2 knockout mice showed that this kinase plays an important role in embryonic cardiac development and function. Homozygous GRK2−/− embryos die during gestation. In contrast, heterozygous GRK2± mice are viable and develop normally.39 GRK2 is required for the proper development and maintenance of heart structures, and none of the other GRKs can compensate for its loss. A histological analysis of GRK2deficient embryos showed a pronounced hypoplasia of the ventricular myocardium and remarkable hypoplasia and dysplasia of the interventricular septum. These cardiac malformations are associated with impairment in heart functions.39 These results clearly demonstrated that myocardial contractility can be modulated by GRK2 activity.44 Recent studies were completed using transgenic mice lacking the GRK2 gene and cardiac GRK2-deleted (GRK2del) mice.45 GRK2 knockout mice showed a retardation and impairment in heart development and died. GRK2del mice, in which GRK2 expression is lower but not absent in the heart, were viable and showed unaltered cardiac structure and myocardial histology. These results suggest that GRK2 knockout mice represent a lethal phenotype because of the impairment of important extracardiac GRK2 functions. However, the hearts of GRK2del mice showed enhanced inotropic sensitivity after isoproterenol infusion and decreased acute tachyphylaxis during sustained isoproterenol stimulation.45 Mice with an endothelial-specific ablation of GRK2 (Tie2Cre-Grk2fl/f l) were viable but characterized by a marked vascular impairment. However, the viability of Tie2Cre-Grk2fl/f l mice further supports that the extravascular functions of GRK2 are involved in the lethality of GRK2 knockout mice.46 Several GPCRs, such as adrenergic, angiotensin, endothelin, and adenosine receptors, are expressed in cardiovascular tissues, and their deregulation is involved in the onset and progression of disorders that lead to heart failure (HF).43 This pathology can develop as a result of different disease conditions, including coronary artery disease, high blood pressure, and diabetes. Increased levels of GRK2 appear to be one of the first detectable alterations in myocytes after cardiac impairment or stress.47 Genetically engineered mice with a 50% reduction in GRK2 expression showed hyper-responsiveness to catecholamines and increased cardiac contractility.44 Kinase overexpression is related to reduced myocardial contractility and β-AR responsiveness, thus representing a possible precipitating factor for the onset of HF.48,49 Animal models of spontaneously hypertensive HF showed that a reduction in β-AR number was preceded by an up-regulation of GRK2.49 Studies performed on hearts derived from patients with HF showed reduced β1-AR density and function and an increase in GRK2 expression and activity.50,51 In addition, a reduction in myocardial contractility by approximately 70% occurred in the failing hearts after isoproterenol stimulation compared with control hearts. This weaker response was due to a decrease in β1-AR number.50 The increase in mRNA levels and the consequent increased protein expression of GRK2 are other factors related to the diminished response of failing hearts to β-receptor agonists. These consistent results should encourage researchers to develop selective GRK2-inhibitors to treat HF.50,51 HF animal models were used to evaluate the effects of a gene therapy based on the peptide GRK2ct, a C-terminal fragment of GRK2 corresponding to the Gβγ-binding domain of the kinase.52,53 GRK2ct competes with GRK2 for binding to the Gβγ subunit, thus blocking Gβγ-mediated membrane trasloca-

2. GRK2 FUNCTIONS AND INVOLVEMENT IN HUMAN DISEASES GRK2 is a prototypic GRK. This cytosolic protein is ubiquitously expressed in many tissues, but it is particularly important for embryonic development and heart function.7,39 GRK2 plays a key role in several signal transduction pathways. As previously described, this protein can trigger receptor desensitization and internalization through β-arrestin binding to activated GPCRs. GRK2 can also phosphorylate different effectors involved in signal transduction. Moreover, the expression and/or function of GRK2 is altered in several pathological conditions,5 including cardiovascular40 and inflammatory pathologies41 and cancer.42 For these reasons, a deep understanding of the molecular mechanisms that regulate GRK2 expression and function is required to modulate the altered kinase activity observed in pathological disorders.43 9279



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tion.54 In addition to cardiac β1-ARs, GRK2ct overexpression influences kinase interactions with other GPCRs.44,48 A recent study demonstrated that GRK2 is responsible for the desensitization of α2-ARs that are implicated in the feedback for catecholamine release from the adrenal gland. These receptor subtypes are dysregulated in HF models and contribute to increased levels of catecholamines.55,56 Patients with ischemic or idiopathic dilated cardiomyopathy,50 cardiac ischemia,57 and left ventricular hypertrophy49 showed increased left ventricular GRK2 mRNA and related activity. Additional studies indicated that increased GRK2 might be an important factor in the impairment of β-adrenergic-mediated vasodilatation, a characteristic of a hypertensive state.7 Furthermore, GRK2 is involved in the regulation of insulin signaling. Excess kinase levels are responsible for serious modifications in cardiac glucose uptake and insulin signaling in vivo and in vitro.58 GRK2 overexpression decreases insulin-stimulated GLUT4 translocation and thus works as an endogenous inhibitor of insulin-induced glucose transport. In contrast, the microinjection of GRK2 antibody into 3T3-L1 adipocytes did not alter the basal GLUT4 levels but did lead to an increase in GLUT4 translocation from the perinuclear cell region to the plasma membrane after cell stimulation with insulin.59 Insulin-stimulated GLUT4 translocation is inhibited by the binding of the GRK2 RGS domain and the Gαq/11 subunit, a protein responsible for the activation of other effectors. This interaction elicits downstream glucose transport stimulation.60 The involvement of the RGS domain in the regulation of insulin signaling suggests a phosphorylation-independent mechanism to control signal propagation. Evaluation of the insulin-stimulated glucose transport in cells expressing a GRK2 vector lacking the RGS domain showed that glucose transport stimulation was unchanged because Gαq/11 activity was not inhibited.59 Insulin administration increases GRK2 cellular levels within 15−30 min.61 Insulin-like growth factor 1 induces GRK2 cellular accumulation by inhibiting mouse double minute 2 (Mdm2), which is responsible for kinase ubiquitination and degradation.62 Chronic insulin treatments are characterized by increased GRK2 levels.47 Increased GRK2 expression was detected in tissue samples from animal models of obesity, human adipocytes, and blood mononuclear cells derived from patients with insulin resistance, which suggests that GRK2 can influence the development of insulin resistance.63 The relationship between obesity and the development of other important pathological conditions, including hyperinsulinemia, glucose intolerance, cardiovascular diseases, and type 2 diabetes, is well-known. The involvement of GRK2 in the regulation of metabolic functions and development of in vivo insulin resistance was supported by a recent study of the effects of high-fat-diet-dependent obesity and insulin resistance on an animal model of tamoxifen-induced GRK2 ablation. Ablation of GRK2 normalized parameters that were characteristic of an obese phenotype, including fasting glycemia, glucose tolerance and homeostasis, and body weight control.64 Overall, these data suggest that GRK2 inhibitors play a key role in the control of the insulin signaling pathway, body weight increase, fatty acid metabolism, and Raven’s syndrome.64 GRK2 is considered as both an intrinsic and extrinsic regulator of cell proliferation43 because it is involved in a network of functional interactions during cell cycle progression and regulates the correct transition from the G2 to M phase.65

Under normal conditions, GRK2 levels are controlled by cellcycle machinery and contribute to cell cycle progression and arrest in a receptor-independent manner. CDK2-mediated phosphorylation of the carboxyl-terminal Ser670 residue of GRK2 is responsible for the down-regulation of this kinase during the G2/M phase because it triggers the binding of GRK2 to prolyl isomerase, which results in degradation. Thus, inhibiting GRK2 phosphorylation at Ser670 prevents GRK2 down-regulation and delays cell cycle progression.43,66 GRK2 levels are critical for the control of cell proliferation. However, distinct results were obtained for the role of GRK2 involvement in different cancer types. In the case of thyroid cancer, GRK2 is up-regulated and reduces cell proliferation.67 Another study showed that the overexpression of GRK2 inhibited the growth of human hepatocellular carcinoma cells (HCC), and elevated GRK2 levels were associated with delayed cell cycle progression; thus, increasing GRK2 expression may be a valid approach for the treatment of human liver cancer.68 GRK2 may be an important factor in Kaposi’s sarcomaassociated herpes virus-induced tumors. Repressing GRK2 caused miR-K3 to promote cell migration and invasion via the activation of CXCR2/AKT signaling, whereas the overexpression of GRK2 significantly abolished miR-K3-induced cell migration.69 Additional studies suggest that specific inhibition of GRK2 increased the mass of malignant tumors, such as squamous cell carcinoma. The enhanced tumor growth was attributed to the activation of the growth-promoting MAPK pathway. Therefore, cardioprotection of the myocardium against death-promoting stimuli, which is conferred by MAPK activity, overlapped with tumor growth promotion.70 Riva et al.46 determined that GRK2 is a key angiogenesis regulator. Mouse embryos with systemic or endotheliumselective Grk2 ablation had marked vascular malformations. Thus, GRK2 down-regulation is a relevant event during the angiogenic switch of tumor development and enhances tumor progression. GRK2 is highly expressed in different cell types of the immune system. GRK2 phosphorylates chemokine and chemotactic receptors, which subsequently induce leukocyte accumulation in the inflamed area and the mobilization of T cells from lymphoid organs. The altered levels of GRK2 in immune cells are not well understood; thus, it is possible that various diseases may develop and respond differently based on the GRK2 changes.71,41 Early down-regulation of GRK2 in immune cells may be the potential mechanism for easy cell activation. In contrast, chronic kinase down-regulation produces a worsening of inflammation. In patients and animal models of rheumatoid arthritis, peripheral blood mononuclear cells showed a reduction in GRK2 protein expression and activity. This result suggests an important role for GRK2 in the regulation of immune cells and development of inflammatory diseases.72 GRK2 plays also a key role in autoimmune disorders. Reduced GRK2 expression was found in rat splenocytes from an experimental model of autoimmune encephalomyelitis73,74 and in both an animal model and patients with multiple sclerosis.75 In contrast, the GRK2 concentration was increased in neutrophils from patients with sepsis.5,76 It is well-known that vascular lesions are involved in the development of Alzheimer’s disease (AD).5 GRK2 may critically contribute to the pathogenesis of AD because it is highly expressed in cardiac, vascular, and cerebral tissues. Indeed, AD patients showed increased levels of GRK2 protein 9280



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and mRNA that correlated with the degree of cognitive impairment.77,78 GRK2 was up-regulated in the endothelial cells of AD patients and in a two-vessel carotid artery occlusion model that mimics cognitive and vascular damage in early AD. These observations suggest that GRK2 is overexpressed in impaired cellular compartments, such as mitochondria and damaged vessel wall cells, during the early stages of AD; therefore, this kinase could serve as an early marker of brain alterations.75,77 Major depressive disorders are characterized by the abnormal activity of GPCRs in the brain and blood cells.79,80 As a consequence, GRKs, which are responsible for GPCR regulation, are involved in the development of depression. A post-mortem analysis of depressed suicide victims showed that GRK2 levels were increased in the prefrontal cortex; however, antidepressant treatments restored the normal GRK2 values.81,82 Notably, GRK2 can be used as biomarker for antidepressant effects because its levels increase prior to clinical symptom improvement.5,79,80 In conclusion, GRK2-mediated modulation of various signaling pathways influences a wide range of cellular processes. Thus, selectively targeting GRK2 might represent an innovative therapeutic strategy to treat human pathologies in which GRK2 overexpression leads to dysregulated signaling pathways.83

Figure 2. Structural features of GRK2, reproduced with permission from Molecular Pharmacology (Thal, D. M.; Yeow, R. Y.; Schoenau, C.; Huber, J.; Tesmer, J. J., Molecular mechanism of selectivity among G protein-coupled receptor kinase 2 inhibitors) 2011, 80, 294−303;88 Copyright 2011 American Society for Pharmacology and Experimental Therapeutics.

3. GRK2 STRUCTURAL FEATURES The crystal structures of bovine GRK2 complexed with Gαq (PDB code 2BCJ84) and Gβγ (PDB codes 1OMW85 and 1YM786) subunits were reported. The crystal structures of GRK2 complexed with its inhibitors have also been published: GRK2-balanol (1) (PDB code 3KRX87), GRK2-CMPD101 (2a) (PDB code 3PVU88), GRK2-CMPD103A (2b) (PDB code 3PVW88), GRK2-RNA aptamer (3a) (PDB code 3UZT89), GRK2-paroxetine (4a) (PDB code 3V5W90), GRK2-CCG206584 (4d)-Gβγ (PDB code 4MK091), and GRK2-GSK180736A (5a) (PDB code 4PNK92). The KD typically adopts an “open conformation” that is presumed to be inactive because the binding site for ATP is not fully assembled. In contrast, the transition to a “closed conformation” activates the kinase. GRK2 inhibitors generally bind to an intermediate open/closed conformation.87 The general structure of the GRK family consists of a wellconserved central catalytic domain (∼270 amino acids), an Nterminal domain (∼185 amino acids), and a C-terminal domain of ranging from ∼100 to 230 amino acids.20,43 The three GRK2 domains, located on the vertices of a triangle, represent the ability of this kinase to block signal transmission by simultaneously binding to GPCRs and Gβγ and Gα subunits via different binding sites.85 The KD of GRK2 is composed of a small lobe or “N” (residues 186−272 and 496−513) and a large lobe or “C” (residues 273−475) and shows ∼32% sequence homology to PKA. The small lobe is characterized by six-stranded antiparallel β-sheets (β1−β5 and β10) and three α-helices (αB, αC, and αK). The large lobe is mainly formed by α-helices and four antiparallel β-strands (Figure 2). As shown in Figure 2, the ATP binding site is located at the interface of the two lobes in proximity to the binding site of polypeptide substrates.85,88 The structural elements that develop around the ATP binding site are the phosphate binding loop (P-loop), the α-C helix, the activation loop, and the hinge connecting the two lobes. These features are common to the catalytic sites of approximately 500 different

kinases. Therefore, small ATP-mimetic molecules are unable to differentiate among the various families and isoforms to produce a selective inhibition.88 The N-terminal domain is important for receptor recognition and anchoring to the intracellular membrane. In addition, this region contains an RH domain (∼120 amino acids) that creates specific interactions with Gαq proteins to block their association with the phospholipase C β effector.43 The RH domain consists of nine α-helices (amino acids 30−185) and two additional α-helices derived from a region between the kinase and PH domains (amino acids 513−547). The RH domain participates in interactions with both the kinase and the PH domains, thus playing an important role in the regulation of protein activity. The RH domain may function as an effector antagonist that prevents interactions between G proteins and GPCRs. Recent studies demonstrated the ability of the GRK2RH domain to attenuate the signaling of numerous receptors even in the absence of phosphorylation.11,33,93 Notably, both receptor phosphorylation- and nonphosphorylation-mediated mechanisms are important for signal attenuation.18,85 The Nterminal RH domain also participates in different interactions with both the small and large lobes of the KD to aid in unique KD conformations.88 The C-terminal domain is arranged in a seven-stranded antiparallel β-barrel and is capped with a C-terminal helix on one end, and it can contain a PH domain (from 553 to 661 residues) that is unique to GRK2 and GRK3. The PH domain has binding sites for phosphatidylinositol 4,5-bisphosphate and Gβγ subunits, with the latter binding to the PH domain and increasing the phosphorylation of activated-GPCRs and recruiting the GRK2 to the plasma membrane.43,85 Moreover, phosphorylation and protein−protein interactions can produce conformational changes of the RH or PH domains and alter catalytic activity.85,33 In summary, the RH and PH domains cooperate to control the intrinsic activity of the KD and promote the translocation 9281



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of the kinase from the cytosol to the plasma membrane, thus allowing its interaction with activated receptors.85

4. GRK2 INHIBITORS Over the past several decades, studies of complex network of signaling pathways involving GRK2 have strengthened the hypothesis that GRK2 can serve as a unique diagnostic marker and therapeutic target. In this context, a significant effort was made to develop selective GRK2 inhibitors. Our manuscript focuses on the most promising GRK2 inhibitors developed in recent years. Particular attention will be devoted to the description of their mechanism of action, structure−activity relationships, and main interactions with the target kinase. 4.1. Polyanions and Polycations. The first compounds examined for their ability to inhibit the phosphorylation of rhodopsin by GRK2 were polyanionic and polycationic compounds; however, the high charge of these compounds makes them inefficient at crossing the plasma membrane. Among all of the tested compounds, the most potent inhibitors were heparin and dextran sulfate with IC50 values of 0.15 μM. Good activity was also obtained with polyaspartic acid (IC50 = 1.3 μM),94 polyglutamic acid (IC50 = 2.0 μM),94 and inositol hexasulfate (IC50 = 13.5 μM).94 The sulfate moiety is important for the inhibition process. In comparison to the corresponding inositol phosphate (IC50 = 3600 μM),94 the anionic character of the sulfate group, with respect to the phosphate moiety, is fundamental for GRK2 inhibition.94 However, the most active inhibitor, i.e., heparin, targets other kinases, including casein kinase II (IC50 = 0.13 μg/mL)95 and low density lipoprotein receptor kinase II, which was completely inhibited with 0.7 μg/mL of heparin.96 4.2. Balanol. Compound 1 is a fungal metabolite synthesized by Verticillium balanoides that acts as a competitive inhibitor of ATP at the GRK2 KD.97 Compound 1 was tested as an inhibitor of various kinases, and competitive inhibition was demonstrated by overcoming the effects of compound 1 by increasing ATP concentrations.97 Compound 1 is cell permeable, and unlike polyanions, it is appropriate for the design of inhibitors to be used in vivo. Structurally, 1 is composed of four ring systems, named A−D, that are linked in a linear fashion. The 4-hydroxybenzoyl moiety (A ring) is connected to the hexahydroazepine ring (B ring) by an amide linkage. Ring B is connected to the benzophenone moiety (C and D rings) by an ester linkage (Figure 3).

Figure 4. Accommodation of the A−D rings of the inhibitor 1, in the various ATP subsites of the GRK2 binding site.

polyphosphate subsite. The D ring occupies a hydrophobic subsite.98 The plane on which the B ring lies is almost perpendicular to the plane of the A ring and the planes formed by the C and D rings.3 Under endogenous conditions, the early interaction of ATP triggers a conformational change from an “open” (inactive) to a “closed” (active) conformation, which allows the various structural elements of the substrate to be arranged in the different subsites. After the allocation of the structural elements of 1, which is a competitive ATP inhibitor, it inhibits GRK2 KD.87 Figure 4 highlights the interactions established by 1 in the catalytic site of GRK2: (i) two H-bonds between the hydroxyl group of the A ring and the hinge of the KD;98 (ii) an H-bond between the endocyclic NH of the B ring and the backbone carbonyl oxygen of Ala321, mimicking the H-bond formed by the 3′-OH of ATP;98 (iii) an H-bond between the amide linkage (between the A and B rings) and Ser334;98 two Hbonds between the C ring and the backbone NH of Gly203 in the P-loop and Asp335;98 and (iv) two H-bonds between the D ring and the side chain of Glu239 and the backbone amide of Gly201 in the P-loop.88,98 Notably, these H-bonds involve highly conserved residues among all AGC kinases; furthermore, the conformational flexibility of this structure confers the possibility for 1 to adapt to many KDs, thus explaining its lack of selectivity.88,98 When tested on various GRK isoforms using biotinylated tubulin dimers and a phosphorylation assay, 1 inhibited GRK2 at the nanomolar level (IC50 = 42 nM). A similar trend was observed against GRK3 (IC50 = 47 nM). 1 was less active on other isoforms (GRK1, IC50 = 340 nM; GRK4, IC50 = 260 nM; GRK5, IC50 = 160 nM; GRK6, IC50 = 490 nM; and GRK7, IC50 = 180 nM). Under similar conditions, 1 inhibited PKA with an IC50 of 110 nM.87 In a rhodopsin phosphorylation assay using bovine rod outer segments (bROS), 1 showed an IC50 of 35 nM toward GRK2. This value was similar to the results obtained by tubulin phosphorylation assay. Compound 1 showed less potency against GRK1 and GRK5 (IC50 of 4.1 μM and 440 nM, respectively) in the rhodopsin phosphorylation assay.88

Figure 3. Structure of 1.

Compound 1 is an ATP mimetic, and its four rings establish interactions with the ATP binding subsites (Figure 4). The A ring occupies the hydrophobic adenine subsite, which can accommodate one or two cyclic rings. The B ring occupies the ribose subsite, and the aromatic C ring binds to the 9282



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Figure 5. Structures of Takeda inhibitors 2a and 2b.

The crystal structures of 1 complexed with PKA3 and GRK287 showed a similar and largely extended conformation of the inhibitor in the active site that stabilized a “slightly more closed” conformation of the KD (the large lobe was rotated toward the small lobe by 4°). Additional assays clearly showed that 1 is a competitive ATP inhibitor at the KD of PKAα (3.9 nM),97 PKGα (1.6 nM),97 PKCα (6.4 nM),97 and PKCβII (1.8 nM).97 Therefore, an attempt to increase its selectivity toward the inhibition of specific GRK isoforms, and hopefully the sole inhibition of GRK2, could be achieved by designing less flexible molecules that adapt to only a single conformation of the catalytic site. 4.3. Takeda Inhibitors. Heterocyclic compounds 2a and 2b were discovered by Takeda Pharmaceutical Company in 2007. Similar to 1, their structures are formed by a four-ring system (A−D) connected in a linear manner (Figure 5). The crystal structures of 2a and 2b complexed with GRK2Gβγ88 showed different interactions in the catalytic site compared with 1. As shown in Figure 6, the A rings of both

carbonyl oxygen of the amide linkage (C and D rings) and the backbone amide nitrogens of Gly201, Phe202, and Gly203 (the same interactions established by the benzophenone moiety of balanol).88 The D ring represents the main difference in the structure of Takeda inhibitors and binds to the hydrophobic subsite through nonpolar interactions with Gly201, Phe202, Leu235, Glu239, Gly337, and Leu338. Notably, the side chain of Leu235 rotates and changes its orientation with respect to apo-GRK2 to accommodate the D ring.88 Analogous to 1, compounds 2a and 2b bind to GRK2 in an open, noncatalytic conformation, thus inducing a slight closure of the GRK2 KD (with the large lobe rotating toward the small lobe by 2.4° and 3.6°, respectively, versus 4° for compound 1). The degree of closure strictly correlates with the potency of the inhibitors.88 Compounds 2a and 2b were tested against GRK2 (Table 1) and showed IC50 values of 290 nM and 54 nM, respectively.88 Table 1. IC50 Values of 1, 2a, and 2b for GRK1, GRK2, and GRK588 IC50, nM compd

GRK2

GRK1

GRK5

2a 2b 1

290 ± 98 54 ± 14 35 ± 8

ni ni 4100 ± 600

ni ni 440 ± 150

Even if compound 1 is slightly more potent (IC50 value of 35 nM) than 2a and 2b, both compounds were inactive against GRK1 and GRK5 isoforms at any of the tested concentrations (up to 125 μM), thus demonstrating their selectivity toward GRK2.88 Compound 2a showed IC50 > 2000 nM when tested against PKA,99 which further highlights its selectivity toward the target kinase. The difference in the selectivity profile of 2a, 2b, and 1 toward GRK subfamilies can be explained by evaluating the structural differences of the C and D rings and their interactions with different kinases. The benzophenone moiety of 1 is characterized by oxygen-rich substituents that produce conformational changes in the active sites of different kinases. In contrast, the interactions generated by the C and D rings of 2a and 2b are mostly nonpolar and thus less able to interact with the KD residues of other GRK isoforms and different types of kinases.88 Compound 2a was used in a recent study to evaluate its effects on agonist-induced μ-opioid receptor (MOPr) desensitization in locus coeruleus neurons.100 In HEK 293 cells, 2a inhibited receptor phosphorylation at Ser375 and caused subsequent arrestin binding and internalization. This finding suggests the involvement of GRK2 in MOPr desensitization. Furthermore, compound 2a inhibited the desensitization of Gprotein-coupled inwardly rectifying potassium currents activated by opioids, which clearly demonstrates the involvement of GRK2 in this process.100

Figure 6. Binding of 2b in the GRK2 binding site.

inhibitors bind at the adenine subsite through an H-bond between the pyridine/pyrimidine N4 atom and the backbone amide nitrogen of Met274 in the hinge of the catalytic core.88 The substituted 1,2,4-triazole moiety binds in the ribose subsite, deeper than ribose, through an H-bond between the N9 atom and the side chain amino group of Lys220. Moreover, the 1,2,4-triazole participates in nonpolar interactions with the side chains of Ile197, Val205, Leu271, and Ser334.88 The C rings bind in the triphosphate subsite through H-bonds between the NH and the side chains of Asp335 and Lys220.88 Additional H-bonds are established between the 9283



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Figure 7. (a) General structure of RNA aptamers: (in red) 20-nucleotide (N20) central random region arranged in a loop structure; (in green) 13nucleotide stem structure with a central A-A mismatch; (in orange and brown) PstI and HindII restriction sites, respectively.104 (b) Structure of 3a.104 (c) Structure of 3b.104 (d) RNA sequences of 3a−d.89,104

4.4. RNA Aptamers. Aptamers are specific oligonucleotides selected from random sequence pools that bind to various targets, such as small molecules, proteins, nucleic acids, and cells.101 When an aptamer is identified as an inhibitor, it can be used as starting point for drug development by turning its chemical features into a small druglike molecule that retains its properties.102,103

Recently, the SELEX process was used to identify a specific RNA aptamer that bound and inhibited GRK2. Thus, the SELEX process is a powerful approach for identifying novel inhibitors.104 The identification of RNA aptamers was performed by designing an RNA library characterized by a region that included 20 random nucleotides in a loop structure (Figure 7a).104 This structure is bordered on both sides by 13 9284



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phosphate via Mg2+ and His280 and the phosphodiester backbone (C52).89 In conclusion, all of the structural studies on truncated aptamers of 3a established that the position of A51 is crucial for the binding of 3a because it strongly contributes to RNA affinity and selectivity.89 Aptamer 3a stabilizes a unique inactive conformation of the kinase, establishes multiple interactions within and outside the catalytic core, and remodels basic regions of the kinase for better interactions with its polyanionic phosphodiester backbone.89 This mechanism may also be representative of the interaction of polyanionic compounds (e.g., heparin) with the GRK2 protein.98 Thus, the aptamer structure represents a promising starting point for understanding GRK2 function inside cells and living organisms using plasmid DNA vectors for cell transfection. However, the main problem related to the use of RNA aptamer derivatives is the high molecular weight of compounds 3a−d with respect to the previously described GRK2 inhibitors 1, 2a, and 2b. These latter compounds, even if they are slightly less active toward GRK2, better match Lipinski’s rules, especially in terms of molecular weight; thus, these compounds are better druglike molecules suitable for further development. Recent studies attempted to convert aptamers into small molecule inhibitors by using the so-called aptamer displacement assay. The identified molecules should exhibit the same features of the aptamer but with improved bioavailability.104 4.5. Paroxetine and Derivatives. Compound 4a (Paxil, Figure 8) is an SSRI approved by the FDA to treat depression,

nucleotides that pair with the complementary bases of the antiparallel strand.104 The stem begins with two consecutive GU base pairs and includes a central A-A mismatch to reduce its stability. The stem is flanked by two restriction sites, i.e., 5′-Pst1 and 3′HindII, that correspond to restriction endonucleases obtained from Providencia startui and Haemophilus inf luenza, respectively. The stem is also flanked by single-stranded constant regions (C20 and C47) for primer hybridization during PCR amplification.104 Subsequently, an in vitro selection process of this library identified RNA aptamers that specifically bind and inhibit GRK2.104 Among all of the analyzed aptamers, RNA aptamer 3a bound the kinase with the highest affinity (KD = 78.2 ± 2.6 nM, Table 2).104 The high selectivity for GRK2 was demonstrated by the Table 2. Dissociation Constants (KD) and IC50 Values of the RNA Aptamers 3a−d toward GRK2 GRK2 compd 104

3a 3b104 3c89 3d89

KD, nM

IC50, nM

78.2 ± 2.6 101.2 ± 7.2 1.2 ± 0.6 35 ± 5

4.1 ± 1.2 nd 11 ± 4 220 ± 40

poor binding affinities (KD > 5000 nM) of 3a against PKA and Erk-2.104 In the rhodopsin phosphorylation assay, GRK2 kinase activity was strongly inhibited by compound 3a (IC50 = 4.1 ± 1.2 nM, Table 2).104 Thus, 3a is a more potent inhibitor than are 1, 2a, and 2b. When tested with GRK5, 3a showed a 20-fold reduction in activity (IC50 = 79.4 ± 20.4 nM),104 which highlights its affinity for GRK2. The structure of 3a (Figure 7b) is characterized by a change in the HindII restriction site sequence from GTTAAC to GCTAC. The stem region is similar to the parent RNA library.104 Compound 3a was also easily truncated by excluding the primer binding and restriction sites to produce a variant of 51 nucleotides named C13.51 (3b, Figure 7c). The latter showed KD = 101.2 ± 7.2 nM for GRK2 (Table 2), which is largely comparable to the KD of the full length aptamer 3a.104 To identify the regions of 3a involved in GRK2 binding, a series of truncations and modifications of the 3b variant of the aptamer were synthesized.89 The most interesting results were obtained with C13.28 (3c, Figure 7d), which was designed by shortening the terminal stem of the parent aptamer 3b to only four base pairs, and C13.18 (3d, Figure 7d), which lacks the terminal stem and two residues from the 5′ end of the 20 nucleotide variable region.89 The truncated forms inhibited GRK2 with IC50 values of 11 ± 4 nM (3c, Table 2) and 220 ± 40 nM (3d, Table 2).89 The crystal structure of the GRK2-3d complex89 allowed the identification of binding regions on the target. The aptamer binds in the cleft between the small and large lobes of the KD. Compound 3d mimics the binding of ATP in the GRK2 active site.104 The aptamer participates in key hydrophobic and electrostatic interactions with the catalytic core of the KD through a small portion of its DNA sequence (49-AUAC-52).89 Aptamer 3d makes a tight turn, thus protruding A51 into the KD so that its AMP moiety and the 5′-phosphate of U50 overlap with the AMP moiety and γ-phosphate of the endogenous substrate.89 Other key interactions occur between Asp335 and the U50

Figure 8. Structure of 4a.

obsessive-compulsive disorder, anxiety, and post-traumatic stress disorders. Compound 4a is a selective GRK2 inhibitor discovered through an aptamer displacement assay aimed at identifying small molecules that selectively target GRK2.90 The crystal structures of 4a complexed with GRK290,91 clearly showed the accommodation of the inhibitor in the KD (Figure 9). The dioxole moiety binds in the adenine subsite through an H-bond between one of the oxygens (mimicking the N1 atom of ATP) and the amide backbone nitrogen of Met274. Furthermore, the carbon of the benzodioxole ring and the backbone carbonyl oxygen of Asp272 in the hinge region establish an unusually low free energy carbon−oxygen Hbond.90,91 The bridging methylene participates in positive van der Waals interactions in the adenine subsite, which is decreased in desmethylene paroxetine (4c, Table 3).90 The piperidine moiety binds in the ribose subsite through a network of Hbonds. The endocyclic NH interacts with the carboxylic acid of Asp278, the carbonyl oxygen of Ala321, and the carboxamide of Asn322 through a water molecule. The fluorophenyl ring fits in a pocket formed by P-loop residues and by the side chains of Lys220 and Leu222 to produce 18 nonpolar interactions that further stabilize GRK2-paroxetine binding. As a consequence, 9285



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the fluoxetine groups showed a reduction of LV function, whereas the group treated with 4a showed improved LV function and structure, and several hallmarks of HF were either inhibited or reversed. In addition, these data clearly demonstrated that the inhibition of GRK2 by 4a improved cardiac function after myocardial infarction. Thus, 4a represents a new starting point for the development of novel GRK2 inhibitors.105 Additional in vivo studies performed by Tang et al.106 provided supplementary unexpected results on the efficacy of 4a. The compound was evaluated for its ability to modulate GRK2 expression in superior cervical ganglion (SCG) neurons using a rat model of limb ischemia−reperfusion (I/R) injury that is associated with complex regional pain syndrome type I.106 The I/R injury decreased GRK2 expression levels in the ipsilateral SCGs, whereas GRK2 levels in the contralateral SCGs were unaffected. The administration of compound 4a restored GRK2 expression in the ipsilateral SCGs within 14 days without affecting the contralateral side.106 In this animal model, GRK2 mRNA levels were decreased in both sides of the SCGs. The administration of compound 4a increased GRK2 levels in both sides within 1 day. These results showed that 4a normalized both altered mRNA levels and protein expression in I/R injury models, even if the duration of the drug administration varied. These data also show the involvement of GRK2 in neuropathic pain syndromes.106 Inhibitor 4a reduced sensory abnormalities, such as allodynia, by inducing GRK2 up-regulation in SCGs, but the mechanism by which 4a modulates these processes is still unclear.106 Among the 4a analogues that have been synthesized to study structure−activity relationships, another derivative, 4b (Table 3), consists of a benzodioxole nucleus replaced with a benzolactam ring.91 The aim of this substitution was to obtain a more potent inhibitor through the formation of a stronger Hbond with the hinge region compared with unconventional carbon−oxygen H-bonds.91 The crystal structure of the GRK24b-Gβγ complex91 clearly showed a highly conserved conformation of the active site that was quite similar to the GRK2-paroxetine-Gβγ complex. Consequently, the main interactions described for 4a are maintained between GRK2 and 4b, with the exception of Met274, which rotates to avoid a steric clash with the benzolactam carbonyl.91 A phosphorylation assay of light-activated bROS showed no significant changes in the affinity of 4b for any GRKs compared with 4a. The benzolactam inhibitor is less selective because it more strongly inhibits PKA and PKC (IC50 values of 2.6 μM and 26 μM, respectively) compared to 4a (IC50 values of 45 μM for PKA and 220 μM for PKC).91 These results clearly indicate that the presence of groups with conventional H-bonds (i.e., NH) increases the activity toward other AGC family kinases and decreases the selectivity toward GRK isoforms. This feature worsens the inhibitory profile of the new derivatives.91 Indazole/dihydropyrimidine compounds were developed to select new scaffolds that maintain the good pharmacokinetic properties and high selectivity of 4a.92 A library of known kinase inhibitors assembled by the Structural Genomics Consortium at Oxford University was screened against GRK2 and GRK5 isoforms.92 Only eight compounds inhibited GRK2, five of which showed a structural similarity to 4a. These five compounds are characterized by three ring systems: an indazole ring, a dihydropyrimidine/dihydropyridone ring, and a variously decorated phenyl ring (Table 4).92

Figure 9. Accommodation of 4a in the various ATP subsites of the GRK2 binding site.90,91

Table 3. Derivatives 4b−d and Related IC50 Values for GRK2-Mediated Phosphorylation of Bovine Rhodopsin

GRK2 IC50, μM 4b91 4c90 4d90

6.3 63.1 316.2

the loss of fluorine is detrimental for kinase inhibition, which is the case for defluoro paroxetine (4d, Table 3).90,91 Crystallographic analysis showed that 4a fits in the active site of GRK2 and stabilizes a unique and atypical conformation of the kinase due to a reorganization of 3.5° after inhibitor binding. This reorganization misaligns the small and large lobes, unlike the effects from the previously described compounds. Thus, 4a represents a unique scaffold for the design of selective GRK2 inhibitors.90 Compound 4a was also tested for its ability to inhibit GRK2mediated phosphorylation of light-activated rhodopsin in rod outer segment (ROS) membranes. In the presence of 5 μM ATP, compound 4a inhibited the kinase with an IC50 of 19.9 μM,90 whereas a 16- and 13-fold lower potency toward GRK1 (IC50 = 316.2 μM)90 and GRK5 (IC50 = 251.1 μM), respectively, was observed.90 In a tubulin phosphorylation assay, 4a showed an 8-fold more potent inhibition of GRK2 phosphorylation with an IC50 of 2.5 μM.90 An improved selectivity toward the target kinase was observed over the other GRK isoforms. Compound 4a was 63 and 50 times more potent toward GRK2 compared with GRK1 (IC50 = 158.4 μM)90 and GRK5 (IC50 = 125.8 μM), respectively.90 Compound 4a inhibits GPCR phosphorylation by GRK2 both in vitro and in living cells with a 60-fold selectivity over other GRK isoforms (10-fold over PKA and 40fold over PKC).90 In vivo studies with 4a were performed by treating wild-type mice 2 weeks after a myocardial infarction. The treated mice were compared with mice treated with fluoxetine, which does not inhibit GRK2. All mice initially exhibited similar left ventricular (LV) dysfunction. After treatment, the control and 9286



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Table 4. Structures and Activities of Derivatives 5a−e92

The crystal structure of GRK2 complexed with the most active inhibitor, 5a, was used to clarify its binding and structure−activity relationships.92 Compound 5a binds in the active site of the KD in a manner similar to 4a.92 As shown in Figure 10, the indazole ring binds to the adenine subsite through two H-bonds with the hinge residues backbone atoms, which is similar to the binding pattern of the benzodioxole ring of 4a.92 The amide linker established a H-bond with Ser334, while the dihydropyrimidine ring formed an H-bond with the backbone carbonyl of Arg199 in the P-loop and van der Waals interactions with residues in the large lobe.92 The fluorophenyl group binds in a pocket formed by the P-loop and the side chain of active site Lys220.92

In the tubulin phosphorylation assay, 5a was the most potent inhibitor toward GRK2 (IC50 = 0.25 μM)92 among all of the tested compounds (see Table 4) and showed improved activity toward the parent compound 4a (IC50 = 1.3 μM under these assay conditions).92 Inhibitor 5a also showed a good selectivity toward GRK2 compared with 4a. Compound 5a was 4000 and 400 times more potent toward GRK2 over GRK1 and GRK5, respectively.92 Uniquely, 4a showed a reduced selectivity because it was only 306 and 97 times more active toward GRK2 compared with GRK1 and GRK5, respectively.92 4.6. Peptides. Another class of GRK2 inhibitors is represented by peptides, which were first designed based on the knowledge that the main function of GRK2ct peptide, the 9287



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Table 5. Synthetic Peptides 6a−d Derived from the First Intracellular Loop of the Hamster β2-AR and Their IC50 Values for GRK2 Inhibition108 GRK2

peptidea

sequence

6a, 56−74 6b, 57−71 6c, 59−69 6d, 60−66

TAIAKFERLQTVTNYFITS AIAKFERLQTVTNYF AKFERLQTVTN KFERLQT

IC50, μM 40 62 1600 2600

The numbers correspond to the first and last position of the protein sequence. a

GRK2. The four residues appear to be critical for inhibitory activity (A and I from the N-terminus; Y and F from the Cterminus). In fact, their removal results in decreased activity (IC50 of 62 μM for 6b and 1600 μM for 6c).108 For this reason, the short sequence of 6c was considered as the inactive core region of the peptide, and subsequent studies attempted to increase peptide/GRK2 interactions through several structural modifications.109 Peptide 6a, which was the most potent inhibitor, was used as the peptide prototype for the development of new inhibitors. The introduction of charged residues (specifically glutamate or lysine) at the N-terminus and/or the C-terminus, together with the truncation of the prototype sequence, was used to evaluate whether these modifications could influence peptide/GRK2 interactions. The truncated forms were used to clarify which peptide end was responsible for the activity. The synthesized peptides 7a−f are shown in Table 6 with their IC50 values (rhodopsin phosphorylation assay) for three GRK isoforms.109 The addition of polar amino acids at both ends of the prototype sequence produced an increased solubility. The new peptides showed a similar or higher potency, and a 9-fold increase in activity was observed for 7b (IC50 = 4.4 μM).109 To study the structure−activity relationship of peptide 7b and evaluate the influence of the C-terminal glutamate and Nterminal lysine, two different peptides, 7d and 7f, were synthesized. The resulting IC50 values suggest that the removal of the N-terminal lysine produces an increase in activity (i.e., 7f, IC50 = 0.6 μM), while the absence of the C-terminal glutamate leads to a slight decrease of potency (7d, IC50 = 5.6 μM) vs 7b.109 To check the selectivity toward GRK2, the peptides were tested for their activity toward GRK3 and GRK5 isoforms. Compound 7f showed the lowest selectivity with IC50 values for GRK3 and GRK5 in the low micromolar range (Table 6).109 Peptide 7f acts as a noncompetitive inhibitor with respect to ATP. In fact, peptides do not interact with the KD of GRK2.108 Instead, peptides establish only one contact point with the kinase by mimicking the first intracellular loop of the receptor.109 Peptide 7f was tested on the human A431 cell line that expresses endogenous β2-AR. Compound 7f inhibited agonistinduced β2-AR desensitization and enhanced receptor signaling via GRK2 inhibition.109 These results support the use of peptide inhibitors as a treatment for heart disease by increasing β-adrenergic signaling pathway in cardiomyocytes. This mechanism is analogous to the effects of cardiac GRK2ct overexpression via gene transfer, which is not currently approved in humans.107 Transgenic mice with the myocardium-targeted expression of a GRK2-specific peptide inhibitor (GRK-Inh, 7g) were used to

Figure 10. Accommodation of 5a in the various ATP subsites of the GRK2 binding site.92

C-terminal fragment of GRK2, is to inhibit endogenous GRK2.54 Transgenic mice with cardiac-restricted overexpression of either the peptide inhibitor GRK2ct or β2-AR were mated to create two different genetic models of murine HF (MLP−/−): MLP−/−/GRK2ct, and MLP−/−/β2-AR, respectively.52 In the MLP−/−/β2-AR model, the cardiac overexpression of the β2-AR did not influence the development of the dilated cardiomyopathy phenotype. In contrast, the overexpression of GRK2ct in the MLP−/−/GRK2ct model was responsible for cardiac-targeted GRK2 inhibition and reduction of β1-AR desensitization, which prevented a worsening of cardiac function.52 Moreover, monitoring of the cardiac function during aging showed a positive effect due to GRK2ct overexpression compared to the MLP−/−/β2-AR model.52 Therefore, GRK2 inhibition could offer a novel therapeutic approach in HF by restoring normal β-AR signaling and avoiding chronic desensitization.52 Another animal model of HF was generated by mating transgenic mice overexpressing the peptide inhibitor GRK2ct with transgenic mice overexpressing the sarcoplasmic reticulum Ca21-binding protein, calsequestrin (CSQ).107 Cardiac overexpression of CSQ was used to better understand how GRK2 inhibition improves cardiac functionality and survival in transgenic mice with aggressive dilated cardiomyopathy.107 The results showed that GRK2 inhibition improved cardiac dilation, cardiac functionality, and survival in a state of severe cardiomyopathy, and it restored the normal percentage of β-AR compared with CSQ and wild-type mice.107 However, gene therapy is not currently approved in humans, and GRK2ct, due to its size (∼200 amino acids), is not suitable for clinical use. Thus, the design and development of shorter peptides are needed.54 To identify the β-AR portions involved in the interaction with GRK2, several synthetic peptides consisting of the intraand extracellular domains of hamster β2-AR were tested as potential GRK2 inhibitors.108 Four of these inhibitors were derived from the first intracellular loop of β2-AR: β2-AR 56−74 (6a), β2-AR 57−71 (6b), β2-AR 59−69 (6c), and β2-AR 60−66 (6d). The sequences of these inhibitors are shown in Table 5. Among the four inhibitors, 6a is the longest and most potent GRK2 inhibitor.108 The IC50 values reported in Table 5 clearly show that the truncated derivatives from both ends are less able to inhibit 9288



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Table 6. New Synthetic Peptide Inhibitors and Their Respective IC50 Values for GRK2 and Recombinant GRK3 and GRK5 Inhibition109 IC50, μM peptide

sequence

GRK2

GRK3

GRK5

7a (K56−74K) 7b (K56−74E) 7c (E56−74K) 7d (K56−69) 7e (59−74K) 7f (59−74E)

KTAIAKFERLQTVTNYFITSK KTAIAKFERLQTVTNYFITSE ETAIAKFERLQTVTNYFITSK KTAIAKFERLQTVTN AKFERLQTVTNYFITSK AKFERLQTVTNYFITSE

36.5 4.4 52.2 5.6 32.2 0.6

30.4 6.1 22.6 11.6 55.5 1.5

61.3 3.9 7.4 2.1 9.4 1.6

the exception of the N-terminal acylation at Gly1, selectively inhibited GRK2 in vitro with an inhibition of 47.6% and 49.6%, respectively.9 On the basis of these results, a head-to-tail cyclization was completed to obtain more stable and selective compounds. Peptides GLLRrHS (10a) and GLLRrHSI (10b) were subsequently identified.8 A second group of peptides was designed based on the GRK2 crystal structure. Given that the side chain of K383 points toward Ser389, a side chain-to-side chain cyclization was attempted to further stabilize the peptide. This method resulted in peptides KLLRrHD (10c) and [KLLRrHD]I (10d), which contain the original Lys383 in place of Gly1 and Asp in place of Ser7, respectively.8 When tested in the rhodopsin phosphorylation assay, the most interesting results were obtained with peptides 9b and 10d, which showed IC50 values of 0.34 μM and 0.12 μM, respectively.8 Both compounds displayed a good selectivity toward GRK2, based on their poor inhibition of GRK5 when tested in the rhodopsin phosphorylation assay (Table 7).8

evaluate the cardioprotective effects of GRK2 inhibition. Compound 7g is a small peptide derived from the first intracellular loop of β2-AR and has a peptide sequence of MAKFERLQTVTNYFITSE.70 The inhibition of GRK2 activity by 7g resulted in a cardioprotective effect due to an increased activation of the MAPK signaling pathway, which oversees the processes of cell growth and proliferation. Thus, treatment with 7g prevented cardiomyocyte apoptosis and the consequent impairment of heart function.70 An analysis of heart tissue showed that transgenic mice overexpressing RAF kinase inhibitor protein (RKIP), which is responsible for the inhibition of GRK2 and MAPK pathways, revealed cardiomyocyte apoptosis, heart dilatation, cardiac lipid overload, and upregulation of lipid metabolism genes.70,110 In contrast, an analysis of C57BL/6J mice hearts expressing 7g showed less cardiomyocyte apoptosis than did mice overexpressing RKIP; moreover, there was no evidence of cardiac dilatation and lipid overload. These data demonstrate the cardioprotective effect from selective GRK2 inhibition rather than the simultaneous inhibition of GRK2 and MAPK cascades.70 GRK2 inhibition by 7g blocked the typical metabolic dysfunctions of late-stage HF.111 Transgenic models with a myocardium-targeted expression of fatty-acid synthase (FASN) were used to reproduce the energetic substrate of HF patients. As a result, FASN was up-regulated.110,111 Endogenous fatty acid β-oxidation under basal conditions showed enhanced oxidation levels in transgenic mice when compared with controls. These results indicated an enhanced substrate availability of cardiomyocytes with available energy for the heart muscle.111 However, FASN up-regulation was also responsible for palmitate accumulation and inhibition of the MAPK cascade,112 which led to cardiomyocyte apoptosis and a worsening of heart functionality. GRK2 inhibition prevented cardiac lipid accumulation and reduced FASN levels, thus delaying HF symptoms.111 In addition to peptides derived from the first intracellular loop of the hamster β2-AR, small peptides derived from GRK2/ 3 KD were tested in animal models of diabetes to investigate the treatments for type 2 diabetes.113 GRK2 inhibition can lead to the restoration of signaling pathways involved in glucose homeostasis control.59,61,63,114 Acylated glycine derivatives of short peptides, such as myristyl GLLRrHS (8a, KRX-683107) and lauryl GLLRrHSI (8b, KRX683124), reduced blood glucose levels in animal models by inhibiting GRK2/3.113 The peptide fragments of these inhibitors are quite similar to the catalytic fragment 383−390 KLLRGHSP of GRK2, which is the last part of an α-helix (residues 383−386) and the first part of a β-strand (residues 387−390).8 Furthermore, 1 μM peptides GLLRrHS (9a) and GLLRrHSI (9b), whose sequences are identical to those of 8a and 8b, with

Table 7. Peptide Inhibitor Structure and Percentage of GRK2/5 Inhibition in a Rhodopsin Phosphorylation Assay8 % inhibition peptide

GRK2

GRK5

8a 8b 9a 9b 10a 10b 10c 10d

nd nd 47.6 49.6 47.8 37.2 36.3 55.3

nd nd

G-Protein-Coupled Receptor Kinase 2 (GRK2) Inhibitors: Current

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