Gene therapy is a novel approach to treat and prevent diseases by changing the expression of target genes. Recently, this technique has been moved from the laboratory research to translational clinical trials for many diseases, such as severe combined immune deficiency, hemophilia, cancer, chronic granulomatous disorder, and neurodegenerative diseases (36).

For CVD, gene therapy has recently been proposed (37–39) mainly for the treatment of coronary artery disease, HF, and arrhythmias (40). In particular, gene therapy targets for HF are sarcoendoplasmic reticulum calcium-ATPase 2a (SERCA2a) and stromal-derived factor-1 (SDF-1), which are the actual objects of ongoing clinical trials (41, 42).

In addition to these targets, it has been demonstrated that several other genes are effective in the treatment of HF in animal models of disease. In this context, gene therapy has been used in preclinical studies to target GRK2 on the plasma membrane and to avoid βAR desensitization. This is achieved through the expression of βARKct that is mediated by the adenovirus (AD) or adeno-associated virus (AAV). βARKct resembles the carboxy-terminal domain of GRK2 that is responsible for its translocation to the plasma membrane and its binding to Gβγ. This strategy is effective in several models of CVD. Raake and colleagues used adeno-associated virus serotype 6 (AAV6) to express βARKct in a porcine model of HF (43). The Authors found that the long-term βARKct expression induced a significant amelioration of left ventricular hemodynamics and contractile function in pigs with HF compared to controls, which showed an impaired cardiac function.

The ventricular delivery of Adeno-βARKct in failing hearts of rabbits, using coronary catheterization, reversed ventricular dysfunction (44). These findings support the idea that gene therapy with βARKct could become an effective therapeutic strategy for HF. Viral vectors are commonly used for cardiovascular applications, including AD and AAV, which can infect non-dividing cells and transduce heart with good efficiency. Differences between the two DNA viruses regard the limited amount of DNA that AAVs can carry, and the high inflammatory response of the ADs, which limits the time of expression of the transgene. Therefore, limitations that prevent the use of AAV expressing βARKct in humans have still to be overcome. First of all, it is not completely known the full range of effects that viruses expressing βARKct can exert on GRK2 and also on other intracellular signalings. Indeed, it is known that βARKct is able to displace GRK2 from plasma membrane allowing its translocation to other compartments (29). In lipopolysaccharide (LPS)-treated macrophages, the adenoviral-mediated gene transfer of βARKct maintains macrophage functionality by inducing an earlier localization of GRK2 to mitochondria (29). Indeed, βARKct also blocks βγ signaling (45) and prevents cellular responses to important extracellular stimulants. These findings clearly demonstrate that βARKct, besides GRK2 inhibition in the plasma membrane, exerts multiple effects within the cell. Indeed, through the interaction with Gβγ, βARKct also inhibits Gβγ signaling that is involved in the regulation of cell proliferation and survival. Moreover, βARKct, by displacing GRK2 from the plasma membrane, allows the kinase to accumulate in other cellular compartments, where GRK2 can interfere with many other cellular functions. Therefore, it is likely that βARKct-based gene therapy in humans could provoke several side effects.

A recent study shows that paroxetine, the selective serotonin reuptake inhibitor (SSRI), can inhibit GRK2 activity (46, 47). Thal and colleagues show that paroxetine binds the active site of GRK2 stabilizing the kinase domain in a novel conformation (46). Both in vitro in isolated cardiomyocytes and in vivo in mice, pretreatment with paroxetine potentiates isoproterenol effects on βAR-mediated contractility (46). Moreover, in wild-type mice with myocardial infarction, paroxetine significantly improves cardiac function (47). Paroxetine seems to be an efficient inhibitor of GRK2 with selectivity over other GRKs even if it is still unknown its selectivity over other kinases and its side effects in vivo in other tissues. A major limitation for the use of this drug is the very high dosage at which it is effective to inhibit the kinase. Indeed, the effective doses exceed those approved for the use of paroxetine in humans, making unavoidable effects on the central nervous system. It is most likely that paroxetine will never be used in humans for the treatment of cardiac dysfunction in HF.

Gallein is a novel small molecule that selectively blocks Gβγ-binding interactions, including the one with GRK2. It has been shown that gallein reduces the recruitment of GRK2 on the plasma membrane and enhances contractility in isolated adult mouse cardiomyocytes in response to a βAR agonist (48). In a mouse model of HF due to isoproterenol injections, the treatment with gallein prevents HF and reduces GRK2 expression (48). These data suggest that gallein could be a promising therapeutic drug for the treatment of HF. However, gallein is a specific inhibitor of Gβγ rather than GRK2. Thus, it is likely that this molecule affects other intracellular signalings like βARKct.

Since it has been shown that the Regulator of G Protein Signaling (RGS) domain of GRK2 interacts with Gαq and inhibits it in vitro, transgenic mice with cardiac-specific expression of the RGS domain of GRK2 have been generated and subjected to cardiac damage in response to pressure overload. These mice show less hypertrophy and less adverse structural remodeling compared with controls (49). In this case, it appears that the beneficial effect is more on Gαq inhibition rather than on GRK2 inhibition. These data confirm previous works of the group of Gerard Dorn, who was the first to exploit Gαq as a mechanism of cardiac hypertrophy (50). Thus, RGS domain of GRK2 could be used as a prototype for the development of effective drugs to prevent cardiac hypertrophy.

RNA aptamers have been developed to inhibit GRK2 through systematic evolution of ligands by exponential enrichment (SELEX). Among them, C13 binds GRK2 with a high affinity and inhibits GRK2-dependent rhodopsin phosphorylation in vitro (51). C13 can stabilize GRK2 in an inactive conformation through multiple interactions in the active site pocket of the kinase domain (52). In particular, the positioning of an adenine nucleotide in the ATP-binding pocket and the interactions with the basic αF–αG helicoidal regions of the GRK2 kinase domain are mainly involved in the kinase inhibition. The use of aptamers is limited to in vitro studies but could be converted into small inhibitors through an aptamer-displacement assay (53). Thus, this approach could be potentially transferred to the clinical scenario, even if further studies are necessary to reach this aim.

Raf kinase inhibitor protein modulates several key intracellular signaling, including the signaling cascades of ERK, NFκB, glycogen synthase kinase-3β (54–56). It has been shown that RKIP is also a physiological inhibitor of GRK2 (57). After the activation of G protein-coupled receptors, RKIP dissociates from Raf-1 to associate with GRK2. This switch is due to RKIP dimerization (58) that is regulated by PKC-mediated phosphorylation at Ser-153 (57). RKIP binds GRK2 in the amino-terminal domain. In cardiomyocytes, the downregulation of RKIP inhibits beta-adrenergic signaling and contractile activity (57). This evidence suggests that this physiological mechanism of inhibition of GRK2 could be useful for the treatment of CVD. However, the enthusiasm of this discovery is cooled by the poor selectivity of this small protein on kinase activity since RKIP also affects several intracellular signaling pathways.

The design and the synthesis of peptide-based compounds have spread in the last decade (59). The use of peptides as therapeutic drugs has some limitations, including the parenteral route of administration since peptides are not well absorbed in the gastrointestinal tract. Moreover, peptides do not usually cross plasma membrane and are rapidly metabolized by proteolytic enzymes. However, compared to synthetic small molecules, peptides are less toxic, more selective, and they do not accumulate in organs. Their rapid degradation makes them less harmful, and their degradation products are simply amino acids and should not have toxic effects (60). Considering these advantages, it is not surprising that there are many peptide-based drugs available on the market (59), such as receptor agonists and antagonists, peptide hormones and analogs, and HIV protease inhibitors (61). Several peptide inhibitors of GRK2 have been developed, modeled on the structure of the kinase. It has been demonstrated that the inhibition of GRK2 by GRKInh, a peptide inhibitor of the kinase (62), could counteract the dysfunctional metabolism of HF in a transgenic model of myocardium-specific expression of fatty acid synthase (FASN) (63). Anis et al. (64) demonstrated that myristyl or lauryl glycine derivatives of short peptides derived from the HJ loop of GRK2, KRX-683₁₀₇, and KRX-683₁₂₄ are potent inhibitors of the kinase and exert hypoglycemic effects in animal models of type 2 diabetes. We have recently found that non-acylated derivatives of KRX-683₁₀₇ and KRX-683₁₂₄ (peptides 2 and 3) selectively inhibit GRK2 in vitro (65). In vivo in hypertensive rats, the infusion of peptide 3 for 30 days ameliorates GRK2-dependent insulin resistance and IRS1 tyrosine phosphorylation (66). Moreover, the intracardiac injection of this peptide reduces phenylephrine- or hypertension-induced left ventricular hypertrophy (24). Thus, it is likely that this peptide could be effective to ameliorate the cardiac morphology and function in the failing heart.

To reach better results, cyclic peptides have recently been designed. These are polypeptide chains taking cyclic ring structure by linking the two ends of the peptide with an amide bond or other chemically stable bonds. Cyclic peptides have a better biological activity compared to their linear counterparts due to their conformational rigidity (67), which allows a selective binding with their targets. Moreover, their cyclic structure makes peptides resistant to hydrolysis by exopeptidases (due to the lack of both amino and carboxyl termini) and endopeptidases (since the structure is less flexible than linear peptides). Some cyclic peptides can autonomously cross plasma membrane, thus avoiding the need of a vehicle for internalization, such as HIV-1 Tat protein and Penetratin, which increases peptide size.

In particular, cyclic compounds have been designed, which are modeled on the conformation of the HJ loop within the X-ray structure of GRK and are based on the structure of the above-described KRX-683₁₂₄. One of these compounds, the cyclic peptide 7, can inhibit GRK2 activity and is more active than its linear precursor. In cultured cells, this peptide confirms its potentiality and specificity as a GRK2 inhibitor (68). Thus, this cyclic peptide has a great potentiality to be translated to clinical trials.

The regulation of the balance between GRK2 degradation and its synthesis could be an effective approach to reducing GRK2 levels in several diseases (69, 70) (Figure 3). The degradation of GRK2, for instance, that occurs via the proteasome (69) can be prevented by its interaction with Hsp90, which stabilizes the correct folding of the protein (71). Thus, NMR analysis of this complex could help to develop new inhibitors that can reduce the interaction between HSP90 and GRK2, leading to kinase degradation in pathological conditions.

The regulation of GRK2 localization within the cell could be a useful target for diseases. Indeed, it is emerging that GRK2 exerts different effects within the cell, which depend on its localization, cell type, stimuli, and physiopathological context (28, 33, 34, 72). In particular, several stressors increase the levels of GRK2 in mitochondria, in an ERK- and HSP90-dependent mechanism (73). The effects of such accumulation are still the object of investigation since opposite results in the literature show either a protective mechanism (28, 29, 74) or the acceleration of unfavorable processes (73). Nevertheless, given the established notion that the accumulation of GRK2 in plasma membrane inhibits GPCR signaling or its binding with cytosolic substrates activates pro-death signaling, the possibility to modulate GRK2 accumulation within specific organelles might in the future pose the strategy to regulate kinase effects in pathological conditions (Figure 4).