Elsevier

Drug Discovery Today

Volume 25, Issue 2, February 2020, Pages 446-455
Drug Discovery Today

Review
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ROCK-2-selective targeting and its therapeutic outcomes

https://doi.org/10.1016/j.drudis.2019.11.017Get rights and content

Highlights

  • Rho-kinase-2 is an important therapeutic target.

  • ROCK-2 plays a pivotal role in the pathogenesis of vascular, neuronal disorders and cancer.

  • ROCK-2 selective targeting produces better therapeutic outcomes.

  • ROCK-2 specific targeting is a better alternative over non-specific inhibitors.

Despite the identification of distinct isoforms of Rho-kinase (ROCK-1 and ROCK-2), their isoform-specific roles in several disorders remain obscure. Recent studies have revealed a vital role of ROCK-2 in various vascular and neuronal disorders, where the potential for disease alleviation is wider with ROCK-2-selective targeting than with nonspecific ROCK inhibition. This approach is also crucial for resolving issues of safety and specificity associated with nonspecific ROCK inhibitors. In this review, we focus on the latest developments concerning ROCK-2 as a therapeutic target and justify the clinical use of ROCK-2-selective inhibitors.

Introduction

The Rho-associated coiled-coil containing protein kinases (ROCKs/Rho-kinase/Rho-associated kinase) are downstream effectors of the small GTPase Rho (Rho A, Rho B, Rho C, and Rho E) and belong to the family of serine/threonine kinases. The active GTP bound form of Rho mediates several biological functions through the action of ROCKs, including smooth muscle contractions, cell motility [1], and cytokinesis [2]. The ROCK proteins were identified in 1996 as proteins that bind to Rho GTPase. Two proteins were independently isolated as p160 and p164 3, 4. Later, these were recognised as ROCK-1 and ROCK-2, respectively and as isoforms of the Rho-associated kinase [5].

The two isoforms, ROCK-1 (ROCK-β/p160) and ROCK-2 (ROCK-α/p164), share 92% similarity in their amino acid sequence [5]. Their structure comprises an N-terminally located catalytic kinase domain, followed by a coiled-coil containing region (∼600 amino acids) with a Rho-binding domain and a pleckstrin homology (PH) domain at the C terminus [3] (Fig. 1). They have varying locations, and distinct physiological roles have been identified for each. The ROCK-1 transcript (gene located on chromosome 18) is ubiquitous, with more prominent expression in liver, kidney, spleen, testis [5], thymus, and blood corpuscles [6], whereas ROCK-2 mRNA (chromosome 2) is expressed more abundantly in skeletal muscles and brain [5[, suggesting that they have specialized roles in these locations.

Genetic approaches, such as the use of transgenic animals with specific ROCK deletion (ROCK-1–/– or ROCK-2–/–) and RNA interference (RNAi) techniques, have helped uncover distinct functions of each isoform, such as ROCK-1 activity during apoptosis [7], eyelid and ventral body wall closure in neonates [8], stress fibre formation [9], and diabetic vascular injury [10], and ROCK-2-mediated negative regulation of insulin signalling [11], cytoskeletal reorganisation [12], blood coagulation, placental development [13],and adipogenesis [14]. Several other studies also recognised ROCK-1 and -2-specific cellular functions, such as the reorganisation of actin cytoskeleton [15], development of cell surface protrusions, cellular elongation [16], keratinocyte differentiation [17], and stress fibre formation [18].

The two isoforms vary in their intracellular locations and activation. ROCK-2 is predominantly found in the cytoplasm [19]. Activated Rho A binding recruits the protein from the cytosol to the membrane, from where it exerts its functions via actin reorganisation [20]. ROCK-1 is primarily centrosome bound [21]. ROCK-2 is also found within the nucleus, where it phosphorylates acetyltransferase and increases its activity [22]. The ROCK proteins are self-suppressed and are activated either by the binding of GTP-bound Rho to the Rho-binding domain, which induces a conformational change, removing the negative regulation and freeing the kinase activity [4], or via cleavage of the autoinhibitory domains, which results in constitutively active enzymes. ROCK-1 cleavage is mediated by caspases [23], whereas ROCK-2 is activated by granzyme B-mediated cleavage [24] (Fig. 1). ROCKs are also activated by arachidonic acid independent of Rho [25]. The activated ROCK then phosphorylates myosin light chain kinase (MLC), which results in smooth muscle contractions [1], and also myosin phosphatase [26], inactivating it. This inactivation prevents the dephosphorylation of MLC, thereby prolonging muscle contraction [27].

Subsequent studies showed that the two proteins also vary in their inactivation processes. Inactivation is mediated via small endogenous GTP-binding proteins (Rad and Gem). Rad negatively regulates ROCK-2, whereas Gem inhibits ROCK-1-induced responses [28]. In addition, Riento et al. revealed a RhoE phosphorylation pathway mediated by ROCK-1 without the involvement of ROCK-2, demonstrating, for the first time, that the two isoforms might have separate targets [29]. A negative feedback loop has also been identified in which the phosphorylated RhoE GTPase is involved in the inactivation of ROCK-1 but not of ROCK-2 [9]. ROCK inactivation and/or inhibition promotes several biological processes, including bone formation [30], neuronal morphogenesis 31, 32, and keratinocyte differentiation [33], while also attenuating hypoxia-induced angiogenesis [34]. These functions, mediated via ROCK inhibition, are significant and form the basis for the development of ROCK inhibitors.

ROCK inhibitors have been considered for use in numerous diseases, such as cerebral ischaemia [35], hypertension 36, 37, erectile dysfunction [38], glaucoma [39], osteoporosis [30], cardiac hypertrophy [40], diabetic cardiomyopathy 41, 42, retinopathy [43], pulmonary hypertension [34], and atherosclerosis [44]. However, their implementation is limited because of a lack of knowledge regarding the involvement of the particular ROCK isoform. Whether isoform-specific targeting or combined ROCK inhibition would provide a better therapeutic outcome is yet to be verified.

Despite their incomplete specificity towards the ROCK isoforms as well as other serine/threonine kinases, such as PRK2, PKC, cAMP-dependent protein kinase, and citron kinase [45], some nonspecific ROCK inhibitors have shown promising results in certain pathological states, such as glaucoma and hypertension. However, further work is required for isoform-selective ROCK inhibitors to be of clinical use, although several research-based studies have helped resolve much ambiguity over ROCK-1 and ROCK-2-specific functions. Thus, understanding the discreet functions of each isoform in a particular disorder could help resolve issues of safety and specificity and expand the therapeutic applications of ROCK inhibitors.

Section snippets

Therapeutic relevance of ROCK-2

The involvement of ROCK in cardiovascular and neurological pathology has been established and reviewed elsewhere 46, 47. However, the specific isoforms involved have remained obscure. Several studies have now shown that ROCK-2 modulation could influence disease progression in these spheres as well as in oncology.

Current developmental status of ROCK-2 inhibitors

So far, two nonspecific ROCK inhibitors, fasudil and ripasudil, have been approved for human use. Fasudil was approved in 1995 in Japan for the prevention and treatment of cerebral vasospasm [124] and is currently being considered for use in hypertension and other cardiovascular disorders. Ripasudil was also approved in Japan in 2014 for the treatment of glaucoma and ocular hypertension [125]. Y-27632 is another nonspecific ROCK inhibitor that is being studied intensively with regard to various

Concluding remarks

Here, we have summarised recent developments concerning the physiological and pathological roles of ROCK-2 and its variant ROCK-2m, with an emphasis on its possible role as a target in the range of diseases in which ROCK-2 regulation might prove beneficial. Despite experimental evidence clearly pointing towards the significance of ROCK-2 as a promising therapeutic target in several disorders, there remains room for further discovery in other diseases where the role of ROCK has been established

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