6-Substituted purines as ROCK inhibitors with anti-metastatic activity

Rho-associated serine/threonine kinases (ROCKs) are principal regulators of the actin cytoskeleton that regulate the contractility, shape, motility, and invasion of cells. We explored the relationships between structure and anti-ROCK2 activity in a group of purine derivatives substituted at the C6 atom by piperidin-1-yl or azepan-1-yl groups. Structure-activity relationship (SAR) analyses suggested that anti-ROCK activity is retained, and may be further increased, by substitution of the parent compounds at the C2 atom or by expansion of the C6 side chain. These inhibitors of ROCK can reach effective concentrations within cells, as demonstrated by a decrease in phosphorylation of the ROCK target MLC, and by inhibition of the ROCK-dependent invasion of melanoma cells in the collagen matrix. Our study may be useful for further optimization of C6-substituted purine inhibitors of ROCKs and of other sensitive kinases identified by the screening of a broad panel of protein kinases.     

Adenosine-oligoarginine conjugate, a novel bisubstrate inhibitor, effectively dissociates the actin cytoskeleton

Aberrant regulation of protein kinases impairs normal cellular functioning and may lead to disease. The protein kinase involved in the regulation of the dynamics of the actin cytoskeleton, Rho-kinase (ROCK), phosphorylates various substrates (e.g. myosin light chain, myosin phosphatase), causing the formation of actin fibers and tension inside cells. Hyperactivation of ROCK, for example, causes hypertension and cardiovascular disorders. Thus, the design of highly specific protein kinase inhibitors is of the utmost importance. To date, the majority of inhibitors investigated have been found to mimic and compete with ATP. However, in the present study we characterized the cellular effects of a novel bisubstrate inhibitor -- adenosine-oligoarginine conjugate (ARC) -- designed to interfere simultaneously with the ATP site and the substrate-binding pocket of basophilic kinases. ARC effectively pulled down ROCK from cell lysates, showed no cytotoxicity and suppressed the assembly of the actin cytoskeleton (especially central actin bundles) as the result of interference with the activity of the kinase. Combination of ARC with chloroquine yielded a stronger inhibitory effect and gave results similar to treatment with Y-27632. However, treatment with ARC produced more actin fragments and yielded a longer-lasting effect than treatment with Y-27632. Additionally, quantification of phosphorylated myosin light chain levels in ARC-treated or Y-27632-treated cells implies that ARC is more effective than Y-27632 in suppressing the phosphorylation of at least one of the substrates of ROCK. We believe that the described bisubstrate strategy could be a useful lead for designing novel, highly specific inhibitors for different protein kinases.     

Rho kinase and hypertension

Arterial hypertension is a multifactorial disease that is characterised by increased peripheral vascular resistance often accompanied by smooth muscle cell hypertrophy and proliferation. Rho kinases (ROCKs) are the most extensively studied effectors of the small G-protein RhoA and abnormalities in RhoA/ROCK signalling have been observed in various cardiovascular disease including hypertension. The RhoA/ROCK-pathway is a key player in different smooth muscle cell functions including contractility, proliferation and migration. Furthermore, there is extensive crosstalk between RhoA/ROCK- and NO-signalling. Therefore, not only ROCK inhibitors but also NO-donators or pleiotropic agents like statins exert their beneficial effects on the cardiovascular system at least in part via Rho/Rho-kinase.     

Rho-kinase: important new therapeutic target in cardiovascular diseases

Rho-kinase (ROCKs) belongs to the family of serine/threonine kinases and is an important downstream effector of the small GTP-binding protein RhoA. There are two isoforms of Rho-kinase, ROCK1 and ROCK2, and they have different functions with ROCK1 for circulating inflammatory cells and ROCK2 for vascular smooth muscle cells. It has been demonstrated that the RhoA/Rho-kinase pathway plays an important role in various fundamental cellular functions, including contraction, motility, proliferation, and apoptosis, leading to the development of cardiovascular disease. The important role of Rho-kinase in vivo has been demonstrated in the pathogenesis of vasospasm, arteriosclerosis, ischemia-reperfusion injury, hypertension, pulmonary hypertension, stroke, and heart failure. Furthermore, the beneficial effects of fasudil, a selective Rho-kinase inhibitor, have been demonstrated for the treatment of several cardiovascular diseases in humans. Thus the Rho-kinase pathway is an important new therapeutic target in cardiovascular medicine.     

Applications for ROCK kinase inhibition

ROCK kinases, which play central roles in the organization of the actin cytoskeleton, are tantalizing targets for the treatment of human diseases. Deletion of ROCK I in mice revealed a role in the pathophysiological responses to high blood pressure, and validated ROCK inhibition for the treatment of specific types of cardiovascular disease. To date, the only ROCK inhibitor employed clinically in humans is fasudil, which has been used safely in Japan since 1995 for the treatment of cerebral vasospasm. Clinical trials, mostly focusing on the cardiovascular system, have uncovered beneficial effects of fasudil for additional indications. Intriguing recent findings also suggest significant potential for ROCK inhibitors in the production and implantation of stem cells for disease therapies.     

Regulation of the actin cytoskeleton by rho kinase controls antigen presentation by CD1d

CD1d molecules are MHC class I-like molecules that present lipid Ags to NKT cells. Although we have previously shown that several different cell signaling molecules can play a role in the control of Ag presentation by CD1d, a defined mechanism by which a cell signaling pathway regulates CD1d function has been unclear. In the current study, we have found that the Rho kinases, Rho-associated, coiled-coil containing protein kinase (ROCK)1 and ROCK2, negatively regulate both human and mouse CD1d-mediated Ag presentation. Inhibition of ROCK pharmacologically, through specific ROCK1 and ROCK2 short hairpin RNA, or by using dendritic cells generated from ROCK1-deficient mice all resulted in enhanced CD1d-mediated Ag presentation compared with controls. ROCK regulates the actin cytoskeleton by phosphorylating LIM kinase, which, in turn, phosphorylates cofilin, prohibiting actin fiber depolymerization. Treatment of APCs with the actin filament depolymerizing agent, cytochalasin D, as well as knockdown of LIM kinase by short hairpin RNA, resulted in enhanced Ag presentation to NKT cells by CD1d, consistent with our ROCK inhibition data. Therefore, our overall results reveal a model whereby CD1d-mediated Ag presentation is negatively regulated by ROCK via its effects on the actin cytoskeleton.     

ROCK与动脉粥样硬化

Rho相关的卷曲螺旋型蛋白激酶（Rho—associated,coiled-coil containing protein kinase,ROCK）是ras同源家族RhoA（ras homolog family memberA）T游的靶蛋白之一,主要功能是调节肌动蛋白细胞骨架的活动,如细胞粘附、细胞运动、细胞迁移及细胞收缩。实验及临床研究表明R0cK可能与多种心血管疾病如高血压、肺动脉高压、动脉粥样硬化以及脑血管疾病有着很大相关性。此篇综述将总结近期对于RhoA／ROCK通路在调控血管功能中的关键作用并探讨其对于动脉粥样硬化相关疾病的潜在治疗价值。     

Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle

The potent vasodilator action of cyclic GMP-dependent protein kinase (cGK) involves decreasing the Ca(2+) sensitivity of contraction of smooth muscle via stimulation of myosin light chain phosphatase through unknown mechanisms (Wu, X., Somlyo, A. V., and Somlyo, A. P. (1996) Biochem. Biophys. Res. Commun. 220, 658-663). Myosin light chain phosphatase activity is controlled by the small GTPase RhoA and its target Rho kinase. Here we demonstrate cGMP effects mediated by cGK that inhibit RhoA-dependent Ca(2+) sensitization of contraction of blood vessels and actin cytoskeleton organization in cultured vascular myocytes. Ca(2+) sensitization and actin organization were inhibited by both 8-bromo-cGMP and sodium nitroprusside (SNP). SNP also caused translocation of activated RhoA from the membrane to the cytosol. SNP-induced actin disassembly was lost in vascular myocytes in culture after successive passages but was restored by transfection of cells with cGK I. Furthermore, cGK phosphorylated RhoA in vitro, and addition of cGK I inhibited RhoA-induced Ca(2+) sensitization in permeabilized smooth muscle. 8-Bromo-cGMP-induced actin disassembly was inhibited in vascular myocytes expressing RhoA(Ala-188), a mutant that could not be phosphorylated. Collectively, these results indicate that cGK phosphorylates and inhibits RhoA and suggest that the consequent inhibition of RhoA-induced Ca(2+) sensitization and actin cytoskeleton organization contributes to the vasodilator action of nitric oxide.     

Rho GTPase/Rho kinase negatively regulates endothelial nitric oxide synthase phosphorylation through the inhibition of protein kinase B/Akt in human endothelial cells

Endothelial nitric oxide synthase (eNOS) is an important regulator of cardiovascular homeostasis by production of nitric oxide (NO) from vascular endothelial cells. It can be activated by protein kinase B (PKB)/Akt via phosphorylation at Ser-1177. We are interested in the role of Rho GTPase/Rho kinase (ROCK) pathway in regulation of eNOS expression and activation. Using adenovirus-mediated gene transfer in human umbilical vein endothelial cells (HUVECs), we show here that both active RhoA and ROCK not only downregulate eNOS gene expression as reported previously but also inhibit eNOS phosphorylation at Ser-1177 and cellular NO production with concomitant suppression of PKB activation. Moreover, coexpression of a constitutive active form of PKB restores the phosphorylation but not gene expression of eNOS in the presence of active RhoA. Furthermore, we show that thrombin inhibits eNOS phosphorylation, as well as expression via Rho/ROCK pathway. Expression of the active PKB reverses eNOS phosphorylation but has no effect on downregulation of eNOS expression induced by thrombin. Taken together, these data demonstrate that Rho/ROCK pathway negatively regulates eNOS phosphorylation through inhibition of PKB, whereas it downregulates eNOS expression independent of PKB.     

ROCK and PRK-2 mediate the inhibitory effect of Y-27632 on polyglutamine aggregation

Polyglutamine expansion in huntingtin (Htt) and the androgen receptor (AR) causes untreatable neurodegenerative diseases. Y-27632, a therapeutic lead, reduces Htt and AR aggregation in cultured cells, and Htt-induced neurodegeneration in Drosophila. Y-27632 inhibits both Rho-associated kinases ROCK and PRK-2, making its precise intracellular target uncertain. Over-expression of either kinase increases Htt and AR aggregation. Three ROCK inhibitors (Y-27632, HA-1077, and H-1152P), and a specific ROCK inhibitory peptide reduce polyglutamine protein aggregation, as does knockdown of ROCK or PRK-2 by RNAi. RNAi also indicates that each kinase is required for the inhibitory effects of Y-27632 to manifest fully. These two actin regulatory kinases are thus involved in polyglutamine aggregation, and their simultaneous inhibition may be an important therapeutic goal.     

Nitric Oxide–Induced F-Actin Disassembly Is Mediated via cGMP, cAMP, and Protein Kinase A Activation in Rat Mesangial Cells

Glomerular mesangial cells contain actin and myosin, and in analogy to vascular smooth muscle cells, they can contract and relax to regulate the glomerular filtration rate. A key molecule that determines hemodynamic properties is nitric oxide, which is produced by nitric oxide synthase isoenzymes located in individual cells of the kidney. The contractility of mesangial cells is based on the interaction of actin microfilament bundles (F-actin) with myosin. We had the notion that nitric oxide influences the shape change of mesangial cells, so we analyzed the signal transduction involved. Chemically unrelated nitric oxide donors induced F-actin dissolution, which was mediated by cGMP but was unrelated to protein kinase G activation. Actin disassembly was achieved with inhibitors of phosphodiesterase-3 and -4 or forskolin-evoked cAMP generation. We assumed that signal transmission involves activation of protein kinase A, and we went on to attenuate F-actin disassembly by protein kinase A inhibition. In conclusion, we found evidence that nitric oxide triggered F-actin dissolution via cGMP generation, inhibition of cAMP-hydrolyzing phosphodiesterase-3, and subsequent protein kinase A activation.     

Nitric oxide-induced F-actin disassembly is mediated via cGMP, cAMP, and protein kinase A activation in rat mesangial cells.

Glomerular mesangial cells contain actin and myosin, and in analogy to vascular smooth muscle cells, they can contract and relax to regulate the glomerular filtration rate. A key molecule that determines hemodynamic properties is nitric oxide, which is produced by nitric oxide synthase isoenzymes located in individual cells of the kidney. The contractility of mesangial cells is based on the interaction of actin microfilament bundles (F-actin) with myosin. We had the notion that nitric oxide influences the shape change of mesangial cells, so we analyzed the signal transduction involved. Chemically unrelated nitric oxide donors induced F-actin dissolution, which was mediated by cGMP but was unrelated to protein kinase G activation. Actin disassembly was achieved with inhibitors of phosphodiesterase-3 and -4 or forskolin-evoked cAMP generation. We assumed that signal transmission involves activation of protein kinase A, and we went on to attenuate F-actin disassembly by protein kinase A inhibition. In conclusion, we found evidence that nitric oxide triggered F-actin dissolution via cGMP generation, inhibition of cAMP-hydrolyzing phosphodiesterase-3, and subsequent protein kinase A activation.     

Determination of potential selective inhibitors for ROCKI and ROCKII isoforms with molecular modeling techniques: structure based docking,ADMET and molecular dynamics simulation

Rho-associated protein kinases (ROCKs) are a member of the serine/threonine protein kinase family and potential therapeutic target for various diseases. This enzyme has two isoforms, Rho-associated protein kinase I (ROCKI) and Rho-associated protein kinase II (ROCKII). They share an overall 65% homology in all amino acid sequence and 92% homology in kinase domains. Since, the kinase domains of ROCKI and ROCKII are highly conserved and similar, the discovery and design of isoform-selective inhibitors are more challenging. Thus, most currently available agents that is against ROCKs exhibit low selectivity and severe side effects. Therefore, this study aimed to elucidate the interaction of compounds that indicated high potential in experimental studies against ROCKI and ROCKII enzymes in the molecular level with molecular modeling techniques. Firstly, we determined the interaction property of catalytic sites of the ROCKs by analyzing with molecular docking. Based on these results, the best ligands (50 compounds) corresponding to experimental studies were selected, and then absorption, distribution, metabolism and excretion – toxicity (ADMET) analysis of these compounds were implemented. According to these study results, the compound 40 for ROCKI and the compound 50 for ROCKII were identified as selective and highly potent inhibitors. And finally, molecular dynamics (MD) simulations were performed for the stability of ROCKs with identified compounds. In the light of this study, it will be possible to treat diseases that ROCKs have a role by developing more effective and specific ROCK inhibitors.

Communicated by Ramaswamy H. Sarma     

Rho kinases regulate corneal epithelial wound healing

We have previously shown that Rho small GTPase is required for modulating both cell migration and proliferation through cytoskeleton reorganization and focal adhesion formation in response to wounding. In the present study, we investigated the role of Rho kinases (ROCKs), major effectors of Rho GTPase, in mediating corneal epithelial wound healing. Both ROCK 1 and 2 were expressed and activated in THCE cells, an SV40-immortalized human corneal epithelial cell (HCEC) line, in response to wounding, lysophosphatidic acid, and heparin-binding EGF-like growth factor (HB-EGF) stimulations. The ROCK inhibitor Y-27632 efficiently antagonized ROCK activities without affecting Rho activation in wounded HCECs. Y-27632 promoted basal and HB-EGF-enhanced scratch wound healing and enhanced cell migration and adhesion to matrices, while retarded HB-EGF induced cell proliferation. E-cadherin- and beta-catenin-mediated cell-cell junction and actin cytoskeleton organization were disrupted by Y-27632. Y-27632 impaired the formation and maintenance of tight junction barriers indicated by decreased trans-epithelial resistance and disrupted occludin staining. We conclude that ROCK activities enhance cell proliferation, promote epithelial differentiation, but negatively modulate cell migration and cell adhesion and therefore play a role in regulating corneal epithelial wound healing.     

Rho-Associated Protein Kinases Play an Important Role in the Differentiation of Rat Adipose-Derived Stromal Cells into Cardiomyocytes In Vitro

Adipose-derived stromal cells (ADSCs) represent a readily available abundant supply of mesenchymal stem cells and have the ability to differentiate into cardiomyocytes in mice and human, making ADSCs a promising source of cardiomyocytes for transplantation. However, there has been no report of differentiation of rat ADSCs into cardiomyocytes. In addition, signaling pathways in the differentiation process from ADSCs to cardiomyocytes are unknown. In this study, we first demonstrated that rat ADSCs spontaneously differentiated into cardiomyocytes in vitro, when cultured on a complete medium formulation MethoCult GF M3534. These differentiated cells possessed cardiomyocyte phenotype and expressed cardiac markers. Moreover, these cells showed open excitation-contracting coupling and Ca²⁺ transient and contracted spontaneously. The role of Rho-associated protein kinases (ROCKs) in the differentiation process was then studied by using ROCK-specific inhibitor Y-27632 and ROCK siRNAs. These agents changed the arrangement of cytoskeleton and diminished appearance of cardiomyocyte phenotype, accompanied by inhibition of c-Jun N-terminal kinase (JNK) phosphorylation and promotion of Akt phosphorylation. Collectively, this is the first study to demonstrate that rat ADSCs could spontaneously differentiate into cardiomyocytes in vitro and ROCKs play an important role in the differentiation of ADSCs into beating cardiomyocytes in conjunction of the PI3K/Akt pathway and the JNK pathway.     

Platelet-derived growth factor-BB induces matrix metalloproteinase-2 expression and rat vascular smooth muscle cell migration via ROCK and ERK/p38 MAPK pathways

Matrix metalloproteinases (MMP) play a pivotal role in the pathogenesis of cardiovascular diseases. Their expressions are altered in response to a variety of stimuli, including growth factors, inflammatory markers, and cytokines. In this study, we demonstrated that platelet-derived growth factor-BB (PDGF-BB) induces a dose- and time-dependent increase in MMP-2 expression in rat vascular smooth muscle cells (VSMC). Treatment with either the Rho-associated protein kinase (ROCK) inhibitor Y-27632 or suppression of ROCK-1/2 by small interfering RNA technology significantly reduced the MMP-2 expression, thus suggesting that ROCK regulates such expression. Similar results were observed when VSMC were pretreated with either U0126 or SB203580, which are selective inhibitors of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase, respectively, thus suggesting that these kinases are important for the induction of MMP-2 expression by PDGF-BB. In conclusion, these results described a novel mechanism in atherosclerosis through PDGF-BB signaling in VSMC, in which MMP-2 expression is induced via extracellular signal-regulated kinases and p38 mitogen-activated protein kinase phosphorylation, as well as ROCK.     

Dynamic compression of chondrocytes induces a Rho kinase-dependent reorganization of the actin cytoskeleton

The signal transduction mechanisms in chondrocytes that recognize applied forces and elicit the appropriate biochemical cellular responses are not well characterized. A current theory is that the actin cytoskeleton provides an intracellular framework onto which mechanosensation mechanisms are assembled. The actin cytoskeleton is linked to the extracellular matrix at multi-protein complexes called focal adhesions, and evidence exists that focal adhesions mediate the conversion of external physical forces into appropriate biochemical signal transduction events. The Rho GTPases affect the arrangement of actin cytoskeletal structures, and enhance the formation of focal adhesions, which link the cytoskeleton to the extracellular matrix. A major effector pathway downstream of Rho is the activation of Rho kinase (ROCK), which phosphorylates and activates Lim kinase, which in turn phosphorylates and inhibits the actin-depolymerizing protein cofilin. The objectives of this study were threefold: first, to quantify the actin reorganization in response to dynamic compression of agarose-embedded chondrocytes. Second, to test whether Rho kinase is required for the actin cytoskeletal reorganization induced by dynamic compression. Third, to test whether dynamic compression alters the intracellular localization of Rho kinase and actin remodeling proteins in chondrocytes. Dynamic compression of agarose-embedded chondrocytes induced actin cytoskeletal remodeling causing a significant increase in punctate F-actin structures. Rho kinase activity was required for these cytoskeletal changes. Dynamic compression increased the amount of phosphorylated Rho kinase. The chemokine CCL20 and inducible nitric oxide synthase (iNOS) were the most highly upregulated genes by dynamic compression and this response was reduced by the Rho kinase inhibitors. In conclusion, we show that dynamic compression induces changes in the actin cytoskeleton of agarose-embedded chondrocytes, and we establish methodology to quantify these changes. Furthermore, we show that Rho kinase activity is required for this actin reorganization and gene expression induced by dynamic compression.     

Recent development in pleiotropic effects of statins on cardiovascular disease through regulation of transforming growth factor-beta superfamily

Background

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors, also known as statins, are a drug class that reduce the level of cholesterol in the blood. As a result, statins are used to suppress the progression of cardiovascular disease. Evidence points to another component of statins involving the non-lipid effects of the drug class in preventing cardiovascular disease. One specific mediator of this action is the transforming growth factor β (TGF-β) superfamily. The TGF-β superfamily consists of proteins that include TGF-β and bone morphogenetic proteins (BMPs). These proteins regulate cellular pathways to mediate effects including immunomodulation, cell cycling, and angiogenesis. One pathway that mediates these effects is Ras. Moreover, within this pathway, different functions are possible depending on the activation of the specific receptor subtype. This review discusses the recent development of the non-lipid effects of statins in preventing cardiovascular disease progression by regulating Ras pathway of the TGF-β superfamily, especially RhoA/ROCK pathway.

Methods

A systematic PubMed database search of all English-language articles up to 2011 was conducted using the following terms: statin, TGF-β, Ras, ROCK, GGPP, inducible nitric oxide synthase, endothelial nitric oxide synthase, actin filament formation, PPARγ, MMP-2, and human trials.

Conclusion

With better understanding of the pathway, various mediators were identified; some of these mediators are important biomarkers producing more specific and accurate assessment of the pleiotropic effects of statins. The review of human trials also highlights that more specific biomarkers are employed in recent studies, and the non-lipid effects on human subjects are more accurately documented. Confirmation of the accuracy of these biomarkers by further large-scale studies and further development of new biomarkers may prove an important path leading to better patient selection for treatment, and thus better cost-effectiveness may be achieved.