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CARDIOVASCULAR
Departments of Investigational Biology (C.D., R.B., R.W.), Vascular Biology and Thrombosis (D.J.B., R.L., Z.C., C.W., E.H.), Urology (E.G.), Drug Metabolism (L.J.), and Medicinal Chemistry (R.S., J.P.M., D.L.), Centres of Excellence for Cardiovascular Urogenital Drug Discovery; Department of Chemistry (M.B., T.P.), Centre of Excellence for Neurology Drug Discovery; and Departments of Gene Expression and Protein Biochemistry (S.K., T.Y., R.K., E.D.), Screening & Compound Profiling (L.L.W., G.K.S.) and Chemistry (D.J.), Discovery Research, GlaxoSmithKline Pharmaceuticals Inc., King of Prussia, Pennsylvania
Received July 11, 2006; accepted October 2, 2006.
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Abstract |
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. Furthermore, both SB-772077-B and GSK269962A induced vasorelaxation in preconstricted rat aorta with an IC50 of 39 and 35 nM, respectively. Oral administration of either GSK269962A or SB-772077-B produced a profound dose-dependent reduction of systemic blood pressure in spontaneously hypertensive rats. At doses of 1, 3, and 30 mg/kg, both compounds induced a reduction in blood pressure of approximately 10, 20, and 50 mm Hg. In addition, administration of SB-772077-B also dramatically lowered blood pressure in DOCA salt-induced hypertensive rats. SB-772077-B and GSK269962A represent a novel class of ROCK inhibitors that have profound effects in the vasculature and may enable us to further evaluate the potential beneficial effects of ROCK inhibition in animal models of cardiovascular as well as other chronic diseases.
). Two isoforms, ROCK1 and ROCK2, have been identified (Ishizaki et al., 1996; Leung et al., 1996; Matsui et al., 1996). They share significant sequence homology in the kinase domain (>90%), whereas the regulatory domains at the C terminus show significant divergence (Riento and Ridley, 2003; Rikitake and Liao, 2005). Both are ubiquitously expressed in various human and rodent tissues. Mouse gene knockout (KO) studies suggest that ROCK1 and ROCK2 have nonoverlapping functions. The ROCK1 KO results in developmental abnormalities in the eyelids, whereas the ROCK2 KO was associated with placenta defects and failed to develop to term (Thumkeo et al., 2003; Shimizu et al., 2005).
ROCK activities play pivotal roles in modulating tonic smooth muscle contraction in various tissues, including blood vessels, bronchial trachea, intestine, carvenosum, and bladder (Leung et al., 1996; Wang et al., 2002; Wibberley et al., 2003). It is a major player in enhancing the calcium sensitivity of smooth muscle by phosphorylating the myosin binding subunit of myosin light chain phosphatase (MYPT), leading to increased phosphorylation of myosin light chain at lower intracellular calcium concentrations, thus maintaining tonic smooth muscle contraction (Amano et al., 2000; Somlyo and Somlyo, 2003). In addition to its role in smooth muscle contraction, ROCK may also participate in diverse biological functions by phosphorylating numerous substrates, such as adducin, ezrin-radixin-moesin, collagpin-response mediator protein 2 and sodium hydrogen exchanger 1, thereby affecting a wide range of cellular activities (Shimokawa, 2002; Riento and Ridley, 2003; Hu and Lee, 2005). The mechanisms associated with these ROCK-mediated biological functions are just beginning to be elucidated.
Increased ROCK activity plays an important role in the blood pressure regulation and the development of hypertension. For example, enhanced ROCK activity, as evidenced by increased ROCK substrate (MYPT) phosphorylation, was observed in spontaneously hypertensive rat (SHR), DOCA salt-induced hypertensive rat (DOCA rat), and other rodent hypertension models (Seko et al., 2003). SHR and DOCA salt-induced hypertensive rats represent two subclass of hypertension: high renin (SHR) and low renin (DOCA) form (Pinto et al., 1998). The enhanced ROCK activity in both of these models suggests that this is a universal feature of the vasoconstrictive state observed in hypertension. Two widely employed ROCK inhibitors, fasudil and Y-27632, have provided preliminary but compelling evidence supporting the potential benefits of ROCK inhibition in animal disease models and in clinical trials. For example, administration of Y-27632 reduced blood pressure in SHR and other models of hypertension (Uehata et al., 1997), improved cardiac function in heart failure (Satoh et al., 2003), and suppressed lesion formation in a model of atherosclerosis (Mallat et al., 2003). However, given the limited potency and kinase specificity for these two compounds (Davies et al., 2000), additional ROCK inhibitors with novel structures and improved potency and selectivity may provide better tools to further evaluate the role of ROCK in various biological functions. Here we describe aminofurazan-based compounds that are novel and potent ROCK inhibitors. Our results demonstrate a good correlation between the potency of ROCK enzyme inhibition with the efficacy of smooth muscle relaxation. These aminofurazan-based ROCK inhibitors also inhibited cytokine production in macrophages. In addition, we further demonstrated that these inhibitors had vasodilatory activity and lowered blood pressure in SHR and DOCA salt-treated hypertensive rats.
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Materials and Methods |
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and IL-6 ELISA kits were from Roche Molecular Biochemicals, Inc. (Indianapolis, IN). Human primary aortic smooth muscle cells were purchased from Cambrex Bio Science (Walkersville, MD). Phenylephrine and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Rhodamine phalloidin was obtained from Invitrogen (Carlsbad, CA). Nitrocellulose membrane for Northern blot was purchased from Schleicher and Schuell Inc. (Keene, NH).
Expression of Recombinant ROCK1 Kinase Domain. Human ROCK1 (amino acid 3–543) was expressed in baculovirus in Sf9 cells. A histidine tag was incorporated at the N terminus. Affinity and conventional chromatography procedures were used to purify the protein to homogeneity. Purified protein was used for subsequent kinase assay. A separate article describing the details of the recombinant expression, purification, and characterization of human ROCK1 has been published elsewhere (Khandekar et al., 2005).
ROCK Enzyme Assays. The enzyme activity and kinetics of the purified ROCK1(3–543) were determined using scintillation proximity assay. In this assay, purified ROCK1 was incubated with peptide substrate (Biotin-Ahx-AKRRLSSLRA-CONH2, purchased from Synpep, Dublin, CA), and 33ATP (Redivue; GE Healthcare, Little Chalfont, Buckinghamshire, UK) and the subsequent incorporation of 33P into the peptide was quantified by streptavidin bead capture.
For IC50 determination, test compounds were dissolved at 10 mM in 100% dimethyl sulfoxide, with subsequent serial dilution in 100% dimethyl sulfoxide. Compounds were typically assayed over an 11-point dilution range with a concentration in the assay of 10 µM to 0.2 nM in 3-fold dilutions. For dose-response curves, data were normalized and expressed as percentage inhibition using the formula 100 x [(U – C1)/(C2 – C1)], where U is the unknown value, C1 is the average of the high signal (0%) control wells, and C2 is the average of the low signal (100%) control wells. Curve fitting was performed with the following equation: y = A + [(B – A)/{1 + (10 x/10 C) D}], where A is the minimal response, B is the maximal response, C is the log10(IC50), D is the Hill slope, and means exponential function. The results for each compound were recorded as pIC50 values (–C in the above equation).
Assays for determining inhibitory activity were performed in opaque, white-walled, 384-well plates in a total assay volume of 10 µl. The assays contained 1 nM ROCK1, 1 µM biotinylated peptide (biotin-Ahx-AKRRRLSSLRA-CONH2), 1 µM ATP, 1.85 kBq/well [
-33P]ATP, 25 mM HEPES, pH 7.4, 15 mM MgCl2, and 0.015% bovine serum albumin. The reactions were incubated at 22°C for 90 min and terminated by the addition of a 10-µl solution containing 150 mM EDTA and streptavidin-coated polystyrene scintillation proximity assay beads (GE Healthcare). The beads were added to a concentration of 0.08 mg/well. The plates were allowed to incubate at 22°C for 10 min before centrifugation at 1500 rpm for 1 min. 33P incorporation was quantified by liquid scintillation counting using a TopCount (PerkinElmer Life and Analytical Sciences, Wellesley, MA).
Cell Culture and Cytokine mRNA and Protein Expression in Human Smooth Muscle Cells and Macrophages. Human aortic smooth muscle cells were purchased from Cambrex Bio Science and grown in smooth muscle cell growth medium according to manufacturer's instructions. For stress fiber formation assay, cells were grown on coverslips, and at approximately 50% confluence, they were serum-starved overnight. Cells were subsequently stimulated with AngII (100 nM) for 2 h before fixing and stained with rhodamine phalloidin (see below).
Human primary macrophages were prepared as follows; leukopacks enriched for monocytes were obtained from Biological Specialty Corporation (Colmar, PA). Cells were washed twice in Hanks' balanced salt solution (without Ca2+, with 1 mM EGTA) and then layered onto 10-ml Histopaque gradient (Sigma-Aldrich, St. Louis, MO). After centrifugation, the harvested interface layer contained both monocytes and lymphocytes. To obtain a pure monocyte population, nonmonocytic cells (i.e., T cells, granulocytes, NK cells, B cells, dendritic cells, and basophils) are labeled using a cocktail of hapten-modified CD3, CD7, CD19, CD45RA, CD56, and anti-IgE antibodies (Miltenyi Biotec, Inc., Auburn, CA). The nonmonocytes are then magnetically labeled using MACS MicroBeads (Miltenyi Biotec Inc., Auburn, CA) coupled to an anti-hapten antibody, and the entire cell population is passed through a magnetized column. Unlabeled monocytes pass through the column and are collected, washed, and plated into Costar 24-well plates (Corning Life Sciences, Acton, MA). Growth medium is RPMI 1640 with 2 mM L-glutamine, 5% human AB serum (Cambrex Inc., East Rutherford, NJ), penicillin-streptomycin (100 U/ml, 100 µg/ml final concentration), and granulocyte-monocyte colony-stimulating factor at 1.0 ng/ml final concentration (Invitrogen, Rockville, MD). Cells are allowed to differentiate in culture for 6 to 8 days before experiments and reach nearly 100% confluence and purity.
Similar to smooth muscle cells, these macrophages were serum-starved overnight and stimulated with LPS (100 ng/ml). Expression of IL-6 mRNA and protein was measured using Northern blot techniques and IL-6 ELISA. Supernatants were also analyzed for TNF- expression using ELISA. ROCK inhibitors were added 30 min before stimulation. Cellular RNA was isolated for Northern analysis, whereas supernatants were used for cytokine measurement. RNA isolation and TaqMan and ELISA measurements were performed as instructed in manufacturer's protocol and published procedures (Sambrook et al., 1989). All cell culture experiments were repeated at least three times, and representative blots are presented.
Immunofluorescence for Actin Stress Fiber Formation and Confocal Microscopy. Detection of actin stress fiber was monitored using rhodamine phalloidin as described by Ueda et al. (2000). Upon the completion of staining, slides were examined using argon laser in a confocal microscope (Olympus, Melville, NY). The images were taken at 40x with a photomultiplier voltage of 664, gain of 1.6, and a 4% offset, with a procedure similar to an earlier study (Grygielko et al., 2005).
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). Male Sprague-Dawley rats (350–400g) were anesthetized with 5% isoflurane in O2 and killed by exsanguination. Aortic rings, approximately 2 to 3 mm in length, were suspended by two 0.1-mm diameter tungsten wire hooks in 10-ml tissue baths containing Krebs of the following composition: 112.0 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM KH2PO2, 1.2 mM MgSO4, 25.0 mM NaHCO3, 11.0 mM dextrose, 0.01 mM indomethacin, and 0.01 mM L-NAME. Krebs was maintained at 37°C and aerated with 95% O2, 5% CO2, pH 7.4,. Changes in isometric force were measured under optimal resting tension (1 g) using FT03 force-displacement transducers (Grass Instruments, Quincy, MA) coupled to model 7D polygraphs. After a 60-min equilibration period, the vessels were treated with standard concentrations of KCl (60 mM) and phenylephrine (1 µM). Cumulative concentration-response curves to phenylephrine were obtained for each tissue by dosing at 0.5-log unit intervals (1 nM to 10 µM). After several washes, each vessel was contracted to equilibrium with an EC80 concentration of phenylephrine, and tone was reversed by adding cumulative amounts of either GSK269962A or SB-772077-B at 0.5-log unit intervals (0.1 nM to 1 µM). Responses were expressed as percentage reversal of the tone established with phenylephrine. SHR Blood Pressure Studies. SHR, obtained from the National Institutes of Health (Bethesda, MD), were bred in the Department of Laboratory Animal Science at GlaxoSmithKline (King of Prussia, PA). Age-matched normotensive rats [Wistar-Kyoto and Sprague-Dawley] were purchased from Charles River Laboratories, Inc. (Wilmington, MA). Experiments were conducted in accordance with the Guide for Care and Use of Laboratory Animals (NIH Publication 85-23), and experimental protocols were reviewed and approved by the GlaxoSmithKline Animal Care and Use Committee.
Blood pressure (BP) measurements were performed using a telemetry system as described previously (Ju et al., 2003). In brief, male SHR (8–10 weeks of age) maintained on a normal powdered diet (Purina Diet 5001; Scott's Distribution, Hudson, NH) were anesthetized with 2% isoflurane anesthesia, and a telemetry transmitter (TA11PA-C40; Data Sciences International, St. Paul, MN) was implanted. The transmitter catheter was inserted into the femoral artery and advanced into the lower abdominal aorta. Baseline measurements of systolic and diastolic blood pressure, heart rate, and activity were obtained 1 week before experiments. Recordings were obtained each week thereafter for a continuous period of 24 h with data acquisition of 10-s averages every 5 min. ROCK inhibitors SB-772077-B and GSK269962A were administered via oral gavage, and BP responses were monitored immediately following drug administration. Four to six animals were examined for each dose in treated and vehicle groups.
Statistical Analysis. Maximal blood pressure changes were analyzed for statistical significance. A two-way analysis of variance was performed. Significance, if indicated, was compared with the vehicle response. P value was described in figure legends. All statistical analyses were performed using Prism (GraphPad Software Inc., San Diego, CA), and P
0.05 was considered significant.
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) and used the purified recombinant protein to screen the GlaxoSmithKline compound collection. Several aminofurazans were identified as putative ROCK inhibitors. Subsequent optimization led to improved potency, selectivity, and pharmacokinetics and resulted in the identification of SB-772077-B and GSK269962A. The structures of SB-772077-B and GSK269962A are depicted in Fig. 1. Both are highly potent toward human ROCK1 with IC50 values of 5.6 nM for SB-772077-B and 1.6 nM for GSK269962A, respectively (Fig. 2). These compounds also potently inhibited human ROCK2 with similar potency (Table 1). In comparison, widely used Rho kinase inhibitors Y-27632 and fasudil inhibited ROCK1 with much lower potency, with an approximate IC50 of 150 nM for Y-27632 and 300 nM for fasudil. Both Y-27632 and fasudil also inhibited human ROCK2 with similar IC50. These data are consistent with previously published results for these two compounds (fasudil and Y-27632) (Uehata et al., 1997). In addition to ROCK1 and ROCK2, SB-772077-B potently inhibits several other kinases (e.g., mitogen and stress-activated protein kinase 1 and ribosomal S6 kinase 1). On the other hand, GSK269962A has a significantly improved kinase selectivity profile with at least >30-fold selectivity against the panel of protein kinase tested. The IC50 value of SB-772077-B and GSK269962A for ROCK1, ROCK2, and profiled kinases are shown in Table 1. These data suggest that SB-772077-B and GSK269962A represent a novel class of ROCK inhibitors with high potency and good protein kinase selectivity.
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Aminofurazan ROCK Inhibitors Disrupt Actin Stress Fiber Formation in Smooth Muscle Cells and Suppress Inflammatory Cytokine Production from Macrophages. To evaluate the cellular effect of novel ROCK inhibitors, we employed actin stress fiber formation as a functional assay. ROCK regulates actin stress fiber formation via the activation of LIM kinase, and ROCK inhibition has been shown to decrease actin fiber content in cells (Amano et al., 1997). SB-772077-B and GSK269962A completely abolished the actin stress fiber formation induced by angiotensin II in human smooth muscles (Fig. 3). Such suppressive effect on actin fiber formation was observed beginning at around 1 µM for both SB-772077-B and GSK269962A. Similar effects were also observed with 30 µM Y-27632 treatment.
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; Aihara et al., 2003; Segain et al., 2003). We evaluated the effects of the aminofurazan ROCK inhibitors in primary human macrophages. When THP-1 macrophages were pretreated with increasing concentrations of SB-772077-B, LPS-induced cytokine production, including TNF- and IL-6, was dose-dependently reduced (Fig. 4). Similar results were obtained with GSK269962A and other structurally related aminofurazans with inhibitory activities toward ROCK. The effect of ROCK inhibitors on cytokine production is likely to act through modulating the transcriptional activation or mRNA stability because the LPS-induced increase in IL-6 mRNA was abolished when macrophages were pretreated with Y-27632 and two structurally related aminofurazan ROCK inhibitors (SB-729743 and SB-742548), but not by an aminofurazan, with no activity toward ROCK1 (SB-739412-B) (Fig. 5, A and B).
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Aminofurazan ROCK Inhibitors Relax Contracted Blood Vessels in Vitro. Inhibition of ROCK has been associated with relaxation of constricted smooth muscles. We examined the effect of SB-772077-B and GSK269962A on contracted rat aortic rings in tissue baths. Both SB-772077-B and GSK269962A profoundly reversed the tonic tension generated by phenylephrine (Fig. 6). The relaxations were highly dose-dependent with IC50 values of 39 and 35 nM for SB-772077-B and GSK269962A, respectively. In comparison, Y-27632 is significantly less potent in this assay, with an IC50 of approximately 1.4 µM (Fig. 6). In addition, the relaxation induced by SB-772077-B and GSK269962A is reversible. Washing the treated aorta will restore the contractility to KCl as well as phenylephrine (data not shown). This is consistent with the reversible nature of ROCK inhibition by these compounds.
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1000 nM) toward ROCK1, and the results revealed a correlation coefficient R2 = 0.65 in linear regression analysis. Because the effects on aortic ring contraction require cell/tissue permeability, some of the variability observed may be due to the differences in cell membrane permeability.
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In addition, we also evaluated the effect SB-772077-B on blood pressure in normotensive SD rat and DOCA salt-induced hypertensive rats. As shown in Fig. 9, oral administration of SB-772077-B at 1 mg/kg profoundly reduced blood pressure in DOCA rats. The reduction in blood pressure was more acute and dramatic in DOCA rats than in SHR, with a maximal decrease of approximately 60 mm Hg reached at approximately 2 h postdosing. The maximal decrease in blood pressure in DOCA-treated rats is more than the maximal decrease (40 mm Hg) in SHR at the same dose (Fig. 9A). An increase in heart rate was observed following oral dosing in DOCA rats similar to that of SHR (Fig. 8). In comparison, oral administration of SB-7720770B in normotensive rat (SD rat) induced only modest decrease in blood pressure (12 mm Hg) (Fig. 9B).
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; Lee et al., 2004). ROCK regulates the calcium sensitization by enhancing the phosphorylation of myosin binding subunit of MYPT of MYPT and inhibiting phosphatase activity and consequently increases the phosphorylation of myosin light chain, leading to sustained smooth muscle contraction without significantly elevated intracellular calcium. This mechanism seems to play a pivotal role in blood pressure homeostasis. Furthermore, there is evidence suggesting that ROCK and ROCK pathway components RhoA, GTP exchange factors, and ROCK are increased in SHR (Mukai et al., 2001; Seko et al., 2003; Moriki et al., 2004; Ying et al., 2004) and human hypertensive patients (Masumoto et al., 2001). Elevated levels of these elements lead to enhanced ROCK activity and further resulted in smooth muscle hyperconstriction, which is one of the hallmarks of hypertension and other cardiovascular diseases. Small molecule ROCK inhibitors may offer an attractive strategy to attenuate the enhanced ROCK activity, leading to reduced hypercontraction, thereby providing potential therapeutic benefits.
Here we describe the identification of a novel class of aminofurazan-based small molecules that are highly potent and selective ROCK inhibitors. Aminofurazan ring has not been used as one of the motifs for ROCK inhibitors, and our results demonstrate that aminofurazan is a key feature of contact between this small molecule and ROCK ATP binding pocket. In this respect, the identification of this structure motif is a novel finding. Two compounds from this chemical series, SB-772077-B and GSK269962A, demonstrated low nanomolar potency toward human ROCK enzyme and relaxed preconstricted blood vessels in vitro. These novel inhibitors showed significantly enhanced potency in vitro and in vivo compared with the widely studied ROCK inhibitors Y-27632 and fasudil (Uehata et al., 1997). A strong correlation between ROCK inhibition and aorta relaxation was observed for this novel class of inhibitors. We also demonstrated that aminofurazan-based ROCK inhibitors suppress the production of IL-6 and TNF- from LPS-stimulated human macrophages. The anti-inflammatory effects of ROCK inhibitors, such as Y-27632 or fasudil, were described previously in several laboratories (Funakoshi et al., 2001; Eto et al., 2002; Segain et al., 2003; Aihara et al., 2004). Our results showed that in macrophages, SB-772077-B and GSK269962A, as members of a distinct class of ROCK inhibitors, suppressed IL-6 mRNA transcription and reduced LPS-induced IL-6 and TNF- protein production. This, combined with our earlier findings demonstrating that Y-27632 administration reduced cytokine production in rat models of ischemia reperfusion-induced myocardial infarction (Bao et al., 2004), strongly suggest that ROCK inhibition per se is sufficient to reduce the inflammatory cytokine expression.
The mechanism responsible for ROCK inhibitor-mediated suppression of cytokine expression is not fully understood. Several investigators (Hippenstiel et al., 2002; Segain et al., 2003) have postulated that nuclear factor-kB may be involved in this process. Indeed, it was demonstrated that p65Rel phosphorylation in vivo was reduced upon treatment of Y-27632 in murine peritoneal macrophages (Mallat et al., 2003). We have failed to observe significant reduction in p65Rel phosphorylation on Ser276, Ser536, or Ser468 residues upon pretreatment of either Y-27632 or aminofurazan-based ROCK inhibitors in TNF--treated primary human macrophages in vitro (data not shown). Whether ROCK-mediated p65Rel phosphorylation is involved in suppression of cytokine production in human primary macrophages warrants further careful analysis.
Aminofurazan ROCK inhibitors exhibited good oral activities in rat models of hypertension. SB-772077-B and GSK269962A potently reduced blood pressure in both SHR and DOCA-treated hypertensive rats, while having a much smaller effect on blood pressure in normotensive animals. These results are consistent with the hypothesis that enhanced ROCK activity contributes to vasoconstriction and elevated blood pressure in these animal models. Concomitant with blood pressure decrease, acute increases in heart rates were observed for both SB-772077-B and GSK269962A in a dose-dependent manner. Interestingly, compared with these two compounds, Y-27632 induced a more dramatic increase in heart rate with doses that had comparable effect on blood pressure (Fig. 8). It is well known that acute decrease in blood pressure induces baroreflex activation and leads to increased heart rate and cardiac output. The observed heart rate is probably due to such baroreflex activation. ROCK inhibitors have been shown to influence baroreflex activation by inhibiting brain-specific ROCK2 and reduce neuronal activation (Ito et al., 2003). The differences in heart rate induced by aminofurazan-based inhibitor versus Y-27632 may result from their ability to penetrate blood-brain barrier and affect the baroreflex activation. Further analysis is needed to examine this possibility.
In summary, we have identified a novel class of potent and selective ROCK inhibitors that possess vasodilatory and anti-inflammatory activity in tissue baths and cultured cells. When administered in vivo, these compounds are potent antihypertensive agents in two models of rat hypertension. Such inhibitors represent additional tools for further exploration of the biological functions of ROCK in smooth muscle biology and for evaluating the usefulness of ROCK inhibitors in various cardiovascular diseases, including hypertension.
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Acknowledgements |
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Footnotes |
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ABBREVIATIONS: ROCK, Rho kinase; KO, knockout; TNF-, tumor necrosis factor-; MYPT, myosin light chain phosphatase; DOCA, deoxycorticosterone acetate; SHR, spontaneously hypertensive rat; L-NAME, N2ù-nitro-L-arginine-methyl-ester; AngII, angiotensin II; BP, blood pressure; LPS, lipopolysaccharide; IL, interleukin; SB-772077-B, 4-(7-{[(3S)-3-amino-1-pyrrolidinyl]carbonyl}-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,2,5-oxadiazol-3-amine; ELISA, enzyme-linked immunosorbent assay; GSK269962A, N-(3-{[2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-1 H-imidazo[4,5-c]pyridin-6-yl]oxy}phenyl)-4-{[2-(4-morpholinyl)ethyl]oxy}benzamide; Y-27632, (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide; SB-729743, 4-(1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl)-1,2,5-oxadiazol-3-amine; SB-742584, 4-{7-[4-(aminomethyl)phenyl]-1-ethyl-1H-imidazo[4,5-c]pyridin-2-yl}-1,2,5-oxadiazol-3-amine hydrochloride.
Address correspondence to: Dr. Erding Hu, Mail Code UW2510, GlaxoSmithKline Pharmaceuticals, 709 Swedeland Road, King of Prussia, PA 19406. E-mail: erding.hu-1{at}gsk.com
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