Inhibition of the Rho/ROCK pathway promotes the expression of developmental and migration-related genes in astrocytes exposed to alcohol.

Astrocytes are an important regulator of alcohol dependence. Furthermore, the downregulation of Rho-associated coiled coil-containing protein kinase 2 (ROCK2) attenuates alcohol-induced inflammation and oxidative stress in astrocytes. On the basis of these findings, we examined the effects of alcohol and a Rho/RACK kinases inhibitor on astrocyte function and investigated their effects on mRNA expression to further explore the protective mechanisms of a Rho/RACK kinases inhibitor in astrocytes after alcohol exposure. CTX TNA2 astrocytes were cultured with alcohol and Rho/RACK kinases inhibitor intervention before undergoing transcriptome sequencing, quantitative reverse transcription polymerase chain reaction (qRT-PCR), and wound healing assays. Alcohol exposure modulated cell morphology and inhibited astrocyte migration, whereas Fasudil improved cell morphology and promoted astrocyte migration after alcohol exposure. Transcriptome sequencing results indicated that alcohol exposure modulates the expression of genes involved in astrocyte development. Fasudil reversed the effects of alcohol exposure on the astrocyte developmental process. Four genes related to the developmental process and migration - Ccl2, Postn, Itga8, and Serpine1 - with the highest protein-protein interaction correlations (node degree >7) were selected for verification by qRT-PCR, and the results were consistent with those of the sequencing and wound healing assays. Our results suggest that the Rho/ROCK pathway is essential for alcohol to be able to interfere with astrocyte development and migration gene expression. The Rho/ROCK pathway inhibitor Fasudil reversed the adverse effects of alcohol exposure on astrocytes and may have clinical applications.

Molecular Basis of Small-Molecule Binding to α-Synuclein.

Intrinsically disordered proteins (IDPs) are implicated in many human diseases. They have generally not been amenable to conventional structure-based drug design, however, because their intrinsic conformational variability has precluded an atomic-level understanding of their binding to small molecules. Here we present long-time-scale, atomic-level molecular dynamics (MD) simulations of monomeric α-synuclein (an IDP whose aggregation is associated with Parkinson's disease) binding the small-molecule drug fasudil in which the observed protein-ligand interactions were found to be in good agreement with previously reported NMR chemical shift data. In our simulations, fasudil, when bound, favored certain charge-charge and π-stacking interactions near the C terminus of α-synuclein but tended not to form these interactions simultaneously, rather breaking one of these interactions and forming another nearby (a mechanism we term dynamic shuttling). Further simulations with small molecules chosen to modify these interactions yielded binding affinities and key structural features of binding consistent with subsequent NMR experiments, suggesting the potential for MD-based strategies to facilitate the rational design of small molecules that bind with disordered proteins.

A Rho kinase inhibitor (Fasudil) suppresses TGF-ß mediated autophagy in urethra fibroblasts to attenuate traumatic urethral stricture (TUS) through re-activating Akt/mTOR pathway: An in vitro study.

AIMS: Transforming growth factor-ß (TGF-ß) mediated super-activation of urethra fibroblasts contributes to the progression of traumatic urethral stricture (TUS), and the Rho-associated kinase inhibitors, Fasudil, might be a novel therapeutic agent for TUS, but the underlying mechanisms had not been studied. MATERIALS AND METHODS: The primary urethral fibroblasts (PUFs) were isolated from rabbit urethral scar tissues and cultured in vitro, and the PUFs were subsequently treated with TGF-ß (10 µg/L) to simulate the realistic conditions of TUS pathogenesis. Next, the PUFs were exposed to Fasudil (50 µM) and autophagy inhibitor 3-methyladenine (3-MA) treatment. Genes expression was examined by Western Blot and immunofluorescence staining, and cellular functions were determined by MTT assay and Transwell assay. KEY FINDINGS: TGF-ß promoted cell proliferation, migration, autophagy, and secretion of extracellular matrix (ECM), including collagen I and collagen III, which were reversed by co-treating cells with both Fasudil and 3-MA. In addition, TGF-ß treatment decreased the expression levels of phosphorylated Akt (p-Akt) and mTOR (p-mTOR) to inactivate the Akt/mTOR pathway in the PUFs, which could be re-activated by Fasudil. Then, the fibroblasts were treated with the Pan-Akt inhibitor (GDC-0068), and we surprisingly found that GDC-0068 abrogated the inhibiting effects of Fasudil on cell autophagy and proliferation in the PUFs treated with TGF-ß. SIGNIFICANCE: Fasudil regulated Akt/mTOR pathway mediated autophagy to hamper TGF-ß-mediated super-activation in PUFs, which supported that Fasudil might be an ideal candidate therapeutic agent for TUS treatment for clinical utilization.

In-silico and Molecular Analysis of Reticulon 4asNovel therapeutic option for axonal regeneration.

OBJECTIVES: To find interactions of the ligand with axonali nhibitors, and to check the optimum interactions of existing drugs as inhibitors for the protein that hinders the growth of injured neurons. METHODS: The study was conducted at Kongju National University, Korea from May 2016 to March 2017. It consisted of two parts. Molecular analysis and bioinformatics analysis. The study comprised a family of six with Charcot-Marie-Tooth phenotypes, recommended by a neurologist for molecular analysis on the clinical symptoms to find the mutations responsible for the disease. Blood samples were collected from each family member and total deoxyribonucleic acid was extracted and it was analysed for Reticulon 4 gene by sequencing the coding and intronic regions. However, a missense mutation was found on exon 2 of the gene in the proband and the whole family was subsequently analysed. Bioinformatics analysis and docking studies were carried out to investigate the potential behaviour of Reticulon 4 as therapeutic agent. Sequencing analysis was performed to find the pathoegenic variant responsible for Charcot-Marie-Tooth type 1. RESULTS: After checking pathogenicity of the mutation, Reticulon 4 gene was found to be not involved in Charcot- Marie-Tooth disease type 1. CONCLUSIONS: Reticulon 4 gene was not found to be involved in causing Charcot-Marie-Tooth disease type 1.

Aldehyde oxidase-dependent species difference in hepatic metabolism of fasudil to hydroxyfasudil.

1. An investigation on the metabolic mechanism of fasudil to hydroxyfasudil was conducted in vitro using liver subcellular fractions of different species. Hydroxyfasudil was generated in large amounts by rat liver S9 and to a similar extent by human liver S9 but was not detected in dog liver S9 incubations. 2. Studies with various molybdenum hydroxylase inhibitors demonstrated that aldehyde oxidase (AO), but not xanthine oxidase (XO), selectively catalyzed fasudil to hydroxyfasudil in both rat and human liver cytosol. In addition, the oxygen atom incorporated into hydroxyfasudil was derived from water rather than atmospheric oxygen, which further corroborated AO involvement. 3. Enzyme kinetics experiments revealed that fasudil had a higher affinity to human hepatic AO than to rat hepatic AO. Besides, significantly different in vivo pharmacokinetic parameters observed between male and female rats indicated that the AO activity in rats was gender-dependent. 4. The present study provided first evidences that AO causes differences in fasudil metabolism between species.

N-[(11)C]-methyl-hydroxyfasudil is a potential biomarker of cardiac hypertrophy.

INTRODUCTION: Pathologic cardiac hypertrophy is one of the leading causes of sudden death from cardiac disease and involves a complex network of bio-signaling mechanisms. To date, the clinical detection and pathologic progression of hypertrophy remains elusive. Here we tested whether imaging Rho kinase activity would serve an accurate proxy for detecting hypertrophy. Specifically, we examine the use of the N-[(11)C]-methylated derivative of hydroxyfasudil, a Rho kinase inhibitor, as a biomarker for accurate identification of cardiomyocyte hypertrophy. METHODS: Both transformed and primary neonatal cardiomyocytes were treated with isoproterenol to induce ß-adrenergic receptor stimulation and hypertrophy. Phenotypic hypertrophy was verified using cytochemical evaluation of cell and nuclear size. Western blot and activity assays were used to detect ERK 1/2 mTOR and Rho kinase activation. N-[(11)C]-methyl-hydroxyfasudil binding was verified using in vitro binding assays with isoproterenol stimulated cells. RESULTS: Isoproterenol induced a rapid and distinct activation of ERK 1/2, mTOR and Rho kinase with negligible cytotoxicity. Subsequent expansion in cell and nuclear size that is typically associated with hypertrophy was also observed. Enhanced retention of N-[(11)C]-methyl-hydroxyfasudil observed after ISO-induced Rho kinase activation in hypertrophic cells was prevented by pre-treatment with unlabeled hydroxyfasudil. CONCLUSIONS: N-[(11)C]-methyl-hydroxyfasudil is able to measure increased Rho kinase activity via specific binding in hypertrophied cardiomyocytes and demonstrates the potential for molecular imaging of altered Rho kinase activity in diseases such as cardiac hypertrophy.

Cell permeable peptide conjugated nanoerythrosomes of fasudil prolong pulmonary arterial vasodilation in PAH rats.

In this study, we tested the hypothesis that a cell permeable peptide, CARSKNKDC (CAR), conjugated nanoerythrosomes (NERs) containing fasudil, a rho-kinase (ROCK) inhibitor, produces prolonged pulmonary preferential vasodilation. CAR conjugated NERs containing fasudil were prepared by hypotonic lysis and extrusion method, and optimized for various physicochemical properties in-vitro. The formulations were then used to study the hemodynamic efficacy in a monocrotaline-induced rodent model of pulmonary arterial hypertension (PAH). CAR-NERs-Fasudil was spherical in shape with an average vesicle size and entrapment efficiency of 161.3 ± 1.37 nm and 48.81 ± 1.96%, respectively. Formulations were stable for ~3 weeks when stored at 4 °C and the drug was released in a controlled fashion for >48 h. The uptake of CAR-NERs-Fasudil by TGF-b activated pulmonary arterial smooth muscle cell was ~1.5-fold greater than the uptake of NERs-Fasudil. CAR-NERs-Fasudil inhibited ROCK activity and 5-hydroxytryptamine induced cell proliferation. In terms of reduction of pulmonary arterial pressure, intratracheal administration of CAR-NERs-Fasudil was ~2-fold more specific to the lungs compared with plain fasudil. Overall,CAR peptide grafted nanoerythrosomes offers a new platform for improving the therapeutic efficacy ofa rho-kinase inhibitor, fasudil, without affecting peripheral vasodilation.

Effect of fasudil hydrochloride on the post-thaw viability of cryopreserved porcine adipose-derived stem cells.

BACKGROUND: Adipose-derived stem cells (ADSCs) are of interest for regenerative medicine as they are isolated easily and can differentiate into multiple cell lineages. Recently, it was reported that a Rho-associated kinase (ROCK) inhibitor Y-27632 could enhance the post-thaw viability and physiological function of cryopreserved BMSC. OBJECTIVE: The present study is to investigate whether Fasudil hydrochloride (FH, a selective Rho-kinase inhibitor like Y-27632) can exert a similar beneficial effect on the post-thaw viability of cryopreserved ADSCs. METHODS: ADSCs were allotted in three cryopreservation solutions [I:10 % (v/v) (DMSO), 30 % (v/v) FBS (as a negative control); II:10µM FH, 10 % (v/v) DMSO, 30 % (v/v) FBS; III:10 µM Y-27632, 10 % (v/v) DMSO, 30 % (v/v) fetal bovine serum, All groups were frozen using a rapid freezing method and stored at -196 degree C in liquid nitrogen for 30 days.After thawing and being cultured 24h,viability of ADSCs were detected by MTT assay. RESULT: The MTT assay showed significant differences in the proportion of adherent viable cells over the concentration of Y-27632 and FH, Additionally, FH did not induce morphological changes in the frozen-thawed ADSCs like Y-27632 did. CONCLUSIONS: FH might represent a promising cryopreservation solution in enhancing the post-thaw viability and physiological function of cryopreserved ADSCs and did not chang the cell appearance.

Starch-coated magnetic liposomes as an inhalable carrier for accumulation of fasudil in the pulmonary vasculature.

In this study, we tested the feasibility of magnetic liposomes as a carrier for pulmonary preferential accumulation of fasudil, an investigational drug for the treatment of pulmonary arterial hypertension (PAH). To develop an optimal inhalable formulation, various magnetic liposomes were prepared and characterized for physicochemical properties, storage stability and in vitro release profiles. Select formulations were evaluated for uptake by pulmonary arterial smooth muscle cells (PASMCs) - target cells - using fluorescence microscopy and HPLC. The efficacy of the magnetic liposomes in reducing hyperplasia was tested in 5-HT-induced proliferated PASMCs. The drug absorption profiles upon intratracheal administration were monitored in healthy rats. Optimized spherical liposomes - with mean size of 170 nm, zeta potential of -35mV and entrapment efficiency of 85% - exhibited an 80% cumulative drug release over 120 h. Fluorescence microscopic study revealed an enhanced uptake of liposomes by PASMCs under an applied magnetic field: the uptake was 3-fold greater compared with that observed in the absence of magnetic field. PASMC proliferation was reduced by 40% under the influence of the magnetic field. Optimized liposomes appeared to be safe when incubated with PASMCs and bronchial epithelial cells. Compared with plain fasudil, intratracheal magnetic liposomes containing fasudil extended the half-life and area under the curve by 27- and 14-fold, respectively. Magnetic-liposomes could be a viable delivery system for site-specific treatment of PAH.

ROCK inhibition facilitates tissue reconstitution from embryonic kidney cell suspensions.

Explanted embryonic kidney tissues, when placed in organ culture, recapitulate key aspects of nephron formation. Yet attempts to dissociate these tissues into single cells to make them more accessible for genetic manipulation and tissue recombination have been disappointing. Now it is reported that inhibitors of Rho-associated kinase (ROCK) facilitate tissue reconstitution and nephron differentiation from single-cell suspensions of the embryonic kidney. This finding illustrates an unexpected potential for self-organization in these cells and introduces a window of single-cell accessibility to the organ culture system.

Expression of the dominant-negative tail of myosin Va enhances exocytosis of large dense core vesicles in neurons.

Regulated exocytosis of secretory vesicles is a fundamental process in neurotransmission and the release of hormones and growth factors. The F-actin-binding motor protein myosin Va was recently shown to be involved in exocytosis of peptide-containing large dense core vesicles of neuroendocrine cells. It has not previously been discussed whether it plays a similar role in neurons. We performed live-cell imaging of cultured hippocampal neurons to measure the exocytosis of large dense core vesicles containing fluorescently labelled neuropeptide Y. To address the role of myosin Va in this process, neurons were transfected with the dominant-negative tail domain of myosin Va (myosinVa-tail). Under control conditions, about 0.75% of the labelled large dense core vesicles underwent exocytosis during 5 min of stimulation. This value was doubled to 1.80% of the vesicles when myosinVa-tail was expressed. Depolymerization of F-actin using latrunculin B resulted in a similar increase in exocytosis in both control and myosinVa-tail expressing cells. Interestingly, the increase in exocytosis caused by myosinVa-tail expression was completely abolished in the presence of KN-62, an inhibitor of calcium-calmodulin-dependent kinase II. We suggest that myosinVa-tail causes the liberation of large dense core vesicles from the actin cytoskeleton, leading to an increase in exocytosis in the cultured hippocampal neurons.

Involvement of Rho-kinase in prostaglandin F2alpha-stimulated interleukin-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts.

We have previously reported that prostaglandin F(2alpha) (PGF(2alpha)) stimulates interleukin-6 (IL-6), a potent bone resorptive agent, through p44/p42 mitogen-activated protein (MAP) kinase in osteoblast-like MC3T3-E1 cells. In the present study, we investigated whether Rho-kinase is implicated in the PGF(2alpha)-stimulated IL-6 synthesis in MC3T3-E1 cells. PGF(2alpha) time-dependently induced the phosphorylation of myosin phosphatase targeting subunit (MYPT-1), a Rho-kinase substrate. Y27632, a specific Rho-kinase inhibitor, significantly reduced the PGF(2alpha)-stimulated IL-6 synthesis as well as the MYPT-1 phosphorylation. Fasudil, another inhibitor of Rho-kinase, suppressed the PGF(2alpha)-stimulated IL-6 synthesis. Y27632 and fasudil failed to affect the PGF(2alpha)-induced phosphorylation of p44/p42 MAP kinase. SB203580 and BIRB0796, potent inhibitors of p38 MAP kinase, suppressed the IL-6 synthesis induced by PGF(2alpha). While SP600125, an inhibitor of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK), failed to reduce the synthesis. Y27632 as well as fasudil attenuated the PGF(2alpha)-induced phosphorylation of p38 MAP kinase. These results strongly suggest that Rho-kinase regulates PGF(2alpha)-stimulated IL-6 synthesis via p38 MAP kinase activation in osteoblasts.

A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation.

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein-ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase-fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA-fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase-fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase-fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase.

Wide therapeutic time window for Rho-kinase inhibition therapy in ischemic brain damage in a rat cerebral thrombosis model.

The aim of this study was to investigate the influence of delayed Rho-kinase inhibition with fasudil on second ischemic injury in a rat cerebral thrombosis model. Cerebral ischemia was induced in rats by injecting 150 mug of sodium laurate into the left internal carotid artery on day 1. In the ischemic group, the regional cerebral blood flow (rCBF) was significantly decreased 6.5 h after the injection. Fasudil (3 mg/kg/30 min i.v. infusion) significantly increased rCBF. The viscosity of whole blood was significantly increased 48 h after the injection of sodium laurate. Fasudil (10 mg/kg, i.p.) significantly decreased blood viscosity. To clarify the therapeutic time window of fasudil, rats received their first i.p. administration of fasudil (10 mg/kg) 6 h after an injection of sodium laurate. Administration of fasudil twice daily was continued until day 4. Fasudil prevented the accumulation of neutrophils within the brain as seen from measurements taken on day 3, and improved neuronal functions and reduced the infarction area as seen on day 5. Fasudil and hydroxyfasudil, an active metabolite of fasudil, concentration-dependently inhibited phosphorylation of myosin binding subunit of myosin phosphatase in neutrophils. The present results indicate that inhibition of Rho-kinase activation with fasudil is effective for the treatment of ischemic brain damage with a wide therapeutic time window by improving hemodynamic function and preventing the inflammatory responses. These results suggest that fasudil will be a novel and efficacious approach for the treatment of acute ischemic stroke.

Negative and positive allosteric modulators of the P2X(7) receptor.

BACKGROUND AND PURPOSE: Antagonist effects at the P2X(7) receptor are complex with many behaving in a non-competitive manner. In this study, the effects of N-[2-({2-[(2-hydroxyethyl)amino]ethyl}amino)-5-quinolinyl]-2-tricyclo[3.3.1.1(3,7)]dec-1-ylacetamide (compound-17) and N (2)-(3,4-difluorophenyl)-N (1)-[2-methyl-5-(1-piperazinylmethyl)phenyl]glycinamide dihydrochloride (GW791343) on P2X(7) receptors were examined and their mechanism of action explored. EXPERIMENTAL APPROACH: Antagonist effects were studied by measuring agonist-stimulated ethidium accumulation in cells expressing human or rat recombinant P2X(7) receptors and in radioligand binding studies. KEY RESULTS: Compound-17 and GW791343 were non-competitive inhibitors of human P2X(7) receptors. Receptor protection studies using decavanadate and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) showed that neither compound-17 nor GW791343 competitively interacted at the ATP binding site and so were probably negative allosteric modulators of the P2X(7) receptor. GW791343 prevented the slowly reversible blockade of the human P2X(7) receptor produced by compound-17 and inhibited [(3)H]-compound-17 binding to the P2X(7) receptor suggesting they may bind to similar or interacting sites. At rat P2X(7) receptors, compound-17 was a negative allosteric modulator but the predominant effect of GW791343 was to increase agonist responses. Antagonist interaction and radioligand binding studies revealed that GW791343 did not interact at the ATP binding site but did interact with the compound-17 binding site suggesting that GW791343 is a positive allosteric modulator of the rat P2X(7) receptor. CONCLUSIONS: Compound-17 was a negative allosteric modulator of human and rat P2X(7) receptors. GW791343 was a negative allosteric modulator of the human P2X(7) receptor but at the rat P2X(7) receptor its predominant effect was positive allosteric modulation. These compounds should provide valuable tools for mechanistic studies on P2X(7) receptors.

Foreign body-type multinucleated giant cell formation requires protein kinase C beta, delta, and zeta.

Multinucleated giant cells are a classic cellular feature of chronic inflammation, although the mechanism of macrophage fusion leading to their formation is not well understood. Here, we investigate the participation of protein kinase C (PKC) in the interleukin (IL)-4-induced fusion of human monocyte-derived macrophages and foreign body giant cell (FBGC) formation in vitro. The PKC inhibitors H-7 and calphostin C attenuated macrophage fusion, whereas H-8, which is more selective for PKA and PKG, did not. Macrophage fusion was also prevented by the phospholipase C inhibitor, Et-18-OCH(3), the PKC isoform inhibitors GO6983 or rottlerin and by peptide inhibitors for PKC (20-28), PKCbeta, or PKCzeta but not by HBDDE or peptide inhibitors for PKCvarepsilon or PKA. In cultures of fusing macrophages/FBGC, we detected only PKCalpha, beta, delta, and zeta by immunoprecipitation and immunoblotting, and we also observed strong expression of these isoforms by immunocytochemistry. Our collective results suggest that the gamma, epsilon, eta, mu, theta, or iota PKC isoforms are not required in the mechanism of IL-4-induced macrophage fusion; whether PKCalpha is required is unclear. However, new evidence is provided that FBGC formation is supported by PKCbeta, PKCdelta, and PKCzeta in combined diacylglycerol-dependent (PKCbeta and PKCdelta) and -independent (PKCzeta) signaling pathways.

Photobleaching reveals complex effects of inhibitors on transcribing RNA polymerase II in living cells.

RNA polymerase II transcribes most eukaryotic genes. Photobleaching studies have revealed that living Chinese hamster ovary cells expressing the catalytic subunit of the polymerase tagged with the green fluorescent protein contain a large rapidly exchanging pool of enzyme, plus a smaller engaged fraction; genetic complementation shows this tagged polymerase to be fully functional. We investigated how transcriptional inhibitors--some of which are used therapeutically--affect the engaged fraction in living cells using fluorescence loss in photobleaching; all were used at concentrations that have reversible effects. Various kinase inhibitors (roscovitine, DRB, KM05283, alsterpaullone, isoquinolinesulfonamide derivatives H-7, H-8, H-89, H-9), proteasomal inhibitors (lactacystin, MG132), and an anti-tumour agent (cisplatin) all reduced the engaged fraction; an intercalator (actinomycin D), two histone deacetylase inhibitors (trichostatin A, sodium butyrate), and irradiation with ultra-violet light all increased it. The polymerase proves to be both a sensitive sensor and effector of the response to these inhibitors.

Neural differentiation of human neuroblastoma GOTO cells via a Rho-Rho kinase-phosphorylation signal transduction and continuous observation of a single GOTO cell during differentiation.

Nerve growth factor, retinoic acid, dibutyryl cAMP, ganglioside G(Q1b), and botulinum C3 exoenzyme were evaluated for their neural differentiating potential on human neuroblastoma GOTO cells. C3 exoenzyme is an ADP-ribosyltransferase inactivating Rho protein (a small GTP-binding protein). C3 exoenzyme caused the fastest differentiation of GOTO cells into neural cells and induced the strongest network of the cells. Fasudil, an inhibitor of Rho-kinase, induced outgrowth of the neurites in the GOTO cells. Calyculin A, an inhibitor of phosphatases including myosin phosphatase, counteracted C3 exoenzyme-induced neurite outgrowth of the cells. These findings suggest that differentiation of GOTO cells triggered by C3 exoenzyme is attained via inactivation of Rho protein, inhibition of Rho-kinase, and activation of myosin phosphatase. Because of the strong differentiating potential of C3 exoenzyme, the transduction pathway consisting of Rho protein, Rho-kinase, and myosin phosphatase seems to be main stream in the neural differentiation of GOTO cells. A single GOTO cell was observed continuously after treatment with C3 exoenzyme. The cell started to change shape from its original morphology only 15 min after treatment with C3 exoenzyme, and it was completely spherical within 60 min. Neurites appeared on the membrane of the cell 2 hr after the treatment and then gradual outgrowth began. These observations are fundamental information in elucidating the mechanism of neural differentiation, especially at an early stage.

Systemic availability of the active metabolite hydroxy-fasudil after administration of fasudil to different sites of the human gastrointestinal tract.

This study evaluated the gastrointestinal absorption of fasudil, a novel Rho kinase inhibitor for the treatment of stable angina, at different sites using remote-controlled capsules and assessed the feasibility of developing an extended-release formulation. Ten healthy male volunteers were enrolled, and 8 subjects completed this single-dose, open-label, randomized, 5-way crossover study. Forty milligrams of fasudil HCl was administered as solution to the distal ileum and ascending colon, as powder to the ascending colon, and orally as an immediate-release tablet and solution. All treatments were well-tolerated and no serious adverse events were observed. The mean systemic availabilities of M3 relative to the oral solution were 1.04 (distal ileum, solution), 1.14 (ascending colon, solution), 1.27 (ascending colon, powder) and 1.04 (oral tablet), indicating similar systemic availability of M3 after administration of fasudil HCl to different gastrointestinal sites. The results suggest that development of a once-a-day extended-release formulation for fasudil HCl should be readily achievable.

Molecular mechanism for the regulation of rho-kinase by dimerization and its inhibition by fasudil.

Rho-kinase is a key regulator of cytoskeletal events and a promising drug target in the treatment of vascular diseases and neurological disorders. Unlike other protein kinases, Rho-kinase requires both N- and C-terminal extension segments outside the kinase domain for activity, although the details of this requirement have been elusive. The crystal structure of an active Rho-kinase fragment containing the kinase domain and both the extensions revealed a head-to-head homodimer through the N-terminal extension forming a helix bundle that structurally integrates the C-terminal extension. This structural organization enables binding of the C-terminal hydrophobic motif to the N-terminal lobe, which defines the correct disposition of helix alphaC that is important for the catalytic activity. The bound inhibitor fasudil significantly alters the conformation and, consequently, the mode of interaction with the catalytic cleft that contains local structural changes. Thus, both kinase and drug conformational pliability and stability confer selectivity.