In vitro activity: Y-39983 is a novel, potent and selective inhibitor of Rho-associated coiled coil-forming protein kinase( ROCK ) with IC50 values of 3.6 nM, 0.42 μM and 0.81 μM for ROCK, PKC and CaMKII, respectively. Y-39983 acts by downregulating RhoA/Rho-associated kinase expression during its promotion of axonal regeneration. Y-39983 also promotes regeneration of crushed axons of retinal ganglion cells into the optic nerve of adult cats. Y-39983 has been reported to relax the ciliary arteries of precontracted isolated rabbit in vitro. Y-27632 2HCl inhibits ROCK-II while displaying little activity against PKC, cAMP-dependent protein kinase and myosin light-chain kinase (MLCK) with Ki of 26 μM, 25 μM and> 250 μM, respectively, as well as PKA activated by another Rho-family GTPase member, Cdc42. Y-27632 2HCl inhibits smooth-muscle contraction induces by various agonists including phenylephrine, histamine, acetylcholine, serotonin, endothelin, and thromboxane with IC50 of 0.3-1 μM, by selectively inhibiting Ca2+ sensitization. Y-27632 2HCl suppresses Rho-induced, p160ROCK-mediated formation of stress fibres in cultured cells. Y-27632 2HCl treatment blocks both Rho-mediated activation of actomyosin and LPA-stimulated invasive activity of MM1 cells in a concentration-dependent manner. Y-27632 2HCl treatment is not only sufficient to initiate formation of exuberant axonal processes but also facilitates axonal maturation during the very early stages of axonogenesis, while largely sparing axon elongation. In human embryonic stem (hES) cells, Y-27632 2HCl treatment at 10 μM markedly diminishes dissociation-induced apoptosis even in serum-free suspension (SFEB) culture, increases cloning efficiency (from ~1% to ~27%), facilitates subcloning after gene transfer, and enables SFEB-cultured hES cells to survive and differentiate into Bf1+ cortical and basal telencephalic progenitors.

Kinase Assay: Recombinant ROCK (ROK α/ROCK II), purified protein kinase C (PKC: mixture of α, β, γ isoforms), and recombinant calmodulin-dependent protein kinase II (CaMK II) are used in the assay. ROCK (0.2 U/mL) is incubated with 1 μM [γ-32P] ATP and 10 μg/mL histone as substrates in the absence or presence of various concentrations of Y-27632, Y-33075, or staurosporine at room temperature for 20 minutes in 20 mM MOPS (3-(N-morpholino)propanesulfonic acid) buffer (pH 7.2) containing 0.1 mg/mL bovine serum albumin (BSA), 5 mM dithiothreitol [DTT], 10 mM β-glycerophosphate, 50 μM Na3VO4, and 10 mM MgCl2 in a total volume of 100 μL. PKC (10 ng/mL) is incubated with 1 μM [γ-32P] ATP and 20 μM PKC substrate in the absence or presence of various concentrations of Y-27632, Y-33075, or staurosporine at room temperature for 30 minutes in 20 mM MOPS buffer (pH 7.5) containing 0.1 mg/mL BSA, 10 mM DTT, 10 mM β-glycerophosphate, 50 μM Na3VO4, 2 mM CaCl2, 20 μg/mL phosphatidyl-l-serine, and 10 mM MgCl2 in a total volume of 100 μL. CaMK II (125 U/mL) is incubated with 1 μM [γ-32P] ATP, 10 μM calmodulin, and 20 μM CaMK II substrate, in the absence or presence of various concentrations of Y-27632, Y-33075, or staurosporine at room temperature for 30 minutes in 20 mM MOPS buffer (pH 7.5) containing 0.2 mg/mL BSA, 0.5 mM DTT, 0.1 mM β-glycerophosphate, 50 μM Na3VO4, 1 mM CaCl2, and 5 mM MgCl2 in a total volume of 100 μL. Incubation is terminated by the addition of 100 μL of 0.7% phosphoric acid. A 160 μL portion of the mixture is transferred to Multiscreen-PH plate. A positively charged phosphocellulose filter absorbs the substrate that binds 32P. The filter is washed with 300 μL of 0.5% phosphoric acid and then twice with purified water and then dried. The radioactivity of the dried filter is measured with a liquid scintillation counter. Results are presented as 50% inhibitory concentrations and 95% confidence intervals (CIs).

Cell Assay: retinal cell suspensions are obtained from dissected retinas of Wistar rats by papain treatment. Retinal ganglion cells (RGCs) are purified by the panning method using anti-rat CD11 antibody for removal of microglia cells and anti-rat Thy-1 antibody for isolation of ganglion cells. The purified RGCs (5000 cells/plate) are seeded into 24-well plates coated by 50 μg/mL of poly-l-lysine and 2 μg/mL of merosin, and are cultured in serum-free neurobasal medium supplemented with 2% B27 supplement, 50 ng/mL BDNF, 50 ng/mL CNTF, 5 μM forskolin, and 1 mM glutamine under a 95% air-5% CO2 atmosphere at 37°C. After completion of 24-hour cultivation, RGCs are cultured in medium with or without 10 μM Y-33075 as the final concentration for 24 hours and morphologically observed by phase-contrast microscopy. The concentration used is determined based on the effect of Y-33075 on trabecular meshwork contraction in vitro. Since this study is conducted in order to confirm whether Y-33075 has a potential of effect on axonal regeneration of RGCs, the effect is unquantitateively evaluated