Novel Rho Kinase Inhibitor and Its Perspective

We have made an effort to develop a highly potent and much more specific inhibitor of Rho kinase (Ikenoya et al. 2002). The characteristics of H1152 are summarized in Table 2. In in vitro systems, H1152 potently inhibited Rho kinase activity in a competitive fashion with respect to ATP. As the introduction of two methyl moieties into the HA1077 molecule drastically increases the inhibitory activity against Rho kinase, we analyzed and compared the conformations of H1152 and HA1077 using a computer molecular modeling program. Figure 8 shows that the introduction of the two methyl moieties produces steric hindrance between isoquinoline and homopiper-azine moieties, thereby preventing some configurations of H1152 and suggesting that this inhibitor assumes a more rigid configuration than HA1077. In vivo, H1152 specifically inhibited the phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) in human neuronal (NT)-2 cells

Fig. 8 Comparison of possible conformations of HA1077 and H1152. Systematic conformation analyses and energy minimization, using Chem-X molecular modeling package (version 2000.1; Accerlys K.K. 2-8-4 Shinkawa Chuo-Ku, Tokyo, Japan), assumed stable conformations of HA1077 and H1152. Bonds were rotated in steps of 30°. Energy minimizations were performed employing MM2 force field and default options. The pictures were drawn using MAESTRO software (Schrodinger, Portland OR, USA). (The figure was drawn by Dr. Masami Shiratsuchi and Dr. Hisashi Nakashima, Kowa, Japan, and is cited with permission)

Fig. 8 Comparison of possible conformations of HA1077 and H1152. Systematic conformation analyses and energy minimization, using Chem-X molecular modeling package (version 2000.1; Accerlys K.K. 2-8-4 Shinkawa Chuo-Ku, Tokyo, Japan), assumed stable conformations of HA1077 and H1152. Bonds were rotated in steps of 30°. Energy minimizations were performed employing MM2 force field and default options. The pictures were drawn using MAESTRO software (Schrodinger, Portland OR, USA). (The figure was drawn by Dr. Masami Shiratsuchi and Dr. Hisashi Nakashima, Kowa, Japan, and is cited with permission)

Fig. 9 Inhibition of MARCKS phosphorylation at Ser159 by H1152 in human neuronal (NT)-2 cells stimulated with 0.3 mM lysophosphatidic acid or 0.1 mM phorbol dibutyrate (left panel). Schematic outline of MARCKS phosphorylation pathways initiated by various stimuli (rightpanel)

stimulated with LPA, but not with phorbol ester (Fig. 9). Previously, we showed that the phosphorylation of MARCKS at Ser159 is catalyzed by Rho kinase as well as PKC (Nagumo et al. 2001). H1152 was used to probe a novel signal transduction pathway for the MARCKS phosphorylation by Rho ki-nase in LPS-stimulated neuronal cells. MARCKS is assumed to participate in neurotransmitter release through the reorganization or modulation of F-ac-tin structure in neuronal terminals (Trifaro et al. 2002). It is plausible that the F-actin cross-linking activity of MARCKS participates in the dynamics of actin meshwork in a so-called active zone under the presynaptic membranes, and then the reorganization of actin meshwork facilitates the approach of synaptic vesicles to the active zone. Taken together, these data raise the important possibility and hypothesis that a Rho kinase inhibitor may modulate reorganization of the actin-meshwork, suppressing the trafficking of synaptic vesicles and subsequently inhibiting the release of neuro-transmitter.

In neuronal cells and the CNS, Rho kinase is becoming recognized as a potent regulator of neurogenic events, such as the axon elongation and retraction cycle, axon guidance and synaptogenesis, and neurite extension in cultured cells (Kozma et al. 1997; Bito et al. 2000). These concepts seem to be based on the results of molecular and pharmacological experiments using cells expressing a Rho kinase mutant and/or treated with Rho kinase inhibitors. However, little is known about the target molecule of Rho kinase in neuronal cells and CNS.

These is a growing body of evidence that Rho kinase is involved in an increasing number of severe pathologies including angina pectoris (Katsumata et al. 1997), myocardial infarction (Yamamoto et al. 2000), bronchial asthma

(Chiba et al. 1999), rheumatoid arthritis, insulin-resistant diabetes mellitus, arteriosclerosis (Fukui et al. 2000), cognitive dysfunction (unpublished data), and astrocyte activation (Abe and Misawa 2003). Thus, with the discovery of novel substrates of Rho kinase, highly potent and specific forms of Rho kinase may be recognized not only in normal but also in pathological conditions. Rho kinase inhibitors may be very important as medicines for such diseases as well as diagnostic probes for Rho kinase-involved physiology (Moolenaar et al. 1997).

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