FSD-C10: A more promising novel ROCK inhibitor than Fasudil for treatment of CNS autoimmunity

Abstract

Rho-Rho kinase (Rho-ROCK) triggers an intracellular signalling cascade that regulates cell survival, death, adhesion, migration, neurite outgrowth and retraction and influences the generation and development of several neurological disorders. Although Fasudil, a ROCK inhibitor, effectively suppressed encephalomyelitis (EAE), certain side effects may limit its clinical use. A novel and efficient ROCK inhibitor, FSD-C10, has been explored. In the present study, we present chemical synthesis and structure of FSD-C10, as well as the relationship between compound concentration and ROCK inhibition. We compared the inhibitory efficiency of ROCKI and ROCK II, the cell cytotoxicity, neurite outgrowth and dendritic formation, neurotrophic factors and vasodilation between Fasudil and FSD-C10. The results demonstrated that FSD-C10, like Fasudil, induced neurite outgrowth of neurons and dendritic formation of BV-2 microglia and enhanced the production of neurotrophic factor brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF) and neurotrophin-3 (NT-3). However, the cell cytotoxicity and vasodilation of FSD-C10 were relatively small compared with Fasudil. Although Fasudil inhibited both ROCK I and ROCK II, FSD-C10 more selectively suppressed ROCK II, but not ROCK I, which may be related to vasodilation insensitivity and animal mortality. Thus, FSD-C10 may be a safer and more promising novel ROCK inhibitor than Fasudil for the treatment of several neurological disorders.

Keywords: FSD-C10; Fasudil; Rho kinase; Rho kinase inhibitor.

Figure 1. Synthesis of FSD-C10

(a) Ligand-binding pocket of ROCK homology model and (b) chemical substructures designated for each division of the ligand-binding pocket that are reproduced from [17]: Takami, A., Iwakubo, M., Okada, Y., Kawata, T., Odai, H., Takahashi, N., Shindo, K., Kimura, K., Tagami, Y., Miyake, M. et al. (2004) Design and synthesis of Rho kinase inhibitors. Bioorg. Med. Chem. 12, 2115–2137. The pocket is divided into three parts: region A, F and D. The pocket D region is cleft-like in shape and a wide range of chemical fragments would fit this cleft [17]. Therefore, we designed and synthesized an isoquinoline FSD-C10 targeting region D of the Fasudil. (c) Synthetic process of FSD-C10. (a) CH₂Cl₂, saturated sodium bicarbonate solution, 0°C. (b) CH₂Cl₂, Et₃N, 2 h, 0°C, 95% yield. (c) CH₂Cl₂, reflux, 7 h, 75% yield.

Figure 2. The relationship between compound concentration and ROCK inhibition

Fasudil and FSD-C10 were diluted to the final desired highest compound concentration (10 μM) by 100% DMSO and serially diluted on 96-well plate by transferring 30–60 μl of 100% DMSO in the next well for a total of 10 concentrations in duplicate. DMSO (100 μl) was used for no compound control and no enzyme control. Percentage of ROCK inhibition was calculated by . ‘Max’ stands for DMSO control; ‘min’ stands for low control.

Figure 3. Fasudil and FSD-C10 ameliorated the severity of EAE

Chronic EAE was induced in C57BL/6 mice with MOG_35–55 peptide. Mice received Fasudil or FSD-C10 by i.p. injection from day 3 to 27 p.i. Mean clinical score and mean body weight were recorded. The comparison in each time point was separately analysed by Mann–Whitney U test after non-parametric Kruskal Wallis test.

Figure 4. The viability and death of primary neurons (a) and BV-2 microglia (b) in vitro

Cell viability was measured using the MTT assay and cell death was detected by LDH release assay. Primary neurons and BV-2 microglia were treated with different concentrations of Fasudil or FSD-C10 (0.6, 3, 15 and 75 μg/ml) for 24 h. The quantitative data are mean±S.E.M. based on three independent experiments with similar results; *P<0.05.

Figure 5. Fasudil and FSD-C10 promoted neurite outgrowth in primary neurons and dendritic formation in BV-2 microglia

The neurite length of primary neurons (a) and the dendritic length of BV-2 microglia (b) was examined with an inverted Olympus microscope. (c) High concentrations of Fasudil (75 and 200 μg/ml), but not FSD-C10, caused significant breakage of neurite outgrowth. The length of the longest neurite outgrown or dendritic formation in the cell body was measured by a public-domain image-processing program The quantitative data are mean±S.E.M. based on three independent experiments with similar results; **P<0.01, ***P<0.001.

Figure 6. The effect of Fasudil and FSD-C10 on neuronal growth factors

Chronic EAE was induced in C57BL/6 mice with MOG_35–55 and treated with Fasudil or FSD-C10. On day 28 p.i., brains were harvested to prepare protein for the expression of BDNF (a), GDNF (b), NT-3 (c) and Nogo (d) by Western blot. The results were expressed as the fold change relative to β-actin as the loading control. Quantitative results are mean±S.E.M. of six mice in each group. *P<0.05.

Figure 7. Fasudil and FSD-C10 shifted M1 to M2 phenotype

Chronic EAE was induced in C57BL/6 mice with MOG_35–55 and treated with Fasudil or FSD-C10. On day 28 p.i., brains were harvested to prepare protein for the expression of iNOS and Arg-1 by western blot. The results were expressed as the fold change relative to β-actin as the loading control. Quantitative results are mean±S.E.M. of six mice in each group; *P<0.05.

Figure 8. The influence of Fasudil and FSD-C10 on vasodilation and mortality

(a) After i.p. injection of FSD-C10 or Fasudil, we observe the vasodilation of mice limbs was observed after 30 and 60 min. A representative of three mice each group is shown. (b) After i.p. injection of FSD-C10 or Fasudil at 800, 1600 and 2000 μg/mouse, animal mortality was recorded with 2 h.