Belumosudil (Rezurock) is an orally available, and specific ROCK2 inhibitor (IC50/Ki: 60/41 nM).

ROCK2:41 nM(Ki), ROCK2:60 nM

Belumosudil在人类PBMCs中下调IL-17和IL-21的分泌，并导致CD4+ T细胞中STAT3磷酸化、IRF4和RORγt表达的下调。[2] Belumosudil抑制了人类外周血单核细胞中IL-21、IL-17和IFNγ的分泌，同时降低了磷酸化STAT3，并减少了IRF4和BCL6的蛋白表达。[3]

在小鼠中，Belumosudil（200 mg/kg，p.o.）通过测定MYPT1磷酸化程度，抑制脑和心脏的ROCK活性。Belumosudil（200 mg/kg，p.o.）显著减少灌注缺陷区域，并减少同侧半球的组织损失。[1] 在胶原诱导的关节炎（CIA）小鼠模型中，Belumosudil（200 mg/kg，i.p.）通过靶向Th17介导的途径，抑制胶原诱导关节炎的进展。[2] Belumosudil（150 mg/kg，i.p. 或 p.o.）在全MHC不匹配模型的多器官系统cGVHD中，以及轻微MHC不匹配模型的硬皮病样GVHD中，有效改善cGVHD，这些模型包括支气管细支气管炎综合症和硬皮病。[3]

Cell-free enzyme assays are performed to determine the selective inhibition of ROCK1 and ROCK2 by SLx-2119. Reactions are performed on non-binding surface microplates. Four mU of human ROCK1 and ROCK2 are used to phosphorylate 30 μM of the synthetic ROCK peptide substrate S6 Long, prepared at American Peptide with the addition of 10 μM ATP, containing?33P-ATP in the presence of 10 mM Mg2+, 50 mM Tris, pH 7.5, 0.1 mM EGTA and 1 mM DTT at room temperature. One unit is the amount of kinase needed to catalyze the transfer of 1 nmol phosphate/min to the peptide. The reactions are allowed to proceed for 45 minutes and then stopped with 3% phosphoric acid to a final concentration of 1%. The reactions are captured on phospho cellulose filtration microplates and washed with 75 mM phosphoric acid and methanol using a vacuum manifold. Phosphorylation is measured on a Perkin-Elmer MicroBeta 1450.

SLx-2119 is dissolved in DMSO to obtain a stock solution of 20 mM. Western blots are used to determine whether HMVEC, NHDF and PASMC express ROCK1 and ROCK2. The cells are incubated for 24 hours in 3 mL culture media containing SLx-2119. All cells are collected at passage 3 and lysed on ice in 25 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% tritonX-100, 10% glycerol, 10 mM NaF and a protease inhibitor cocktail. Protein concentration is determined using a BCA protein assay reagent. Cell lysates (35 μg) are separated on 7.5% or 12.5% SDS-PAGE polyacrylamide gels and transferred to PVDF membrane filters. Membranes are blocked in 5% non-fat milk in TBS containing 0.1% Tween 20. Blots are probed with antibodies to ROCK1, ROCK2 or actin and washed well before incubation with HRP-conjugated secondary antibodies and visualization with an enhanced chemiluminescence (ECL) kit.