Belumosudil (KD025)

KD025 down-regulates the IL-17 and IL-21 secretion in human PBMCs, and leads to down-regulation of STAT3 phosphorylation, IRF4, and RORγt expression in CD4+ T cells. ^[2]

KD025 inhibits the secretion of IL-21, IL-17, and IFNγ along with decreasing phosphorylated STAT3 and reduced protein expression of IRF4 and BCL6 in human peripheral blood mononuclear cells. ^[3]

In mice, KD025 (200 mg/kg, p.o.) inhibits ROCK activity in brain and heart, as measured by the degree of MYPT1 phosphorylation. KD025 (200 mg/kg, p.o.) significantly reduces the area of perfusion defect, and reduces tissue loss in the ipsilateral hemisphere. ^[1]

In a collagen-induced arthritis (CIA) mouse model, KD025 (200 mg/kg, i.p.) downregulates the progression of collagen-induced arthritis via targeting of the Th17-mediated pathway. ^[2]

KD025 ()150 mg/kg, i.p. or p.o.) effectively ameliorates cGVHD in a full MHC-mismatch model of multi-organ system cGVHD with bronchiolitis obliterans syndrome and a minor MHC-mismatch model of sclerodermatous GVHD. ^[3]

• Recombinant ROCK1 and ROCK2 assays

  Compound dilutions and reactions are performed in 96-well polystyrene low-binding plates. Filtration is done in 96-well filter plates containing hydrophilic phospho-cellulose cation exchanger membranes. Enzymatic activity of the recombinant ROCK1 and ROCK2 is measured radiometrically in 50 μL of reaction mixture containing assay buffer (50 mmol/L Tris, pH 7.5, 0.1 mmol/L ethyleneglycoltetraacetic acid, 10 mmol/L magnesium acetate and 1 mmol/L dithiothreitol). Long S6 peptide (KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK, 30 μmol/L), ROCK (4 mU per reaction) and ATP (10 μmol/L; 1 μCi [[gamma]-³³P]ATP) and test compound are diluted to a final dimethylsulfoxide concentration of 1%. The reaction is incubated for 45 min at room temperature and stopped with 25 μL of 3% phosphoric acid. Phosphorylated long S6 peptide is separated from unreacted [[gamma]-³³P]ATP by filtration of the quenched reaction contents through a P30 phosphocellulose filter plate. Each filter is washed three times with 75 μL of 75 mmol/L phosphoric acid and one time with 30 μL of 100% methanol. Filter plates are allowed to dry and 30 μL of OptiPhase ‘SuperMix’ scintillation fluid is added to each well. ³³Phosphorous is quantified in an I450 MicroBeta scintillation counter and corrected by subtracting the radioactivity associated with the background samples. Data are analyzed and expressed as percent inhibition using the formula ((U − B)/(C − B)) × 100 where U is the unknown value, B is the average of staurosporine background wells, and C is the average of control wells. Curve fitting is performed by GraphPad Prism software using sigmoidal dose-response (variable slope) equation type analysis to generate IC50 values. Ki values are calculated from an equation of Ki = IC50/(1 + [S]/Km)), where [S] and Km are the concentration of ATP and the Km value of ATP, respectively.

• Cell lines

  A7r5 cells

• Concentrations

  10 µM

• Incubation Time

  2 h

• Method

  Cells were treated with KD025 for 2 hours.

• Animal Models

  Young adult, aged, or type 2 diabetic mice

• Dosages

  300 mg/kg

• Administration

  p.o.