CHEMBL5738657

ROCK-I(h) In Vitro Inhibition Activity-Method A: Table 2: ROCK-I(h) kinase assays were conducted using the KinaseProfiler™ service of Eurofins Pharma Discovery Services UK Limited. ROCK-I(h) was incubated with the test compound in assay buffer containing 8 mM MOPS pH 7.0 (VWR cat #441644J) 0.2 mM EDTA (Sigma cat #E5134), 30 μM KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK (Pepceuticals), 10 mM magnesium acetate (Merck Millipore cat #105819) and [γ-33P-ATP] (Perkin Elmer cat #NEG602K). Test compound in 100% DMSO (Calbiochem cat #317275) was spotted into a 96-well assay plate prior using a Mosquito X1 (TTP Labtech) prior to addition of the reaction mix. The reaction was initiated by the addition of the Mg/ATP mix using a Matrix Wellmate. After incubation for 40 minutes at room temperature, the reaction was stopped by the addition of a 3% phosphoric acid solution (Fisher Scientific cat #O/0500/PB17) using a Matrix Wellmate. An aliquot of the reaction was then spotted onto a filtermat and washed in phosphoric acid followed by a rinse in methanol prior to drying and scintillation counting in a Trilux Wallac Microbeta detector (Perkin Elmer). Results were expressed in relation to controls containing DMSO only in place of test compound. Where applicable, IC50 curve analysis was performed using XLFit version 5.3 (ID Business Solutions). Sigmoidal dose-response (variable slope) curves are fitted using non-linear regression analysis.