CHEMBL5735367

Kinase Assay: Human baculovirus-expressed Janus kinase (JAK) 1, 2, 3 and tyrosin kinase (TYK) 2 were purchased from Carna Biosciences, Inc (#08-144, -045, -046, -147 resp.). All four purified enzymes contain only the catalytic domain. JAK1 (aa 850-1154) and TYK2 (aa 871-1187) are expressed with an N-terminally fused GST-tag, and JAK2 and JAK3 with an N-terminally fused His-tag. Inhibition of phosphorylation of a synthetic peptide was measured in an HTRF-based assay (CisBio #62TKOPEC). First, 75 nL of test compound solution (100% DMSO) was added to a white shallow 384-well plate (NUNC #264706) using a Labcyte ECHO 550 liquid handler. Thereafter, 1 μL of compound dilution buffer (50 mM HEPES, 0.05% bovine serum albumin) and 2 μL of TK solution (TK substrate-biotin in kinase buffer [1× enzymatic buffer from HTRFKinEASE TK kit, 1 mM DTT]) was added. Then, 5 μL kinase-ATP mix (prepared in kinase buffer) was added to the wells and the plates were incubated at RT for 20 (JAK2, 3 and TYK2) to 40 (JAK1) min. For all four kinases a concentration of ATP that corresponded to the Km for ATP was used. The final concentrations of buffers, substrate, kinase and ATP were: JAK1: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 10 mM MgCl2, 1 mM DTT, 7 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 5 ng/well JAK1; JAK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 4 μM ATP, 1 μM TK Substrate-Biotin and 0.1 ng/well JAK2; JAK3: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 2 μM ATP, 1 μM TK Substrate-Biotin and 0.3 ng/well JAK3; TYK2: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 5 mM MgCl2, 1 mM DTT, 13 μM ATP, 50 nM SEB, 1 μM TK Substrate-Biotin and 0.8 ng/well TYK2. Thereafter, the kinase reaction was stopped by adding 4 μL detection mix (final concentrations: 50 mM Hepes buffer pH 7.0, 0.01% BSA, 0.8 M KF, 20 mM EDTA, 42 nM Streptavidin-XL665 and 1:400 STK Ab Cryptate) and the plates were incubated overnight in the dark.