| Assay Method Information | |
| | Competitive Fluorescence Polarization (FP) Assay |
| Description: | Binding affinities (Kd) with the JAK2 JH2 domain were evaluated using the previously developed FP assay and compound 1 as a control. An initial screening is conducted at 50 μM, and binding affinities (Kd's) are measured for those exhibiting >50% binding at 50 μM (Table 1). Replacement of the oxazole in 2 with 6-membered aromatic rings produced compounds with a binding range of 0.129 to 1.4 μM. Conversion of the carboxylate of compound 7 to a methyl ester (8) or just hydrogen (9) resulted in large loss of binding affinity, and the addition of a benzyloxy group gave compounds (11-14) with an affinity range of 0.033 to 0.075 μM. Selectivity measurements were conducted subsequently for the most potent JAK2 JH2 ligands. In a flat black bottom 96 well plate (Corning), 200 μL of FP buffer were added to column 1 (blank), 150 μL to column 2, and 140 μL to columns 3-12. 10 μL of 2.96 μM of JAK2-JH2 WT (3.52 μM for JAK2-JH2-VF, and 6.93 μM for JAK2-JH1), were added to columns 3-12, followed by the addition of 2 μL of DMSO to columns 1-3. 2 μL of inhibitor in DMSO at different concentrations were added from column 4 to 12. 50 μL of 24 nM of tracer were added to columns 2-12. Fluorescence polarization was measured at λexc=485±20 nm, λem=535±25 nm for 1 hour. Experiments were carried out by quadruplicates in three independent experiments. |
| Affinity data for this assay | |
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