Effects of compounds on CKIδ and CKIα activity in vitro were analyzed, as described previously (15). The CK2, PIM2, and CLK2 kinase assays were performed in a 384-well plate (10-μl volume). The reaction mixture contained the following: for CK2, CK2 (1.5 ng/μl; New England Biolabs, P6010), 50 μM CK2 substrate peptide (Millipore, 12-330), and CK2 buffer [40 mM tris, 10 mM MgCl2, 0.5 mM dithiothreitol (DTT), and 150 mM NaCl (pH 7.5)]; for PIM2, PIM2 (3 ng/μl; Sigma-Aldrich, K3518), 100 μM S6K substrate peptide (SignalChem; S05-58), and PIM2 buffer [25 mM tris, 10 mM MgCl2, 2 mM DTT, and 6.25 mM NaCl (pH 7.5)]; for CLK2, CLK2 (3 ng/μl; Invitrogen, PV4201), 100 μM S6K substrate peptide (SignalChem, S05-58), and CLK2 buffer [25 mM tris, 10 mM MgCl2, 0.5 mM EGTA, and 2.5 mM DTT (pH 7.5)]. Five hundred nanoliters of compound was added to the mixture (final 5% DMSO), and the reaction was started by adding ATP (final 3 μM). After incubation at 37°C for 1.5 hours (CK2) or 30°C for 3 hours (PIM2 and CLK2), 10 μl of Kinase-Glo Luminescent Kinase Assay reagent (Promega) was added, and luminescence was detected to determine the amount of the remaining ATP. None of the tested compounds inhibited luciferase activity directly. IC50 values were obtained by sigmoidal dose-response fitting with Prism software (GraphPad Software).

Profiling of 60 kinases, as well as DYRK, HIPK, and PIM family kinases, was done using KinaseProfiler (Millipore). PER2(680-705) peptide RKKKPQPELETVEDMASGPESLDGAAGGL (underlined residues were mutated to Ala where indicated) and CKI control peptide RKKKAEpSVASLTSQCSYSS (pS represents phosphorylated Ser), corresponding to human PER2(659-674), were synthesized and used for CK2 and CKIδ assays.

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