Longdaysin Circadian Assay
The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ~120,000 uncharacterized compounds on human U2OS cells containing a circadian reporter Bmal1-dLuc. The more ..
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The circadian clock underlies daily rhythms of diverse physiological processes, and alterations in clock function have been linked to numerous pathologies. To apply chemical biology methods to modulate and dissect the clock mechanism with new chemical probes, we performed a circadian screen of ~120,000 uncharacterized compounds on human U2OS cells containing a circadian reporter Bmal1-dLuc. The analysis identified a small molecule (compound 1) that potently lengthens the circadian period in a dose-dependent manner. A preliminary structure-activity relationship study helped to identify a derivative of compound 1 that is 3-times more potent and able to generate >10 h period change at a concentration of 10 uM. We termed this derivative longdaysin, based on its prominent period lengthening effect. Longdaysin was amenable for chemical modification to perform affinity chromatography coupled with mass spectrometry analysis to identify target proteins. This analysis yielded 10 proteins including the protein kinases CKIdelta, CKIalpha, ERK2, CDK7, and p38alpha. In vitro kinase assays revealed that longdaysin inhibited CKIdelta, CKIalpha, ERK2, and CDK7 activities, while it had much less effect on p38alpha. In contrast, compound 1 inhibited CKIdelta, CKIalpha, and ERK2 with ~3-times less potency than longdaysin and inhibited CDK7 similarly to longdaysin. The difference in potency between longdaysin and compound 1 against CKIdelta, CKIalpha, and ERK2 was consistent with their cellular period effects, suggesting an involvement of these three kinases in the period regulation. Among the 10 longdaysin-interacting proteins identified by the affinity chromatography, only CKIdelta, CKIalpha, and ERK2 showed period lengthening by siRNA-mediated gene knockdown, supporting important roles for these kinases in period regulation as longdaysin targets. Although individual knockdown of CKIdelta, CKIalpha, and ERK2 had small period effects, their combinatorial knockdown dramatically lengthened the period similar to longdaysin treatment. We characterized the role of CKIalpha in the clock mechanism and found that CKIalpha-mediated phosphorylation stimulated degradation of a clock protein PER1, similar to the function of CKIdelta. Longdaysin treatment inhibited CKIdelta and CKIalpha-mediated PER1 degradation, providing insight into the mechanism of longdaysin-dependent period lengthening. Using larval zebrafish, we further demonstrated that longdaysin drastically lengthened circadian period in vivo. Taken together, the chemical biology approach not only revealed CKIalpha as a clock regulatory kinase but also identified a multiple kinase network conferring robustness to the clock. Longdaysin provides novel possibilities in manipulating clock function due to its ability to simultaneously inhibit several key components of this conserved network across species. This work was done in collaboration with Genomics Institute of the Novartis Research Foundation and The Scripps Research Institute.
U2OS cell-based circadian assay
Human osteosarcoma U2OS cells stably expressing Bmal1-dLuc reporter were suspended in the culture medium (DMEM supplemented with 10% fetal bovine serum, 0.29 mg/ml L-glutamine, 100 units/ml penicillin, and 100 ug/ml streptomycin) and plated onto 384-well white solid-bottom plates at 20 ul (2,000 cells) per well. They were cultured for 2 days to reach confluence. Then, 50 ul of the explant medium [DMEM supplemented with 2% B27, 10 mM Hepes (pH 7.2), 0.38 mg/ml sodium bicarbonate, 0.29 mg/ml L-glutamine, 100 units/ml penicillin, 100 ug/ml streptomycin, 0.1 mg/ml gentamicin, and 1 mM luciferin] was dispensed to each well, followed by the application of 500 nl of compounds (arrayed on 384-well plates at 0.5 uM - 10 mM in DMSO; 10 points of 3-fold dilution series). The plate was covered by an optically clear film, and the luminescence was recorded every 100 min for 5 days by using a microplate reader (Infinite M200, Tecan). The period parameter was obtained from the luminescence rhythm by curve fitting program MultiCycle (Actimetrics). The concentration of compound which causes 5 h, 10 h, or 15 h period lengthening is indicated in column 5h_period, 10h_period, or 15h_period.
In vitro kinase assay
The CKIdelta, CKIalpha, CDK7, and ERK2 kinase assays were performed on 384-well plate (10 ul volume). The reaction mixture was as follows: for CKIdelta, 2 ng/ul CKIdelta (Millipore, 14-520), 50 uM peptide substrate RKKKAEpSVASLTSQCSYSS corresponding to human PER2 Lys659-Ser674, and CKI buffer (40 mM Tris, 10 mM MgCl2, 0.5 mM DTT, 0.1 mg/ml BSA, pH7.5); for CKIalpha, 1 ng/ul CKIalpha (Invitrogen, PV3850), 50 uM CKI peptide substrate (Anaspec, 60547-1), and CKI buffer; for CDK7, 5 ng/ul CDK7 (Millipore, 14-476), 100 uM Cdk7/9 peptide substrate (Millipore, 12-526), and CKI buffer; for ERK2, 1.5 ng/ul ERK2 (Millipore, 14-550), 0.8 ug/ul MBP (Millipore, 13-104), and ERK buffer (50 mM Tris, 10 mM MgCl2, 0.5 mM DTT, 1 mM EGTA, pH7.5). Five hundred nl of compound (arrayed on 384-well plates at 56 nM - 10 mM in DMSO; 12 points of 3-fold dilution series) was added to the mixture (final 5% DMSO), and the reaction was started by adding ATP (final 5 uM). After incubation at 30 degrees C for 3 h, 10 ul of Kinase-Glo Luminescent Kinase Assay reagent (Promega) was added, and the luminescence was detected to determine remaining ATP amount. All of the tested compounds did not inhibit luciferase activity directly. IC50 value was obtained by using Prism software (GraphPad Software) and indicated in column CKId_vitro, CKIa_vitro, ERK2_vitro, or CDK7_vitro.
Cell-based PER1 degradation assay
HEK293T cells (60,000 cells) were reverse transfected on 96-well white solid-bottom plates by Lipofectamine 2000 with 40 ng each of expression vectors for C-terminally luciferase-fused PER1 (in p3XFLAG-CMV-14) and N-terminally HA-tagged CKIdelta or CKIalpha (in p3XFLAG-CMV-14). For luciferase (in p3XFLAG-CMV-14), 2 ng of expression vector with 38 ng of empty vector was used because of its efficient expression. After 24 h, the cells were treated with 0, 5, 10, or 20 M longdaysin for 24 h, and then the medium was supplemented with luciferin (final 1 mM) and HEPES-NaOH (pH7.2; final 10 mM). After 1 h, cycloheximide (final 20 ug/ml) was added to the medium, and the plate was covered with optically clear film. The luminescence was recorded every 10 min for 8 h by using the Tecan luminometer. Half-life was obtained by using Prism software (GraphPad Software). Half-life of PER1-LUC was divided by that of LUC to cancel out the effect of LUC degradation on PER1-LUC stability. EC50 value is indicated in column CKId_cell or CKIa_cell.
The data represent the effects of longdaysin and compound 1 on period length in U2OS cell-based circadian assay (5h_period, 10h_period, and 15h_period) and kinase activity in in vitro kinase assay (CKId_vitro, CKIa_vitro, ERK2_vitro, and CDK7_vitro) and cell-based PER1 degradation assay (CKId_cell and CKIa_cell). Compound 1 was not tested in cell-based PER1 degradation assay.
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* Activity Concentration.
Data Table (Concise)