Fig 2: Physical interaction between M47 and CRYs.A, B HEK293T cells transfected with pcDNA4A-Cry1-His-Myc, pcDNA4A-Cry2-His-Myc, or pcDNA4A-Cry1-T5-His-Myc plasmids then lysates were subjected to pull-down assay. Lysates were treated with solvent (DMSO), 50 µM bM47, and 50 µM bM47 with 100 µM M47 (competitor). While M47 binds to the PHR of CRY1 (A, B) it does not bind to CRY2 (A) (n = 3). C χ1 dihedral angle distribution M47 interacting residues of CRY1 and CRY2 throughout 300 ns MD simulation. D Two superimpose images from two different angles of CRY1 and CRY2 structures. M47 binding residues (Trp399, Trp292, Arg293, and Ser396) in CRY1 and corresponding residues in CRY2 (Trp310, Ser414) are shown in sticks.

Fig 3: The effect of M47 on the half-life of CRYs.A M47 increased the degradation rate of CRY1::LUC dose-dependently. Normalized half-life is shown with ± SEM (n = 4 with triplicates). *p = 0.01, ***p = 0.0008 versus DMSO treated cells with one-way ANOVA with Dunnet’s multiple comparison test. B M47 did not affect the degradation rate of CRY2::LUC. Normalized half-life is shown with ± SEM (n = 5 with triplicates one-way ANOVA with Dunnet’s multiple comparison test). C The representative figure for the effect of M47 on the bioluminescence rhythm of U2OS CRY1 knockout (KO) Bmal1-dLuc. M47 did not affect on the circadian rhythm of the CRY1 knockout U2OS Bmal1-dLuc in dose-dependently (Data represent the mean ± SEM, n = 3 with duplicates **p = 0.002, wild-type U2OS Bmal1-dLuc versus CRY1 KO U2OS Bmal1-dLuc by two-tailed student’s t-test; ns = statistically not significant one-way ANOVA in CRY1 KO U2OS Bmal1-dLuc DMSO vs M47 treatment). D The representative figure for the effect of M47 on the bioluminescence rhythm of CRY2 knockout U2OS Bmal1-dLuc. M47 increased the period length of the circadian rhythm in U2OS CRY2 KO Bmal1-dLuc in dose-dependently (Data represent the mean ± SEM, n = 3 with duplicates **p = 0.0072 and ****p < 0.0001 versus DMSO control by one-way ANOVA with Dunnet’s post hoc test). E Binding pose of M47 on the simulated CRY1 structure predicted by AutodockVina with −11.2 kcal/mol binding energy. Protein structure is shown in surface. FBXL3 binding residues are colored as red. M47 interacting residues are shown in sticks. F M47 did not affect the half-life of mutant CRY1R293AW399L-LUC degradation Normalized half-life is shown with ± SEM (n = 4 with triplicates with one-way ANOVA test). G The ubiquitination of the CRY1 in the presence of M47. (Data represent the mean ± SEM, n = 3; *p = 0.0297 versus DMSO control with t-test with two-tailed).

Fig 4: Dose-dependent effect of M47 on the circadian rhythm of U2OS cells.A Structure of M47. B The representative figure for the effect of M47 on the bioluminescence rhythm of U2OS cells stably expressing Bmal1-dLuc. M47 lengthened the circadian rhythm dose-dependently (Data represent the mean ± SEM, n = 4 with duplicates ***p = 0.0002, *****p < 0.0001, versus DMSO control by one-way ANOVA with Dunnet’s post hoc test). C Effect of M47 on the CRY1/CRY2 double knockout (DKO) U2OS cell line (Data represent the mean ± SEM, n = 3 with duplicates ns=not significant versus DMSO control by one-way ANOVA with Dunnet’s multiple comparison test; ****p < 0.001 versus M47 treated cells by one-way ANOVA with Dunnet’s multiple comparison test).

Fig 5: CRY1 cistrome mapping reveals interplay with oncogenic transcription factors.a Heatmap and profile plots showing number and intensity of binding sites. Right—Genomic traces of CRY1 binding at core circadian clock genes. b Genomic distribution analysis representing genomic binding regions. Percent of total binding is shown in indicated regions. c De novo motif enrichment of CRY1 binding in vehicle treated C4-2 cells within 50 bp binding window on each side of the center of binding with the cutoff of p < 10−20. d Known motif enrichments of CRY1 binding in vehicle treated C4-2 cells within 500 bp binding window on each side of the center of binding with the cutoff of p < 10−20.