Fig. 2.

PHA767491 binds to and inhibits CKL family kinases. (A) Structure–activity relationship study of PHA767491 with period lengthening. Clock period change was determined compared with a DMSO treatment control [each concentration was 250 µM, n = 8, except for (1)], with Student’s t test P compared with DMSO control, Upper). Period changes by (5) or (6) (n = 8, with one-way ANOVA P, Lower). (B) Structure of PHA beads (7) and the procedure for screening proteins bound by PHA beads (Left), and binding between PHA beads and recombinant CKLs in vitro (Right). PHA767491 was added as competitor at 50 μM. GST-fusion proteins in PHA-bead fractions were analyzed by Western blotting (WB) with anti-GST antibody. (C) Inhibition of CKL kinase activity by PHA767491. (D) The IC₅₀ of PHA767491 on CKL4 was determined from three separate experiments. (See also SI Appendix, Fig. S3.) (E) Inhibition of CKL4 by PHA767491 analogs. Arrowheads and asterisks indicate phosphorylated GST-fused CKL proteins and phosphorylated casein, respectively, from C to E. Signals around 25 kDa in lanes loaded with GST-fused CKL analyzed by WB were considerable truncated GST-fused CKL from B to E.