Fig. 1: Characterization of CCM binding to human BMAL1(PASB).
From: Pharmacological targeting of BMAL1 modulates circadian and immune pathways
a, Hit-to-lead optimization of BMAL1 ligand. The Kd values for SPR were determined using equilibrium analysis. b, CCM enhances the Tm of hBMAL1(PASB) by 9.7 °C in a PTS assay. c, ITC detects CCM binding to hBMAL1(PASB) protein with Kd of 1.99 ± 0.38 μM, stoichiometry (n) value of 1.201 ± 0.028 and enthalpy (ΔH) of −29.67 ± 0.826 (kJ mol−1). d, SPR demonstrates the binding of CCM to hBMAL1(PASB) with affinity of 4.05 μM calculated from kinetic analysis. e, CCM stabilizes WT hBMAL1(PASB) but not Y340F/Y404F double mutant in CETSA (left). Isothermal dose–response curves of the effects of CCM on both WT and double mutant at 48 °C to obtain EC50 values reveal key residues necessary for compound stabilization (right). Four technical replicates were applied. Data are presented as mean values ± s.d. f, Volcano plot displays the thermal profile of the U2OS proteome with CCM treatment (50 μM) compared to DMSO treatment. U2OS cell lysate (n = 3) was heated at a temperature gradient (37–60.9 °C) for 3 min, and then denatured proteins were removed by centrifugation. The combined stabilization of the proteins in the supernatant across the heat gradient was detected by MS. The P value was generated by two-tailed t-test. g, CCM stabilization of HiBiT–hBMAL1(PASB) in the same U2OS cells as in f. The y axis shows its remaining protein levels after heating/denaturation steps, detected via the intensity of the Nano-Glo HiBiT signal. Error bars denote the mean ± s.d., and unpaired two-tailed t-test was used for statistical analyses. M.W., molecular weight; Rn, normalized reporter value.
