Extended Data Fig. 9: Cyclin A/B RxL Inhibitors Induce a Neomorphic Cyclin B-Cdk2 Interaction.
From: Targeting G1–S-checkpoint-compromised cancers with cyclin A/B RxL inhibitors
a, Predicted effects of cyclin B mutations identified from the base editor screen on CIRc-004 ligand binding determined by free energy perturbation (FEP) calculations (see Methods). Mutating residues at the RxL binding site, including Ile204 and Trp208 were predicted to have the most significant effect, reducing binding affinity by 1 to 3 kcal/mol relative to cyclin B WT. Mutations of Met214Val and Val227Ala/Ser228Pro mutations showed medium impact on ligand binding 0.5-0.6 kcal/mol. *The energy convergence for the Trp208Arg mutations was poor possibly due to a conformational change from the Arg where Arg’s side chain became solvent-exposed instead of being buried under the ligand. To supplement this, the Trp208Ala mutation was examined. Consistently, both Trp208Ala and Trp208Arg mutations were predicted to have a high effect on ligand binding. b, Mass spectrometry analysis after IP of endogenous Cyclin B1 in NCI-H1048 cells treated with CIRc-004 (50 nM), CIRc-005 (50 nM) (inactive enantiomer of CIRc-004, I.E) or DMSO for 2 h. Plots show the relative abundance of the indicated proteins as Intensity (MaxLFQ, 3VN 1 group, log 2 Median centred, missing values imputed). Data points indicate values obtained from n = 3 biological replicates. Bottom right panel: Ranking plot of the log10 average of the 3 groups (CIRc-004, CIRc-005, DMSO) of raw protein abundance where proteins of interest (POI) are indicated within the total dataset. c, AlphaFold2 model of cyclin B1:Cdk2 complex is shown. Amino acids 169-177 of cyclin B are highlighted in yellow showing Cdk interacting region (pink). Solid lines indicate hydrogen bonds between the two proteins with interacting amino acid residues of cyclin B indicated. d, Immunoblot analysis after IP of endogenous cyclin B1 in NCI-H1048 cells treated with CIRc-004 (300 nM), CIRc-005 (I.E), or DMSO for 2 h. Note that this is the same immunoblot from Fig. 3h but now includes the supernatant. e, Immunoblot analysis after IP of endogenous cyclin B1 in NCI-H446 cells treated with CIRc-004 (300 nM), CIRc-005 (I.E), or DMSO for 2 h. f, Immunoblot analysis after IP of endogenous cyclin B1 in NCI-H1048 cells treated with CIRc-004 (300 nM), CIRc-005 (I.E), cyclin A RxL inhibitor (CIRc-018, 300 nM), cyclin B RxL inhibitor (CIRc-019, 300 nM) or DMSO for 2 h. g, Immunoblot analysis after IP of endogenous cyclin B1 in RPE1 cells treated with CIRc-004 (2000 nM), paclitaxel (60 nM) or DMSO for 2 h. For d-h, Cdk2 band intensity is normalized to cyclin B1 shown at the bottom. h, Immunoblot analysis after IP of endogenous cyclin A in NCI-H1048 cells treated with CIRc-004 (300 nM), CIRc-005 (I.E), cyclin A RxL inhibitor (CIRc-018, 300 nM), cyclin B RxL inhibitor (CIRc-019, 300 nM) or DMSO for 2 h. Cdk2 band intensity is normalized to cyclin A shown at the bottom. i-j, Immunoblot analysis of NCI-H1048 (i) or NCI-H446 (j) cells treated cyclin A/B RxL inhibitor (CIRc-004) at 200 nM, Cdk1 inhibitor (RO-3306), Cdk2 inhibitor (PF-07104091) at the concentrations indicated or DMSO for 4 h. k, Immunoblot analysis after IP of endogenous Cdk2 in HEK-293T cells expressing CCNB1 WT-HA, CCNB1 triple mutant-(E169K/Y170H/Y177C)-HA, or a negative control vector, and treated with CIRc-004 (300 nM) or DMSO for 2 h. HA band intensity is normalized to Cdk2 shown at the bottom. For f, g, i, representative immunoblots from n = 3 biological independent experiments are shown. For d, e, h, j, k, representative immunoblots from n = 2 biological independent experiments are shown.
