Extended Data Fig. 7: Additional characterization of CCNE1:CDK2 2,6-diazaspiro[3.4]octane ligands.

a, Gel-ABPP data showing concentration-dependent blockade of YZ-01-A engagement of N112C-CCNE1 by I-125A or I-198 in 30 min or 90 min reactions. Experiment was performed as described in Fig. 3c. Data are from a single experiment. b, Gel-ABPP data showing that the residual YZ-01-A reactivity with N112C-CCNE1 in the presence of I-125A or I-198 (1 µM) is stereoselective and site-specific. Experiments were performed as described in Fig. 3c. Data are from a single experiment representative of two independent experiments with similar results. c, NanoBRET data showing concentration-dependent blockade of CDK2 active site-directed tracer K-10 (0.2 µM) binding to Flag-N112C-(left) or Flag-WT-CCNE1(right):CDK2-NLuc complexes by dinaciclib, but not I-125A, as performed in HEK293T cell lysates recombinantly expressing CCNE1:CDK2-NLuc complexes. Data are average values ± s.e.m. from three biological replicates. d, Gel-ABPP data showing the concentration-dependent reactivity of WX-03-348 with WT-CCNE2. Purified WT- or C109A-CCNE2:CDK2 complexes (0.2 µM each) were doped into HEK293T cell lysates (1 mg mL⁻¹) and treated with WX-03-346 or WX-03-348 for 100 min. Right, quantification of data from two biological replicates. e, Gel-ABPP data showing that I-125A does not block the reactivity of WX-03-348 with WT-CCNE2. Purified WT- or C109A-CCNE2:CDK2 complexes (0.2 µM each) were doped into HEK293T cell lysates (1 mg mL⁻¹) and treated with WX-03-57, WX-03-59 (both at 20 µM), or I-125A (1 or 10 µM) for 2 h followed by WX-03-348 (3 µM) for 100 min at RT. Data are from a single experiment representative of two independent experiments with similar results.

Source data