Fig. 2: ASP3082 demonstrates selective degradation of KRAS(G12D) via VHL-mediated proteasomal degradation and inhibits KRAS signaling pathway. | Communications Chemistry

Fig. 2: ASP3082 demonstrates selective degradation of KRAS(G12D) via VHL-mediated proteasomal degradation and inhibits KRAS signaling pathway.

From: Discovery of KRAS(G12D) selective degrader ASP3082

Fig. 2

a KRAS degradation and p-ERK inhibitory activity using In-Cell ELISA assay (24 h treatment) and cell viability using CellTiter-Glo assay (6 days treatment) of ASP3082 in AsPC-1 pancreatic cancer cells harboring KRAS(G12D) mutation. Fluorescence signals of KRAS were normalized with that of β-actin for each well, and the KRAS degradation activity was converted to a percentage, where 0% is defined by average intensity of the DMSO-treated group, and 100% is defined by that of the treatment without anti-RAS(G12D) antibody staining. Fluorescence signals of p-ERK were quantified, and p-ERK levels were converted to a percentage, where 100% is defined by DMSO treatment, and 0% is defined by 1 μM trametinib treatment. Cell viability of AsPC-1 cells treated with ASP3082 was normalized as 100% (average luminescence intensity with DMSO treatment) and 0% (average luminescence intensity without cells). Each point represents the mean ± SEM (n = 3). b KRAS degradation activity of ASP3082 in human cancer cell lines. Immunoblot analysis of cancer cell lines treated with the indicated concentrations of ASP3082 or DMSO as a control for 24 h was performed. c Cell viability of human cancer cells with KRAS(G12D) mutation (black) and wild-type (WT) KRAS (white) treated with ASP3082 for 6 days using round-bottom white plates. Cell viability was normalized as 100% (DMSO) and 0% (average luminescence intensity without cells). Each point represents the mean ± SEM (n = 1, quadruplicate). d Multiplexed quantitative proteomics analysis of ASP3082. Scatter plots for individual protein ratio of 1 μM ASP3082 versus DMSO-treated AsPC-1 cells for 4 h and 24 h treatment. Both vertical and horizontal axes are expressed as a percentage. e Proteasome-dependent KRAS degradation by ASP3082. Immunoblot analysis of AsPC-1 cells treated with the indicated concentrations of ASP3082 or DMSO as a control for 24 h was performed. Proteasome inhibitor MG-132 was pre-treated with 10 μM for 1 h prior to ASP3082 or DMSO treatment. f VHL-mediated KRAS degradation by ASP3082. KRAS(G12D) degradation in AsPC-1 cells treated with ASP3082 or compound 5 (structure shown) for 24 h was examined by In-Cell ELISA assay. Data are presented as the geometric mean (n = 3).

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