Journal: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Potent and selective cytotoxicity of TAPTAC1 correlates with degradation of BET proteins and reactivation of the p53 pathway (A) To assess the relative potency and selectivity of TAPTAC1, we compared its anti-cancer activity in culture with A1874, a 2-in-1 PROTAC comprising the selective HDM2 inhibitor RG7388 and JQ1, and the TAPTAC1 F19A point mutant, which abrogates interaction of the stapled p53 peptide component of the chimera with HDM2 and HDMX. (B–D) In SJSA-1 cells that have genetic amplification of HDM2 but little to no HDMX expression, TAPTAC1 exhibits marginally increased potency compared to A1874, as assessed by viability assay (B). In SJSA-X cells, engineered to express HDMX, the dual-targeting capability of TAPTAC1 results in markedly enhanced potency compared to A1874 (C). In Saos-2 cells, which lack p53, no p53/HDM2/HDMX-based activity or selectivity is evident, consistent with the compounds functioning similarly based on residual BET inhibitor activity alone (D). In each cell line, F19A point mutagenesis likewise abrogates the p53-dependent activity of TAPTAC1, further highlighting its specificity of action. Data are mean ± SEM for experiments performed in technical quadruplicate and repeated three times using independent cultures with similar results. (E) Upon treatment of SJSA-X cells with 100 nM TAPTAC1, we observed prompt degradation of BRD4 within 8 h, coinciding with time-dependent upregulation of p53, which peaked at 12 h and triggered both a surge in p21 and counter-elevation of HDM2 and HDMX by 24 h. The reduction of p53 levels observed between 12 and 24 h is consistent with the characteristic negative feedback loop of the p53 pathway. Time-dependent western blot analyses were performed twice using independent SJSA-X cell cultures and compound treatment. NT, no treatment. (F) Quantitative proteomics revealed that TAPTAC1 treatment (1 μM, 24 h) of SJSA-X cells caused a striking reduction of BET protein levels (cyan, e.g., BRD2-4) and marked upregulation of p53 pathway proteins (red, e.g., p53 [TP53], p21 [CDKN1A], HDM2 [MDM2], and HDMX [MDM4]). Quantitative proteomic analysis was performed using three biological replicates representing independent cultures and treatment. ∗, isoform. See also , , and .

Article Snippet: RG7388 , MedChemExpress , Cat# HY-15676.

Techniques: Activity Assay, Mutagenesis, Amplification, Expressing, Viability Assay, Western Blot, Quantitative Proteomics

Journal: Cell Reports Medicine

Article Title: A triple-action PROTAC for wild-type p53 cancer therapy

doi: 10.1016/j.xcrm.2025.102467

Figure Lengend Snippet: Comparative cellular activity and specificity of TAPTAC1 (A and B) The A1874 chimera of RG7388 and JQ1 is no more cytotoxic to SJSA-1 cells than the combination of its individual components, as evaluated by viability assay (A). In contrast, TAPTAC1 is over 30-fold more potent than its individual stapled p53 peptide and JQ1 components in impairing the viability of cultured SJSA-X cells (B). Data are mean ± SEM for experiments performed in technical quadruplicate and repeated three times using independent cultures with similar results. (C) Comparative viability of SJSA-X cells in response to treatment with TAPTAC1, its stapled p53 peptide component, dBET6 (CRBN-based degrader of BET proteins), and JQ1 (BET inhibitor). Data are mean ± SEM for experiments performed in technical quadruplicate and repeated three times using independent cultures with similar results. (D) Comparative effect of TAPTAC1 on WT and p53-null HCT116 cells, demonstrating the selective cytotoxic activity of TAPTAC1 in HCT116 cells that express WT p53. Data are mean ± SEM for experiments performed in technical quadruplicate and repeated two times using independent cultures with similar results. (E) Dose- and time-responsive effect of TAPTAC1 treatment on caspase 3/7 activation in SJSA-X (WT p53) and Saos-2 (p53 −/− ) cells, demonstrating the WT p53-dependence of apoptosis induction by TAPTAC1. Data are mean ± SEM for experiments performed in technical quadruplicate and repeated two times using independent cultures with similar results. (F) Dose- and time-responsive effect of TAPTAC1 treatment on senescence-associated β-galactosidase (SA-β-gal) activity in SJSA-X and Saos-2 cells, demonstrating little to no effect on Saos-2 cells and a decrement of SA-β-gal activity in SJSA-X cells, consistent with cellular loss from apoptosis induction (E). Data are mean ± SEM for experiments performed in technical quadruplicate and repeated two times using independent cultures with similar results.

Article Snippet: RG7388 , MedChemExpress , Cat# HY-15676.

Techniques: Activity Assay, Viability Assay, Cell Culture, Activation Assay