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. 2024 Aug 22;67(16):14633-14648.
doi: 10.1021/acs.jmedchem.4c01497. Epub 2024 Aug 6.

Design, Synthesis, and Evaluation of p53Y220C Acetylation Targeting Chimeras (AceTACs)

Affiliations

Design, Synthesis, and Evaluation of p53Y220C Acetylation Targeting Chimeras (AceTACs)

Xiaoping Hu et al. J Med Chem. .

Abstract

The well-known tumor suppressor p53 is mutated in approximately half of all cancers. The Y220C mutation is one of the major p53 hotspot mutations. Several small-molecule stabilizers of p53Y220C have been developed. We recently developed a new technology for inducing targeted protein acetylation, termed acetylation targeting chimera (AceTAC), and the first p53Y220C AceTAC that effectively acetylated p53Y220C at lysine 382. Here, we report structure-activity relationship (SAR) studies of p53Y220C AceTACs, which led to the discovery of a novel p53Y220C AceTAC, compound 11 (MS182). 11 effectively acetylated p53Y220C at lysine 382 in a time- and concentration-dependent manner via inducing the ternary complex formation between p300/CBP acetyltransferase and p53Y220C. 11 was more effective than the parent p53Y220C stabilizer in suppressing the proliferation and clonogenicity in cancer cells harboring the p53Y200C mutation and was bioavailable in mice. Overall, 11 is a potentially valuable chemical tool to investigate the role of p53Y220C acetylation in cancer.

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Figures

Figure 1.
Figure 1.
Chemical structures of representative small molecules targeting p53Y220C.
Figure 2.
Figure 2.
Design of putative p53Y220C AceTACs. (A) Cocrystal structure of p53Y220C in complex with PK9328 (PDB ID: 6GGF). The cross section of the occupied binding pocket is highlighted, and the chemical structure of PK9328 is shown. (B) Cocrystal structure of the CBP bromodomain in complex with compound 17 (PDB ID: 4NR5). The cross section of the occupied binding pocket is highlighted, and the structures of compounds 17 and 33 are also shown. (C, D) Chemical structures of designed putative carbon-based AceTACs (C) and PEG-based AceTACs (D).
Figure 3.
Figure 3.
Effect of compounds 1–13 on inducing p53Y220C K382 acetylation in NCl-H1299 (p53Y220C) cell line. NCl-H1299 (p53Y220C) cells were treated with the indicated compound at 1 or 10 μM for 24 h. Cell lysates were analyzed by using Western blotting to examine the p53 and p53K382ac levels. Vinculin was used as a loading control. WB results are representative of at least two independent experiments.
Figure 4.
Figure 4.
Cellular thermal shift assay (CETSA) results of compound 11 in NCI-H1299 (p53Y220C) cells. Representative WB results of CETSA in NCI-H1299 (p53Y220C) stable cells treated with DMSO or 100 μM of compound 11 for 1 h and heat treated from 32 to 50 °C. The results shown are representative of at least two independent experiments.
Figure 5.
Figure 5.
Compound 11 is more effective than MS78 at inducing p53Y220C K382 acetylation and suppressing clonogenicity. (A) WB results of the p53K382ac level in NCI-H1299 (p53Y220C) cells treated with DMSO, PK9328, MS78, or compound 11 at the indicated concentration at 24 h. Results shown are representative of two independent experiments. Cell lysates were analyzed using Western blotting to examine the p53 and p53K382ac protein levels. Vinculin was used as the loading control. (B) Quantification of the normalized p53K382ac level (p53Y220C K382ac level over total p53Y220C protein level) following the treatment of NCI-H1299 (p53Y220C) cells with the indicated compound at 1, 3, and 10 μM for 24 h from panel A and its repeat. Error bars represent ± SD from two independent experiments. (C) Clonogenicity assay results of PK9328, compound 33, and MS78 in NCI-H1299 (p53Y220C) cells treated with DMSO or 1, 3, or 10 μM of the indicated compound for 14 days. Cells were fixed and stained with crystal violet, and the images are representative of two independent experiments. (D) Clonogenicity assay results of PK9328, compound 33, and compound 11 in NCI-H1299 (p53Y220C) cells treated with DMSO or 1, 3, or 10 μM of the indicated compound for 14 days. Cells were fixed and stained with crystal violet, and the images are representative of two independent experiments.
Figure 6.
Figure 6.
Compound 11 induces p53Y220C K382 acetylation in a concentration- and time-dependent manner. (A) Left: WB results of the p53K382ac level in NCl-H1299 (p53Y220C) cells treated with DMSO or compound 11 at 0.1, 0.3, 1, 3, or 10 μM for 24 h. Results shown are representative of three independent experiments. Cell lysates were analyzed using Western blotting to examine the p53 and p53K382ac protein levels. Vinculin was used as the loading control. Right: quantification of the normalized p53K382ac level in NCl-H1299 (p53Y220C) cells from panel A and its repeats. Error bars represent ± SD from three independent experiments. (B) WB results of the p53K382ac level in NCl-H1299 (p53Y220C) cells treated with compound 11 for 2, 4, 8, 16, or 24 h. Results shown are representative of three independent experiments. Cell lysates were analyzed using Western blotting to examine the p53 and p53K382ac protein levels. Vinculin was used as the loading control.
Figure 7.
Figure 7.
Compound 11 induces the formation of the ternary complex between p53Y220C and p300, and the p53Y220C K382 acetylation induced by compound 11 is dependent on p53Y220C and p300. (A) WB results of compound 11-mediated p53Y220C-p300 interaction via p53-FLAG pull-down in NCI-H1299 (p53-null) and NCI-H1299 (p53Y220C) cells treated with DMSO, PK9328, or compound 11 at 10 μM for 24 h. Results shown are representative of two independent experiments. (B) Left: representative WB results of the p53K282ac level induced by PK9328 or compound 11 at 0, 1, and 10 μM in NCI-H1299 p53-null and NCI-H1299 p53Y220C cells (24 h treatment); right: quantification of the p53K382ac protein level normalized to Vinculin from three independent experiments. (C) WB results of p53-FLAG pull-down after treatment of NCI-H1299 (p53Y220C) cells with DMSO, PK9328, or compound 11 at 10 μM for 24 h and with control siRNA or p300 siRNA. Results shown are representative of two independent experiments. (D) Left: representative WB results of the p53K282ac level in NCI-H1299 p53Y220C cells treated with control siRNA or p300 siRNA and then treated with PK9328 or compound 11 at 0, 1, and 10 μM for 24 h; right: quantification of the p53K382ac protein level normalized to Vinculin from two independent experiments. (E) Left: WB results of p53-FLAG pull-down after treatment of NCI-H1299 (p53Y220C) cells with PK9328 alone at 10 μM for 18 h, compound 33 alone at 10 μM for 18 h, compound 11 alone at 3 μM for 18 h, PK9328 at 10 μM for 2 h followed by compound 11 at 3 μM for 18 h, or compound 33 at 10 μM for 2 h followed by compound 11 at 3 μM for 18 h. Results shown are representative of two independent experiments. Right: quantification of the p300 and p53K382ac levels (normalized to total p53 protein level) from two independent experiments shown on the left (***P < 0.001, ns: no significance).
Figure 8.
Figure 8.
Compound 11-induced cell growth inhibition is dependent on p53Y220C and p300. (A, B) Cell viability of PK9328, compound 33, and compound 11 in (A) NCI-H1299 p53-null and (B) NCI-H1299 p53Y220C cells. The cells were treated with the indicated compound at the indicated concentrations for 72 h. The data shown represent the mean ± SD from three independent experiments. (C, D) Cell viability of PK9328, compound 33, and compound 11 in NCI-H1299 p53Y220C cells treated with (C) control siRNA or (D) p300 siRNA. The cells were treated with the indicated siRNA for 72 h and then treated with the indicated compound at the indicated concentration for 72 h. The data shown represent the means ± SD from three independent experiments.
Figure 9.
Figure 9.
RT-qPCR analysis of the relative mRNA levels of p53-target genes. NCI-H1299 p53Y220C cells were treated with DMSO, PK9328, or compound 11 at 3 μM for 24 h. The mRNA expression for each target gene was first normalized to GAPDH and then calculated relative to that of the DMSO control. The data shown represent the means ± SD from three independent experiments.
Figure 10.
Figure 10.
Compound 11 effectively inhibits cell growth in cancer cell lines that endogenously express p53Y220C. (A) Cell viability results of PK9328, compound 33, and compound 11 in BxPC3 cells treated with DMSO or the indicated compound at the indicated concentration for 72 h. The mean value ± SD for each concentration point (in technical triplicate from three biological experiments) is shown in the curves. GraphPad Prism 8 was used in analysis of raw data. (B) WB results of the p53K382ac protein level in BxPC3 cells treated with DMSO, PK9328, compound 33, or compound 11 at 1, 3, or 10 μM for 24 h. Results shown are representative of two independent experiments. Cell lysates were analyzed using Western blotting to examine the p53 and p53K382ac protein levels. Vinculin was used as the loading control. (C) Cell viability results of PK9328, compound 33, and compound 11 in NUGC-3 cells treated with DMSO or the indicated compound at the indicated concentration for 72 h. The mean value ± SD for each concentration point (in technical triplicates from three biological experiments) is shown in the curves. GraphPad Prism 8 was used in analysis of raw data. (D) WB results of the p53K382ac protein level in NUGC-3 cells treated with DMSO, PK9328, compound 33, or compound 11 at 1, 3, or 10 μM for 24 h. Results shown are representative of two independent experiments. Cell lysates were analyzed using Western blotting to examine the p53 and p53K382ac protein levels. Vinculin was used as the loading control.
Figure 11.
Figure 11.
Effect of compound 11 on the growth of WT p53-harboring cancer cells and normal cells and pharmacokinetic (PK) assessment of compound 11. (A) Cell viability assay results of PK9328, compound 33, and compound 11 in U2OS (WT p53) cells treated with the indicated compound at the indicated concentration for 72 h. The mean value ± SD for each concentration point (in technical triplicates from two biological experiments) is shown in the bar graph. (B) WB results of the p53K382ac protein level in U2OS cells treated with DMSO, PK9328, compound 33, or compound 11 at 1, 3, and 10 μM for 24 h. WB results shown are representative of two independent experiments. (C) Cell viability assay results of PK9328 and compound 11 in normal prostate PNT2 cells treated with the indicated compound at the indicated concentration for 72 h. The mean value ± SD for each concentration point (in technical triplicate from two biological experiments) is shown in the bar graph. (D) Plasma concentrations of compound 11 over 8 h in Swiss albino mice were determined following a single 50 mg/kg IP injection. Experiments were performed in biological triplicates. Each point represents the mean concentration ± standard error of the mean (SEM) from three mice.
Scheme 1.
Scheme 1.
Synthetic Routes for Putative p53Y220C AceTACs 1–13a aReaction and conditions: (a) tert-butyl (2-bromoethyl)carbamate, KI, K2CO3, N,N-dimethylformamide (DMF), 60 °C, overnight; (b) LiOH, THF/H2O, room-temperature (rt), 1 h; (c) I4-. HATU, diisopropylethylamine (DIPEA), DMF, rt; (d) AcOH, reflux, overnight; (e) TFA/dichloromethane (DCM), rt; (f) AcOH, NaBH3CN, MeOH, rt, 12 h; (g) DIPEA, (Boc)2O, acetonitrile (ACN), 30 min, rt; (h) linkers 1–2, HATU, DIPEA, DMF, rt; (i) linkers 3–13, HATU, DIPEA, DMF, rt (j) HCl, MeOH, rt, 30 min.

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