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. 2023 Jan 9;13(1):56-69.
doi: 10.1158/2159-8290.CD-22-0381.

A Small Molecule Reacts with the p53 Somatic Mutant Y220C to Rescue Wild-type Thermal Stability

Keelan Z Guiley  1 , Kevan M Shokat  1
Affiliations

A Small Molecule Reacts with the p53 Somatic Mutant Y220C to Rescue Wild-type Thermal Stability

Keelan Z Guiley et al. Cancer Discov. .

Abstract

The transcription factor and tumor suppressor protein p53 is the most frequently mutated and inactivated gene in cancer. Mutations in p53 result in deregulated cell proliferation and genomic instability, both hallmarks of cancer. There are currently no therapies available that directly target mutant p53 to rescue wild-type function. In this study, we identify covalent compsounds that selectively react with the p53 somatic mutant cysteine Y220C and restore wild-type thermal stability.

Significance: The tumor suppressor p53 is the most mutated gene in cancer, and yet no therapeutics to date directly target the mutated protein to rescue wild-type function. In this study, we identify the first allele-specific compound that selectively reacts with the cysteine p53 Y220C to rescue wild-type thermal stability and gene activation. See related commentary by Lane and Verma, p. 14. This article is highlighted in the In This Issue feature, p. 1.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest:

Dr Shokat reports grants from The Emerald Foundation, grants from Samuel Waxman Cancer Research Foundation, and grants from Howard Hughes Medical Institute during the conduct of the study; personal fees from Apertor, personal fees from BioTheryX, personal fees from BridGene Biosciences, personal fees from Black Diamond, personal fees from Denali Therapeutics, personal fees from eFFECTOR Therapeutics, personal fees from Erasca, personal fees from G Protein Therapeutics, personal fees from Genentech/Roche, personal fees from Ikena, personal fees from Initial Therapeutics, personal fees from Janssen Pharmaceuticals, personal fees from Kumquat Biosciences, personal fees from Kura Oncology, personal fees from Merck, personal fees from Mitokinin, personal fees from Nested, personal fees from Nextech, personal fees from Radd Pharma, personal fees from Revolution Medicines, personal fees from Rezo, personal fees from Totus, personal fees from Turning Point, personal fees from Type6 Therapeutics, personal fees from Wellspring Biosciences (Araxes Pharma), personal fees from Vevo, and personal fees from Vicinitas outside the submitted work; in addition, Dr Shokat has a patent for the KG series of molecules owned by UCSF pending and licensed to Nested; and I consult for Nextech which is a venture capital firm that has investments in PMV pharma which is developing p53 (Y220C) activating molecules. I do not consult for PMV pharma.

Dr Guiley reports personal fees from Nested Tx outside the submitted work; in addition, Dr Guiley has a patent for WO2021087096A1 issued and licensed to Nested Tx.

Figures

Figure. 1:
Figure. 1:. Development of a covalent PhiKan083
(A) Chemical structures of the carbazole series. (B) 1 μM p53 Y220C-CL was incubated with 10 μM of the carbazole compounds at 4°C for 24 hr. Adduct formation was observed by LC/MS. (C) HepG2 (p53 WT/+) or Huh-7 (p53 Y220C/−) cells were treated with the indicated KG5 concentration for 1 hr at 37°C and target engagement was observed by gel shift following a copper click reaction with TAMRA-azide. (D) Crystal structure of p53 Y220C-CL bound to KG3 at 2.4 Å resolution. (E) Crystal structure of the p53 Y220C-PhiKan083 complex PDB 2VUK (8).
Figure. 2:
Figure. 2:. A methacrylic indole compound induced a novel p53 conformational state
(A) Chemical structures of methacrylic indole compound series. (B) 1 μM p53 Y220C-CL was incubated with 10 μM indole series at 4°C for 24 hr. Adduct formation was analyzed by LC/MS. (C) Crystal structure of p53 Y220C-CL bound to KG6 in a novel conformational state at 1.6 Å resolution. (D) Crystal structure of p53 Y220C-CL bound to KG10 at 2.0 Å resolution.
Figure. 3:
Figure. 3:. Azaindole compounds stabilized p53 Y220C to WT levels
(A) Chemical structures of the azaindole series. (B) p53 Y220C-CL was fully labeled by the compounds, purified by size-exclusion chromatography, and its thermal stability was analyzed by differential scanning fluorimetry. The difference in Tm relative to apo p53 Y220C-CL is displayed. (C) Crystal structure of p53 Y220C-CL bound to KG13 at 1.7 Å resolution. (D) 1 μM of p53 Y220C-CL or p53 Y220C-CL D228A were incubated with 10 μM compound at 4°C for 24 hr. Adduct formation was analyzed by LC/MS.
Figure. 4:
Figure. 4:. KG13 stabilized cellular p53 Y220C and activated target gene expression
(A) 1 μM of p53 WT or p53 Y220C were incubated with 10 μM of compound at 4°C for 24 hr. Adduct formation was analyzed by LC/MS. (B) Cellular thermal shift assay intensity values were plotted with calculated Tagg values where the KG13 treated sample showed an increase in thermal stability of cellular p53 Y220C compared to DMSO control. (C) RT-qPCR results for 25 μM KG13 treated H1299 cells stably expressing p53 R273C or Y220C. (D) RT-qPCR results for 10 μM Nutlin-3a or 25 μM KG13 treated U2OS cells stably expressing p53 Y220C. (E) Western blot for DMSO, 10 μM Nutlin-3a, and 10 μM KG13 treated cell panel. (F) Viability and caspase 3/7 activity for NUGC-4, NUGC-3, or BxPC-3 cells treated with KG13.
See this image and copyright information in PMC

Comment in

  • Covalent Rescue of Mutant p53.
    Lane DP, Verma CS. Lane DP, et al. Cancer Discov. 2023 Jan 9;13(1):14-16. doi: 10.1158/2159-8290.CD-22-1212. Cancer Discov. 2023. PMID: 36620883

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