Abstract 3162: New hit compounds for p53 identified with a CETSA based high throughput screen

Abstract The p53 tumor suppressor protein is of central importance in tumor biology because of its role as guardian against neoplastic growth. The importance of p53 is highlighted by the frequency of mutations that have been identified in cancers. Most of these mutations are inactivating the original functions of the protein but some mutations also provide p53 with new functions that act pro-tumorigenic. Therefore, therapeutic strategies and initiatives that either restore the original function of p53 or limit gained functions have been applied. Numerous compounds have been reported in the literature to modulate p53 function. Still, to date, no project has proven successful in the clinical setting, which highlights the difficulty of finding compounds to manipulate p53. Reasons for the undruggable nature of p53 comprise the absence of accessible deep pockets and its lack of enzymatic activity. To understand the nature of the interaction between a set of reference compounds and p53, we subjected these molecules to a high throughput (HT) assay based on the Cellular Thermal Shift Assay (CETSA). Three different breast cancer cell lines with different p53 mutational status were used for this study: SK-BR-3 (p53R175H), BT-474 (p53E285K), and MCF-7 (p53WT). We generated ten-point concentration - response curves for all compounds in each of the three cell lines. In addition, knowing the complex nature of both p53 and its central role in the cell, we ran the assay both in lysate and intact cells. Target engagement could be established for some of the compounds in intact cells but was lacking in lysate. There was also a cell type specific variation in effects of the compounds. As an example, Butein exhibited single digit micromolar potency in intact SK-BR-3 cells, whereas there was very little effect in MCF-7 cells, which could be attributed to the difference in p53 mutational status between these cells. To learn more about the reference compounds and to investigate if their effects could be attributed to other cellular protein interactions, we subjected them to a proteome wide CETSA profiling, using mass spectrometry as a read out. The target engagement profile of the tested compounds was as diverse as their chemical structures, with very little overlap. Next, we again turned to the CETSA HT platform to screen a library of low molecular weight compounds aiming to identify new hit compounds for mutant p53. The screen was successful in identifying compounds that induced a thermal shift of mutant p53 in intact SK-BR-3 cells. These hits were then confirmed by QPCR based efficacy assays which showed effects on p53 regulated transcription. Here we highlight the applicability of CETSA in investigating the relevance of annotated tool compounds and how the properties of CETSA make it well-suited for screening for new hit matter, also when aiming for notoriously hard to drug targets. Citation Format: Tomas Friman, Merve Kacal, Laurence Arnold, Stina Lundgren, Daniel Martinez Molina. New hit compounds for p53 identified with a CETSA based high throughput screen [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3162.