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Abstract The confirmation of target engagement for candidate drugs is a fundamental requisite in drug discovery. A significant portion of projects fail to reach the clinic due to lack of efficacy or failure to show lead candidates interacting with the intended target in a more complex environment. The utility of using the CETSA® (CEllular Thermal Shift Assay) technology to measure target engagement in intact cells is increasingly exploited in high throughput screening, where examples demonstrate low false positive rates in screens of up to 500K compounds. The p53 tumor suppressor protein, a crucial guardian against cancer, functions as a transcription factor that mediates cancer suppression by regulating genes involved in a wide range of cellular processes such as DNA repair, cell cycle and apoptosis. Unfortunately, frequent mutations in the TP53 gene hinder p53's tumor suppressor role in human cancers, thus designating it as a promising target for anticancer therapy. However, attempts to restore p53 function for therapeutic purposes have not yet advanced to clinical approval, highlighting the complexities in leveraging p53 for cancer treatment. Its undruggable nature, attributed to the absence of accessible deep pockets, lack of enzymatic activity, and its intracellular location, further complicates targeted interventions with both low and high molecular weight drugs. Here, we demonstrate the analysis of a high-throughput plate based CETSA® HT screen on the challenging target p53, performed using AlphaLISA® SureFire® Ultra™ technology and our newly developed high-throughput screening platform. Hits identified from this screen are privileged from the onset by binding to the target in a physiologically relevant cellular matrix and in a binding-mode agnostic way. This is in contrast to traditional screening methods targeting activity or in vitro interaction where cellular activity is addressed in later stages. This assay cascade highlights a rapid target-to-hit lead generation strategy placing the emphasis on finding chemical matter that interacts with endogenous target in living cells first, followed by analysis of hit material activity. The ‘shortcut’ to chemical matter active in living cells represents significant potential cost savings in comparison to assay development time on recombinantly produced proteins and/or modified cell lines for traditional HTS screening approaches that still rely on target engagement assessment downstream. Citation Format: Merve Kacal, Tomas Friman, Laurence Arnold, Victoria Brehmer, Daniel Martinez Molina, Stina Lundgren. CETSA HT: Employing rapid high-throughput target engagement technology in primary screening of P53 abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts) ; 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84 (6Suppl): Abstract nr 3097.
Kacal et al. (Fri,) studied this question.