Abstract 3166: High-throughput screening and triage assays for the identification of PPI disruptors in cancer cells

Abstract Epigenetic and transcriptional dysregulations play pivotal roles in cancer development, progression aggressiveness, and recurrence of malignancies. In collaboration with a pioneering stealth company sprouted from the academic world, we leveraged an innovative drug discovery platform designed to target the disruption of the target protein-protein interaction (PPI). Using a luminescence complementation assay, we identified small molecules capable of selectively disrupting the target PPI within cells (native conformation, i.e. target assay) as well as disrupting the interacted proteins in cellular extracts. Over 4.105 compounds were screened using the target assay, successfully adapted to a 384-well plate format, in the search of a specific compound disrupting the native conformation of the PPI. An overall statistical hit rate of 0.4% (cut-off ranging between 46%-57% inhibition, depending on the collection tested) was obtained at the completion of the compounds’ libraries screening tested at one concentration (20 µM). Up to 1% of screened compounds were selected for further validation via dose response assessment on both the target and the counter-screening assays (i.e. unrelated protein-protein interaction related to cancer progression). Among the 5, 000 compounds promoted, 184 compounds were identified as putative selective disruptors of the target, while 107 were selected for further assessments due to a > 0.7 log shift in IC50. Similarity search using Tanimoto coefficient from confirmed hits was performed to select additional analogs compounds from the original pool. A total of 1, 279 confirmed hits and their analogs were promoted to potency evaluations on the target and three counter-screening assays. These assays included dynamic interactions in cellular assays and protein disturbance in cell lysates, confirmed the activity on the target assay of more than 60% of the compounds. Notably, 27 hits were confirmed as specific in the native conformation assays, while 88 compounds demonstrated specificity in lysate-based assays. Among these, seven compounds were validated as selective and specific disruptors, and one compound showing a >1 log shift in IC50 between target PPI and unrelated cancer PPI. A subset of compounds has undergone further validation through co-immunoprecipitation and target-specific in vitro growth arrest models, showing encouraging results. Following a well-defined screening cascade that integrates bioactivity in cancer-specific cell lines with cytotoxicity assays in non-transformed cells, the compounds will be further triaged to select the best qualified hits. Targeting PPI directly or indirectly has been a challenging and long-standing goal in cancer research. By integrating diverse expertise and resources, from academia and industry, this drug development program has not only been accelerated but it was enhanced by the robustness of the findings. Citation Format: Aurelie Dos Santos, Roberta De Gioia, Alice Segnali, Debora Zian, Cinzia Nucci, Maria Pia Catalani, Angela Molteni. High-throughput screening and triage assays for the identification of PPI disruptors in cancer cells [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 3166.