Abstract 5547: High resolution target identification by unbiased forward genetics

Abstract Forward genetic screens have revolutionized the field of target discovery, target deconvolution, and target validation, particularly in the oncology field. However, most approaches are limited to the interrogation of non-essential factors and identify gene networks rather than direct target structures. Therefore, the identification of druggable target structures and understanding an active molecules target space remains a challenge in drug discovery and development. To address this, we established a chemical mutagenesis approach in relevant cell types that allows entirely unbiased identification of small molecule targets at amino acid resolution, literally mapping compound-target interaction surfaces. Chemical mutagenesis randomly introduces single nucleotide variants into the entire genome resulting in cell populations which carry substitutions in statistically more than 90% of all amino acids. Challenging such mutagenized cell populations with a drug candidate of interest, followed by next generation sequencing and analysis, reveals direct drug target interactions, drug resistance mechanism, and potential off-targets or concealed efficacies. The alteration of single amino acids thereby allows interrogation of non-essential as well as most essential factors and increases screening resolution to the amino acid level. Several case studies will be shown. Citation Format: Moritz Horn, Georg Michlits, Andreia Rocha, Christoph Stelzer, Josef M. Penninger. High resolution target identification by unbiased forward genetics [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 5547.