Abstract 1665: A comprehensive platform for the screening and assessment of IRAK4-targeting PROTACs

Abstract Interleukin-1 receptor-associated kinase 4 (IRAK4) serves as a pivotal mediator in the signaling cascades of both the interleukin 1 receptor (IL-1R) and toll-like receptor (TLR) pathways. It is crucial for myddosome signaling, as its kinase and scaffolding activities are indispensable for the proper formation and signaling of the myddosome complex. This complex, in turn, initiates the activation of key downstream pathways, including NFkB and interferon-responsive factors 5/7 (IRF 5/7), culminating in the release of inflammatory cytokines and chemokines such as TNF-α, IL-6, IL-1β, and IL-23. These molecules are associated with the development of various autoimmune diseases, such as pyogenic tonsillitis, psoriasis, and atopic dermatitis, positioning IRAK4 as a promising therapeutic target for dampening inflammation mediated by TLR/IL-1R. In this context, we have developed a comprehensive screening and evaluation platform for IRAK4 that spans from in vitro to in vivo studies. The platform encompasses an array of assays at both the biochemical and cellular levels. It features tests for binary and ternary complexes, intracellular detection of ternary complex formation, and a suite of methods for detecting target protein degradation, including flow cytometry, enzyme-linked immunosorbent assay (ELISA), and HiBiT. The platform also offers cellular function assays, such as cytokine release assays utilizing peripheral blood mononuclear cells (PBMCs) and whole blood, as well as assays focused on proteins associated with signal transduction pathways. It also includes off-target and selectivity testing, and extends to in vivo efficacy assessments within the IMQ-induced psoriasis model. Collectively, these assays are designed to facilitate a swift and thorough discovery and evaluation process for IRAK4-targeting PROTACs. Citation Format: Zhaoxia Yin, Xiaojian Wang, Tj (Tiejun) Bing. A comprehensive platform for the screening and assessment of IRAK4-targeting PROTACs [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 1665.