Abstract 4493: Comprehensive assay approaches for PRMT5 targeted drug discovery.

Abstract Protein arginine methyltransferase 5 (PRMT5) is a type II methyltransferase that symmetrically dimethylates arginine residues on histone and non-histone proteins, an essential epigenetic modification that shapes chromatin structure and regulates gene expression. Through these activities, PRMT5 controls key cellular processes, including transcription, RNA splicing, DNA repair, and signal transduction. PRMT5 operates in complex with MEP50, which is required for efficient catalytic activity. Dysregulated PRMT5 is strongly associated with diseases, particularly cancer. PRMT5 is frequently overexpressed in breast, lung, prostate, and hematologic malignancies, where it promotes tumor growth, metastasis, and therapy resistance. Small-molecule PRMT5 inhibitors have emerged as promising agents that block methyltransferase activity, reverse aberrant epigenetic marks, and disrupt oncogenic pathways. Here, we established comprehensive assay platforms for PRMT5-targeted drug discovery and validated them using five known inhibitors (LLY-283, JNJ-64619178, GSK591, EPZ015666, and GSK33326595). Our biochemical FlashPlate assay demonstrated potent inhibition of PRMT5/MEP50 activity, with IC50 values in the low nanomolar range (0.6-17 nM). Surface Plasmon Resonance (SPR) revealed distinct binding profiles: substrate-competitive inhibitors (EPZ015666, GSK33326595, GSK591) showed weak affinity for apo PRMT5, enhanced by cofactors (MTA, SAH, SAM), whereas cofactor-competitive LLY-283 bound tightly to apo PRMT5 but exhibited 50-fold reduced affinity with cofactors. JNJ-64619178 displayed reduced signal upon cofactor binding, driven by a slow off-rate consistent with pseudo-irreversible inhibition. NanoBRET target engagement intracellular assay results indicated that the PRMT5 inhibitors engaged with the PRMT5/MEP50 complex within one hour of incubation in live HEK293 cells, and Western blot confirmed inhibition of histone H4R3me2s methylation, a key substrate of PRMT5, in MV4-11, Jeko-1, 22RV1, and PC3 cancer cell lines. Collectively, these platforms enable identification, optimization, and mechanistic characterization of PRMT5 inhibitors, accelerating development of selective and potent therapeutic candidates. Citation Format: Jianghong Wu, Charles Schmidt, Jamin Steffen, Joseph J. Ferry, Li Liang, Shawn McGinley, Joshua Rettew, Yong Wan, Haiching Ma. Comprehensive assay approaches for PRMT5 targeted drug discovery abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 4493.