PO.ET09.01 · 实验与分子治疗
面向PRMT5靶向药物发现的综合实验方法
Comprehensive assay approaches for PRMT5 targeted drug discovery
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
蛋白精氨酸甲基转移酶5（PRMT5）是一种II型甲基转移酶，可对组蛋白和非组蛋白上的精氨酸残基进行对称二甲基化，这是一种塑造染色质结构并调控基因表达的重要表观遗传修饰。通过这些活动，PRMT5控制着包括转录、RNA剪接、DNA修复和信号转导在内的关键细胞过程。PRMT5与MEP50形成复合物发挥作用，MEP50是高效催化活性所必需的。PRMT5失调与疾病密切相关，尤其是癌症。PRMT5在乳腺癌、肺癌、前列腺癌和血液系统恶性肿瘤中经常过表达，促进肿瘤生长、转移和治疗耐药。小分子PRMT5抑制剂已成为有前景的药物，可阻断甲基转移酶活性、逆转异常的表观遗传标记并破坏致癌通路。在此，我们建立了面向PRMT5靶向药物发现的综合实验平台，并使用五种已知抑制剂（LLY-283、JNJ-64619178、GSK591、EPZ015666和GSK33326595）对其进行了验证。我们的生化FlashPlate实验显示对PRMT5/MEP50活性的强效抑制，IC₅₀值处于低纳摩尔范围（0.6-17 nM）。表面等离子体共振（SPR）揭示了不同的结合特征：底物竞争性抑制剂（EPZ015666、GSK33326595、GSK591）对apo PRMT5的亲和力较弱，被辅因子（MTA、SAH、SAM）增强，而辅因子竞争性的LLY-283与apo PRMT5紧密结合，但在辅因子存在下亲和力降低>50倍。JNJ-64619178在辅因子结合时显示信号降低，这由与假不可逆抑制一致的缓慢解离速率驱动。NanoBRET细胞内靶点结合实验结果表明，PRMT5抑制剂在活HEK293细胞中孵育一小时内即与PRMT5/MEP50复合物结合，Western印迹证实了在MV4-11、Jeko-1、22RV1和PC3癌细胞系中对组蛋白H4R3me2s甲基化（PRMT5的关键底物）的抑制。总的来说，这些平台能够对PRMT5抑制剂进行鉴定、优化和机制表征，加速选择性强效治疗候选物的开发。
查看英文原文 English 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.
利益披露 Disclosure
J. Wu, None..
C. Schmidt, None..
J. Steffen, None..
J. J. Ferry, None..
L. Liang, None..
S. McGinley, None..
J. Rettew, None..
Y. Wan, None..
H. Ma, None.