School of Chemistry, University of Nottingham, Nottingham NG7 2RD, U.K.
Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham NG7 2RD, U.K.
Biochem J. 2020 Feb 28;477(4):787-800. doi: 10.1042/BCJ20190826.
Attenuating the function of protein arginine methyltransferases (PRMTs) is an objective for the investigation and treatment of several diseases including cardiovascular disease and cancer. Bisubstrate inhibitors that simultaneously target binding sites for arginine substrate and the cofactor (S-adenosylmethionine (SAM)) have potential utility, but structural information on their binding is required for their development. Evaluation of bisubstrate inhibitors featuring an isosteric guanidine replacement with two prominent enzymes PRMT1 and CARM1 (PRMT4) by isothermal titration calorimetry (ITC), activity assays and crystallography are reported. Key findings are that 2-aminopyridine is a viable replacement for guanidine, providing an inhibitor that binds more strongly to CARM1 than PRMT1. Moreover, a residue around the active site that differs between CARM1 (Asn-265) and PRMT1 (Tyr-160) is identified that affects the side chain conformation of the catalytically important neighbouring glutamate in the crystal structures. Mutagenesis data supports its contribution to the difference in binding observed for this inhibitor. Structures of CARM1 in complex with a range of seven inhibitors reveal the binding modes and show that inhibitors with an amino acid terminus adopt a single conformation whereas the electron density for equivalent amine-bearing inhibitors is consistent with preferential binding in two conformations. These findings inform the molecular basis of CARM1 ligand binding and identify differences between CARM1 and PRMT1 that can inform drug discovery efforts.
抑制蛋白质精氨酸甲基转移酶（PRMTs）的功能是研究和治疗多种疾病（包括心血管疾病和癌症）的目标。同时针对精氨酸底物和辅因子（S-腺苷甲硫氨酸（SAM））结合位点的双底物抑制剂具有潜在的应用价值，但需要了解其结合的结构信息才能开发它们。通过等温滴定微量热法（ITC）、活性测定和晶体学报告了评估具有等排胍替代物的双底物抑制剂对两种主要酶 PRMT1 和 CARM1（PRMT4）的研究结果。主要发现是 2-氨基吡啶是胍的可行替代品，提供了一种比 PRMT1 更强地结合 CARM1 的抑制剂。此外，鉴定了活性位点周围的一个残基，该残基在 CARM1（Asn-265）和 PRMT1（Tyr-160）之间不同，它影响了晶体结构中催化重要相邻谷氨酸的侧链构象。突变数据支持该残基对观察到的这种抑制剂结合差异的贡献。与一系列七种抑制剂结合的 CARM1 的结构揭示了结合模式，并表明具有氨基酸末端的抑制剂采用单一构象，而具有等效胺的抑制剂的电子密度则与两种构象的优先结合一致。这些发现为 CARM1 配体结合的分子基础提供了信息，并确定了 CARM1 和 PRMT1 之间的差异，这些差异可为药物发现工作提供信息。
