Department of Medicinal Chemistry and Molecular Pharmacology, Center for Cancer Research, Institute for Drug Discovery, Purdue University, West Lafayette, IN, 47907, United States.
Department of Medicinal Chemistry and Molecular Pharmacology, Center for Cancer Research, Institute for Drug Discovery, Purdue University, West Lafayette, IN, 47907, United States.
Anal Biochem. 2020 Sep 1;604:113833. doi: 10.1016/j.ab.2020.113833. Epub 2020 Jul 2.
Methylation-mediated pathways play important roles in the progression of various diseases. Thus, targeting methyltransferases has proven to be a promising strategy for developing novel therapies. Nicotinamide N-methyltransferase (NNMT) is a major metabolic enzyme involved in epigenetic regulation through catalysis of methyl transfer from the cofactor S-adenosyl-l-methionine onto nicotinamide and other pyridines. Accumulating evidence infers that NNMT is a novel therapeutic target for a variety of diseases such as cancer, diabetes, obesity, cardiovascular and neurodegenerative diseases. Therefore, there is an urgent need to discover potent and specific inhibitors for NNMT to assess its therapeutical potential. Herein, we reported the design and synthesis of a fluorescent probe II138, exhibiting a K value of 369 ± 14 nM for NNMT. We also established a fluorescence polarization (FP)-based competition assay for evaluation of NNMT inhibitors. Importantly, the unique feature of this FP competition assay is its capability to identify inhibitors that interfere with the interaction of the NNMT active site directly or allosterically. In addition, this assay performance is robust with a Z'factor of 0.76, indicating its applicability in high-throughput screening for NNMT inhibitors.
甲基化介导的途径在各种疾病的进展中发挥着重要作用。因此，靶向甲基转移酶已被证明是开发新型治疗方法的一种有前途的策略。烟酰胺 N-甲基转移酶（NNMT）是一种主要的代谢酶，通过将辅助因子 S-腺苷甲硫氨酸上的甲基转移到烟酰胺和其他吡啶上，参与表观遗传调控。越来越多的证据推断，NNMT 是癌症、糖尿病、肥胖症、心血管疾病和神经退行性疾病等多种疾病的新的治疗靶点。因此，迫切需要发现强效和特异的 NNMT 抑制剂来评估其治疗潜力。在此，我们报道了荧光探针 II138 的设计和合成，其对 NNMT 的 K 值为 369±14nM。我们还建立了基于荧光偏振（FP）的竞争测定法来评估 NNMT 抑制剂。重要的是，这种 FP 竞争测定法的独特之处在于它能够识别直接或变构干扰 NNMT 活性部位相互作用的抑制剂。此外，该测定法的性能稳健，Z'因子为 0.76，表明其适用于 NNMT 抑制剂的高通量筛选。
