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 Kd 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.
Keywords: Fluorescence polarization; High-throughput screening; Inhibitor; Nicotinamide N-Methyltransferase; Protein methyltransferase.
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