Discovery of Bisubstrate Inhibitors of Nicotinamide N-Methyltransferase (NNMT)

Abstract

Nicotinamide N-methyltransferase (NNMT) catalyzes the N-methylation of pyridine-containing compounds using the cofactor S-5'-adenosyl-l-methionine (SAM) as the methyl group donor. Through the regulation of the levels of its substrates, cofactor, and products, NNMT plays an important role in physiology and pathophysiology. Overexpression of NNMT has been implicated in various human diseases. Potent and selective small-molecule NNMT inhibitors are valuable chemical tools for testing biological and therapeutic hypotheses. However, very few NNMT inhibitors have been reported. Here, we describe the discovery of a bisubstrate NNMT inhibitor MS2734 (6) and characterization of this inhibitor in biochemical, biophysical, kinetic, and structural studies. Importantly, we obtained the first crystal structure of human NNMT in complex with a small-molecule inhibitor. The structure of the NNMT-6 complex has unambiguously demonstrated that 6 occupied both substrate and cofactor binding sites. The findings paved the way for developing more potent and selective NNMT inhibitors in the future.

Probing the Plasticity in the Active Site of Protein N-terminal Methyltransferase 1 Using Bisubstrate Analogs

“…1 Protein Expression and Purification. Expression and purification of NTMT1, G9a, SETD7, PRMT1, PRMT3, TbPRMT7 and NNMT was performed as previously described [6], [18], [35], [37]- [40] NTMT1 Biochemical Assays and Enzyme Kinetics Study. A fluorescence-based SAHH-coupled assay was applied to study the IC50 values of compounds with both SAM and the peptide substrate at their For NNMT, a noncoupled fluorescence assay was performed in a final well volume of 40 µL as described before [41], [42] : 5 mM potassium phosphate buffer (pH = 7.6), 0.4 µM NNMT and 20 µM SAM.…”

Development of fluorescence polarization-based competition assay for nicotinamideN-methyltransferase

“…Both assays require additional coupling enzymes and reagents to produce a detectable signal. In the SAHH-coupled fluorescence assay, AdoHcy is hydrolyzed by SAHH to generate homocysteine, which is then reacted with a thiol-specific dye to form a strong fluorescent signal [17,18]. The MTase-Glo TM assay needs several coupling enzymes to convert AdoHcy to ADP and produce a luciferase signal [17,19].…”

“…In the SAHH-coupled fluorescence assay, AdoHcy is hydrolyzed by SAHH to generate homocysteine, which is then reacted with a thiol-specific dye to form a strong fluorescent signal [17,18]. The MTase-Glo TM assay needs several coupling enzymes to convert AdoHcy to ADP and produce a luciferase signal [17,19]. Direct assay to measure the methylation product like MNAM has been developed through the use of high-pressure liquid chromatography (HPLC) [20].…”

“…In addition, we also checked the relative binding of the cofactor and substrate for NNMT. The cofactor AdoMet (Km of 24 ± 6.8 µM) was determined to have a Ki of 20.27 ± 2.9µM[17]. The substrates NAM and 1-…”

Synthesis of Adenine Dinucleosides 2′,5′‐Bridged by Sulfur‐Containing Linkers as Bisubstrate SAM Analogues for Viral RNA 2′‐O‐Methyltransferases

“…In the same way, several bisubstrates for diverse methyltransferases (DNA MTases, catechol MTase, protein MTases, nicotinamide MTase) have been previously reported in the literature. Nevertheless, it is noteworthy some relevant examples of the use of bisubstrate nucleosidic analogues for the study of nucleic acids methyltransferases.…”