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.

Figure 1

Chemical structures of NNMT inhibitors.

Figure 2

Design of the bisubstrate NNMT inhibitors.

Figure 3

Docking analysis of compound 6 in the binding sites of hNNMT. (A) Docking of compound 6 (green) in the hNNMT (cyan) structure (PDB 2IIP). H-bond interactions are shown in yellow dotted lines. Water molecules are illustrated as red balls. (B) Overlay of the docking model with the hNNMT (gray)–nicotinamide (magenta)–SAH (yellow) complex (PDB 3ROD).

Figure 4

Biochemical and biophysical characterization of 6 and 7 and selectivity assessment of 6. (A) IC₅₀ determination for 6 (14 ± 1.5 μM; HS: 1.1) and 7 (160 ± 1 μM) against hNNMT (n = 3). (B) ITC was used to assess binding of hNNMT to 6 (left) and 7 (right). (C) Selectivity of 6 was determined against a panel of 34 MTs and 1 HAT at two compound concentrations (10 μM (blue) and 50 μM (red)). (D) IC₅₀ determination for 6 against DOT1L (1.3 ± 0.2 μM; HS: 1.1), PRMT7 (20 ± 2 μM; HS: 1.1), BCDIN3D (40 ± 2 μM; HS: 1.3), and SMYD2 (62 ± 7 μM; HS: 0.6) were performed in triplicate. HS: Hill slope.

Figure 5

MOA study of NNMT inhibition with 6. (A) Lineweaver–Burk plot of 1/rate versus 1/[SAM] with varying concentrations of inhibitor 6 showing competitive inhibition and (B) Lineweaver–Burk plot of 1/rate versus 1/[nicotinamide] with varying concentrations of inhibitor 6 showing noncompetitive inhibition. Eight concentrations of inhibitor 6 (from 2 to 250 μM) were used for both experiments: 2 μM (●), 4 μM (○), 8 μM (▲), 16 μM (△), 31 μM (■), 63 μM (□), 125 μM (◆), and 250 μM (⋄). ITC was used to assess binding of (C) SAM and (D) nicotinamide to hNNMT.

Figure 6

X-ray crystal structures. (A) The X-ray crystal structure of a binary complex of 6 (green) and hNNMT (cyan) (PDB 6B1A). The surface of the protein is shown in gray. (B) Key interactions between 6 and hNNMT. H-bond interactions are shown in yellow dotted lines. Water molecules are illustrated as red balls. (C) Overlay of the crystal structure of the hNNMT (cyan)–6 (green) binary complex (PDB 6B1A) with the crystal structure of hNNMT (not shown)–nicotinamide (magenta)–SAH (yellow) complex (PDB 3ROD). (D) Overlay of the crystal structure of the hNNMT (cyan)–6 (green) binary complex (PDB 6B1A) with the crystal structure of monkey NNMT (not shown)–1 (blue)–SAH (yellow) complex (PDB 5XVQ).

Scheme 1. Synthetic route for 6 and 7^a

^aReagents and conditions: (a) NaBH(OAc)₃, AcOH, DCM, rt, 16 h, 61–71%; (b) K₂CO₃, DMSO, H₂O₂, 0 °C to rt, 3 h; (c) HCl in dioxane, 0 °C to rt, 4 h; then 2 drops of water, rt, 4 h, 46–57% over 2 steps.