Macrocyclic peptides as allosteric inhibitors of nicotinamide N-methyltransferase (NNMT)

Abstract

Nicotinamide N-methyltransferase (NNMT) methylates nicotinamide to form 1-methylnicotinamide (MNA) using S-adenosyl-l-methionine (SAM) as the methyl donor. The complexity of the role of NNMT in healthy and disease states is slowly being elucidated and provides an indication that NNMT may be an interesting therapeutic target for a variety of diseases including cancer, diabetes, and obesity. Most inhibitors of NNMT described to date are structurally related to one or both of its substrates. In the search for structurally diverse NNMT inhibitors, an mRNA display screening technique was used to identify macrocyclic peptides which bind to NNMT. Several of the cyclic peptides identified in this manner show potent inhibition of NNMT with IC₅₀ values as low as 229 nM. The peptides were also found to downregulate MNA production in cellular assays. Interestingly, substrate competition experiments reveal that these cyclic peptide inhibitors are noncompetitive with either SAM or NA indicating they may be the first allosteric inhibitors reported for NNMT.

Fig. 1. Schematic overview of the RaPID mRNA display system used to translate a random DNA library (>10¹² library members, 17 residues), affording a large peptide library whose members were selected for binding affinity against NNMT. Selections initiated with either N-chloroacetyl-l-tyrosine or N-chloroacetyl-d-tyrosine were performed to introduce additional structural diversity in the library. The four cyclic peptides indicated at the bottom of the figure are the most potent NNMT inhibitors identified in the present study (see Table 1).

Scheme 1. General synthesis route for the preparation of cyclic peptides identified from the mRNA display screen. The identified peptides identified all contain 18 amino acids (including a single additional C-terminal glycine for efficient synthesis).

Fig. 2. Results of the substrate competition experiment. The data is normalized per compound by setting the IC₅₀ under normal assay conditions at 1. Data is based on duplicate data of at least 7 different concentrations. Structures of control compounds X and Y are presented on the left. Full IC₅₀ curves are presented in Fig. S6 and S7 in the ESI.

Fig. 3. Cellular activity of cyclic peptides 4 and 13 and small molecule reference compound 6-methylamino-nicotinamide (6-MANA) against A549 lung carcinoma cells (left) and human aortic endothelial cells (HAEC, right). Data is based on six replicates. The results indicate a significant reduction of MNA concentration compared to untreated cells.