Structure-Based Drug Discovery of Non-SAM-Mimetic Bisubstrate Inhibitors against Nicotinamide <i>N</i> -Methyltransferase

Nicotinamide N -methyltransferase (NNMT) has emerged as a regulator of cellular methylation, epigenetic remodeling, and energy homeostasis. Its aberrant expression has been implicated in cancer, metabolic disorders, and renal diseases. Although several NNMT inhibitors have been reported, most lack selectivity, cellular activity, or favorable pharmacokinetic properties. Here, we describe the discovery and optimization of a novel non-SAM-mimetic bisubstrate inhibitor of NNMT originating from high-throughput screening. Guided by structure-based design, systematic modifications of hit compound 1 yielded lead compound 16 with over 1000-fold improved potency (IC 50 for compounds 1 and 16 = 10 μM and 0.0084 μM, respectively). Compound 16 exhibited sub-micromolar cell-based activity and high selectivity for a subfamily of methyltransferases. In rodents, compound 16 exhibited pronounced renal distribution and moderate bioavailability, while achieving dose-dependent renal NNMT inhibition in a renal fibrosis model. These results highlight compound 16 as a promising probe and a starting point for NNMT-targeted drug discovery.