Identification of novel Piperazine based bisubstrate inhibitors of human nicotinamide N-methyltransferase (hNNMT) with potential anticancer activities

Abstract

Overexpression of human Nicotinamide N-methyltransferase (hNNMT), nictotinamide metabolizing enzyme, is associated with numerous diseases including metabolic disorders and several cancers. Hence, development of novel inhibitors against hNNMT activity would result in New Chemical Entities (NCEs). Further, identification of novel bisubstrate inhibitors which bind to substrate and co-factor binding pockets of hNNMT is more challenging and warranted. In this present study, we methodically investigated piperazine derivatives as novel bisubstrate inhibitors of hNNMT with potential anticancer activities. Initially, studies on the piperazine derivatives were undertaken to determine their binding affinities with hNNMT in silico. The molecules, which displayed potent binding affinity in silico, were synthesized and further subjected to biochemical, biophysical and computational studies. Thus, we have identified a novel bisusbtrate inhibitor of hNNMT with nanomolar IC₅₀. Computational studies demonstrated that incorporation of quinolone pharmacophore enables the binding in the pocket of cofactor “SAM” and pyridine moiety at the substrate “nicotinamide”. The MOA studies suggest that compound 7b functions as a bisubstrate competitive inhibitor of hNNMT, exhibiting competitive inhibition with respect to both SAM and quinoline, without altering V_max. These observations indicate the identification of novel bisubstrate inhibitors which can be further diversified to improve their DMPK properties. Furthermore, cellular assays showed that compounds 7b, 7c, and 7d selectively suppressed the proliferation of U87 and PANC-1 cancer cells, and low cytotoxicity in HEK293 normal cell lines. Notably, compound 7b exhibited the strongest antiproliferative activity, underscoring the therapeutic potential of this scaffold in targeting NNMT-associated cancers.