With the aid of receptor-oriented pharmacophore-based in silico screening, we established three pharmacophore maps explaining the binding model of hPNMT and a known inhibitor, SK&F 29661 (Martin et al., 2001). The compound library was searched using these maps. Nine-teen selected candidate inhibitors of hPNMT were screened using STD-NMR and fluorescence experiments. An enzymatic activity assay based on HPLC was additionally performed. Consequently, three potential hPNMT inhibitors were identified, specifically, 4-oxo-1,4-dihydro-quinoline-3,7-dicarboxylic acid, 4-(benzo[d][1,3]dioxol-5-ylamino)-4-oxobutanoic acid, and 1,4-diaminonaphthalene-2,6-disulfonic acid. These novel inhibitors were retrieved using Map II comprising one hydrogen bond acceptor, one hydrogen bond donor, one lipophilic feature, and shape constraints, including a hydrogen bond between Lys57 of hPNMT and a hydrogen bond donor of the inhibitor, and stacked hydrophobic interactions between the side-chain of Phe182 and an aromatic region of the inhibitor. Water-mediated interactions between Asn267 and Asn39 of hPNMT and the amide or amine group of three potent inhibitors were additional important features for hPNMT activity. The binding model presented here may be applied to identify inhibitors with higher potency. Moreover, our novel compounds are valuable candidates for further lead optimization of PNMT inhibitors.

Keywords: adrenaline, in silico screening, PNMT, pharmacophore, STD-NMR