Discovery of novel non-competitive inhibitors of mammalian neutral M1 aminopeptidase (APN).

Neutral metallo-aminopeptidase (APN) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of protein or peptide substrates. APN expression is dysregulated in inflammatory diseases as well as in several types of cancer. Therefore, inhibitors of APN may be effective against cancer and inflammation. By virtual screening and enzymatic assays, we identified three non-competitive inhibitors (α > 1) of the porcine and human APN with Ki values in the μM range. These non-peptidic compounds lack the classical zinc-binding groups (ZBG) present in most of the APN inhibitors. Molecular docking simulations suggested the novel inhibitors suppress APN activity by an alternative mechanism to Zn coordination: they interacted with residues comprising the S1 and S5' subsites of APN. Of note, these compounds also inhibited the porcine aminopeptidase A (pAPA) using a competitive inhibition mode. This indicated differences in the binding mode of these compounds with APN and APA. Based on sequence and structural analyses, we predicted the significance of targeting human APN residues: Ala-351, Arg-442, Ala-474, Phe-896 and Asn-900 for improving the selectivity of the identified compounds. Remarkably, the intraperitoneal injection of compounds BTB07018 and JFD00064 inhibited APN activity in rat brain, liver and kidney indicating good bio-distribution of these inhibitors in vivo. These data reinforce the idea of designing novel APN inhibitors based on lead compounds without ZBG.