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 K 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.
Cancer is the second leading cause of death worldwide. Peptidases participate in tumor development and growth. Mammalian neutral aminopeptidase (APN, EC 3.4.11.2, M1 family) catalyzes the cleavage of neutral and basic amino acids from the N-terminus of substrates. APN expression is dysregulated in several types of cancer, being a target for the development of new anticancer agents. Recently, we identified three new non-competitive inhibitors of soluble porcine APN (pAPN) by virtual screening (BTB11079, JFD00064, BTB07018, from Maybridge). In the present contribution we assayed their effect on the activity of APN in a microsomal preparation of porcine kidney cortex, a model of the physicochemical environment of the enzyme. These classical inhibitors had an IC50 value of 3–5 µM. Additionally, using a kinetic approach and a specific substrate, we quantified APN activity on the cell surface of human and murine lung, colon, prostate, and skin tumor cells. APN inhibitors reduced tumor cells viability, more efficiently in the higher APN activity tumor cell lines, but not in non-tumoral cells. BTB11079, JFD00064, BTB07018 effects on cell viability were stronger than that of bestatin, a positive control. Thus, these non-competitive APN inhibitors may be useful tools for cancer treatment.
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