ABSTRACT
Dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) is an abundant serine aminopeptidase that preferentially cleaves N-terminal Xaa-Pro or Xaa-Ala dipeptides from oligopeptides. Inhibitors of DPP-IV activity are used for treating type 2 diabetes mellitus and other diseases. DPP-IV is also involved in tumor progression. We identified four new non-peptide tight-binding competitive inhibitors of porcine DPP-IV by virtual screening and enzymatic assays. Molecular docking simulations supported the competitive behavior, and the selectivity of one of the compounds in the DPP-IV family. Since three of these inhibitors are also aminopeptidase N (APN) inhibitors, we tested their impact on APN+/DPP-IV+ and DPP-IV+ human tumor cells' viability. Using kinetic assays, we determined that HL-60 tumor cells express both APN and DPP-IV activities and that MDA-MB-231 tumor cells express DPP-IV activity. The inhibitors had a slight inhibitory effect on human HEK-293 cell viability but reduced the viability of APN+/DPP-IV+ and DPP-IV+ human tumor cells more potently. Remarkably, the intraperitoneal injection of these compounds inhibited DPP-IV activity in rat brain, liver, and pancreas. In silico studies suggested inhibitors binding to serum albumin contribute to blood-brain barrier crossing. The spectrum of action of some of these compounds may be useful for niche applications.
Subject(s)
Dipeptidyl Peptidase 4/chemistry , Dipeptidyl-Peptidase IV Inhibitors/chemistry , Dipeptidyl-Peptidase IV Inhibitors/pharmacology , Drug Discovery , Models, Molecular , Animals , Binding Sites , Cell Survival/drug effects , Enzyme Activation , Humans , Hydrolysis , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Protein Binding , Substrate Specificity , SwineABSTRACT
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.
