Marine Invertebrates: A Promissory Still Unexplored Source of Inhibitors of Biomedically Relevant Metallo Aminopeptidases Belonging to the M1 and M17 Families.

Proteolytic enzymes, also known as peptidases, are critical in all living organisms. Peptidases control the cleavage, activation, turnover, and synthesis of proteins and regulate many biochemical and physiological processes. They are also involved in several pathophysiological processes. Among peptidases, aminopeptidases catalyze the cleavage of the N-terminal amino acids of proteins or peptide substrates. They are distributed in many phyla and play critical roles in physiology and pathophysiology. Many of them are metallopeptidases belonging to the M1 and M17 families, among others. Some, such as M1 aminopeptidases N and A, thyrotropin-releasing hormone-degrading ectoenzyme, and M17 leucyl aminopeptidase, are targets for the development of therapeutic agents for human diseases, including cancer, hypertension, central nervous system disorders, inflammation, immune system disorders, skin pathologies, and infectious diseases, such as malaria. The relevance of aminopeptidases has driven the search and identification of potent and selective inhibitors as major tools to control proteolysis with an impact in biochemistry, biotechnology, and biomedicine. The present contribution focuses on marine invertebrate biodiversity as an important and promising source of inhibitors of metalloaminopeptidases from M1 and M17 families, with foreseen biomedical applications in human diseases. The results reviewed in the present contribution support and encourage further studies with inhibitors isolated from marine invertebrates in different biomedical models associated with the activity of these families of exopeptidases.

Bufadienolides preferentially inhibit aminopeptidase N among mammalian metallo-aminopeptidases; relationship with effects on human melanoma MeWo cells.

Bufadienolides are steroids that inhibit Na+/K+-ATPase; recent evidence shows that bufalin inhibits the activity of porcine aminopeptidase N (pAPN). We evaluated the selectivity of some bufadienolides on metallo-aminopeptidases. Among the enzymes of the M1 and M17 families, pAPN and porcine aminopeptidase A (pAPA) were the only targets of some bufadienolides. ѱ-bufarenogin, telocinobufagin, marinobufagin, bufalin, cinobufagin, and bufogenin inhibited the activity of pAPN in a dose-dependent manner in the range of 10-7-10-6 M. The inhibition mechanism was classical reversible noncompetitive for telocinobufagin, bufalin and cinobufagin. Bufogenin had the lowest Ki value and a non-competitive behavior. pAPA activity was inhibited by ѱ-bufarenogin, cinobufagin, and bufogenin, with a classical competitive type of inhibition. The models of enzyme-inhibitor complexes agreed with the non-competitive type of inhibition of pAPN by telocinobufagin, bufalin, cinobufagin, and bufogenin. Since APN is a target in cancer therapy, we tested the effect of bufadienolides on the MeWo APN+ human melanoma cell line; they induced cell death, but we obtained scant evidence that inhibition of APN contributed to their effect. Thus, APN is a selective target of some bufadienolides, and we suggest that inhibition of APN activity by bufadienolides is not a major contributor to their antiproliferative properties in MeWo cells.

Discovery of tight-binding competitive inhibitors of dipeptidyl peptidase IV.

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.

Bacitracin is a non-competitive inhibitor of porcine M1 family neutral and glutamyl aminopeptidases.

Membrane alanyl and glutamyl aminopeptidases (APN and APA, respectively) are established targets for the development of biomedical tools in human pathologies. APN overexpression correlates with the progression of tumours, including melanoma. Bacitracin, widely used as a topical antibiotic, inhibits subtilisin-like serine peptidases and disulphide isomerases. In the present contribution, we demonstrate that bacitracin is a non-competitive α = 1 and α < 1 inhibitor of porcine kidney APN and APA, respectively, with Ki values in the micromolar range. To test a potential application of this result, we assayed the effect of bacitracin on murine melanoma MB16F10 cell line viability. We demonstrated the cell line expresses an APN-like activity inhibited by bacitracin and bestatin. Additionally, we identified a cytotoxic effect of bacitracin. Further experiments are required to understand in depth the mechanisms of action of bacitracin on melanoma cells. They will clarify the therapeutic potential of bacitracin for melanoma treatment.

Bestatin and bacitracin inhibit porcine kidney cortex dipeptidyl peptidase IV activity and reduce human melanoma MeWo cell viability.

Bestatin and bacitracin are inhibitors of metallo aminopeptidases and bacterial proteases. However, their effects on other human peptidases, like dipeptidyl peptidase IV (DPP-IV, EC 3.4.14.5) are not established. Inhibitors of DPP-IV activity are used for treating type 2 diabetes mellitus, cancers and immune system diseases. Bacitracin and bestatin inhibited porcine membrane-bound DPP-IV (pDPP-IV) activity. Mechanisms were different, i.e. non-competitive with α > 1 (α = 3.9) and Ki value of 75 µM for bestatin, and competitive with Ki value of 630 µM for bacitracin. The binding mode in the tertiary complex enzyme:substrate:bestatin suggested the structural basis of the inhibitory effect and that bestatin is potentially selective for DPP-IV, ineffective vs. S9 family members dipeptidyl peptidase 8/9 and fibroblast activation protein. In the human melanoma MeWo cell line, bestatin and bacitracin inhibited aminopeptidase N (APN) and DPP-IV activities, reduced cell viability and increased DNA fragmentation, suggesting induction of apoptosis. Since bacitracin and bestatin are already marketed drugs, studying in depth the molecular mechanisms underlying their effects on melanoma cells is warranted. Additionally, bestatin emerges as a new lead compound for the development of DPP-IV inhibitors, and a promising dual APN/DPP-IV inhibitor for the treatment of pathologies in which both enzymes are upregulated.

Biochemical evidences for M1-, M17- and M18-like aminopeptidases in marine invertebrates from Cuban coastline.

Metallo-aminopeptidases (mAPs) control many physiological processes. They are classified in different families according to structural similarities. Neutral mAPs catalyze the cleavage of neutral amino acids from the N-terminus of proteins or peptide substrates; they need one or two metallic cofactors in their active site. Information about marine invertebrate's neutral mAPs properties is scarce; available data are mainly derived from genomics and cDNA studies. The goal of this work was to characterize the biochemical properties of the neutral APs activities in eight Cuban marine invertebrate species from the Phyla Mollusca, Porifera, Echinodermata, and Cnidaria. Determination of substrate specificity, optimal pH and effects of inhibitors (1,10-phenanthroline, amastatin, and bestatin) and cobalt on activity led to the identification of distinct neutral AP-like activities, whose biochemical behaviors were similar to those of the M1 and M17 families of mAPs. Additionally, M18-like glutamyl AP activities were detected. Thus, marine invertebrates express biochemical activities likely belonging to various families of metallo-aminopeptidases.