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.

JM-20 treatment prevents neuronal damage and memory impairment induced by aluminum chloride in rats.

The number of people with dementia worldwide is estimated at 50 million by 2018 and continues to rise mainly due to increasing aging and population growth. Clinical impact of current interventions remains modest and all efforts aimed at the identification of new therapeutic approaches are therefore critical. Previously, we showed that JM-20, a dihydropyridine-benzodiazepine hybrid molecule, protected memory processes against scopolamine-induced cholinergic dysfunction. In order to gain further insight into the therapeutic potential of JM-20 on cognitive decline and Alzheimer's disease (AD) pathology, here we evaluated its neuroprotective effects after chronic aluminum chloride (AlCl3) administration to rats and assessed possible alterations in several types of episodic memory and associated pathological mechanisms. Oral administration of aluminum to rodents recapitulates several neuropathological alterations and cognitive impairment, being considered a convenient tool for testing the efficacy of new therapies for dementia. We used behavioral tasks to test spatial, emotional- associative and novel object recognition memory, as well as molecular, enzymatic and histological assays to evaluate selected biochemical parameters. Our study revealed that JM-20 prevented memory decline alongside the inhibition of AlCl3 -induced oxidative stress, increased AChE activity, TNF-α and pro-apoptotic proteins (like Bax, caspase-3, and 8) levels. JM-20 also protected against neuronal damage in the hippocampus and prefrontal cortex. Our findings expanded our understanding of the ability of JM-20 to preserve memory in rats under neurotoxic conditions and confirm its potential capacity to counteract cognitive impairment and etiological factors of AD by breaking the progression of key steps associated with neurodegeneration.

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.

Sticholysin II-mediated cytotoxicity involves the activation of regulated intracellular responses that anticipates cell death.

Sticholysin II (StII) is a pore-forming toxin of biomedical interest that belongs to the actinoporin protein family. Sticholysins are currently under examination as an active immunomodulating component of a vaccinal platform against tumoral cells and as a key element of a nucleic acids delivery system to cell cytosol. These proteins form pores in the plasma membrane leading to ion imbalance and cell lysis. However, the intracellular mechanisms triggered by actinoporins upon binding to membranes and its consequences for cell death are barely understood. Here, we have examined the cytotoxicity and intracellular responses induced by StII upon binding to human B-cell lymphoma Raji in vitro. StII cytotoxicity involves a functional actin cytoskeleton, induces cellular swelling, lysis and the concomitant release of cytosol content. In addition, StII induces calcium release mainly from the Endoplasmic Reticulum, activates Mitogen-Activated Protein Kinase ERK and impairs mitochondrial membrane potential. Furthermore, StII stimulates the expression of receptor interacting protein kinase 1 (RIP1), normally related to different forms of regulated cell death such as apoptosis and necroptosis. In correspondence, necrostatin-1, an inhibitor of this kinase, reduces StII cytotoxicity. However, the mechanism of cell death activated by StII does not involve caspases activation, typical molecular features of apoptosis and pyroptosis. Our results suggest that, beyond pore-formation and cell lysis, StII-induced cytotoxicity could involve other regulated intracellular mechanisms connected to RIP1-MEK1/2 -ERK1/2- pathways. This opens new perspectives and challenges the general point of view that these toxins induce a completely unregulated mechanism of necrotic cell death. This study contributes to a better understanding of the molecular mechanisms involved in toxin-cell interaction and the implications for cell functioning, with connotation for the exploitations of these toxins in clinical settings.