Serine protease inhibitors decrease metastasis in prostate, breast, and ovarian cancers.

Targeted therapies for prostate, breast, and ovarian cancers are based on their activity against primary tumors rather than their anti-metastatic activity. Consequently, there is an urgent need for new agents targeting the metastatic process. Emerging evidence correlates in vitro and in vivo cancer invasion and metastasis with increased activity of the proteases mesotrypsin (prostate and breast cancer) and kallikrein 6 (KLK6; ovarian cancer). Thus, mesotrypsin and KLK6 are attractive putative targets for therapeutic intervention. As potential therapeutics for advanced metastatic prostate, breast, and ovarian cancers, we report novel mesotrypsin- and KLK6-based therapies, based on our previously developed mutants of the human amyloid ß-protein precursor Kunitz protease inhibitor domain (APPI). These mutants, designated APPI-3M (prostate and breast cancer) and APPI-4M (ovarian cancer), demonstrated significant accumulation in tumors and therapeutic efficacy in orthotopic preclinical models, with the advantages of long retention times in vivo, high affinity and favorable pharmacokinetic properties. The applicability of the APPIs, as a novel therapy and for imaging purposes, is supported by their good safety profile and their controlled and scalable manufacturability in bioreactors.

Unmasking BACE1 in aging and age-related diseases.

The beta-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) has long been considered a conventional target for Alzheimer's disease (AD). Unfortunately, AD clinical trials of most BACE1 inhibitors were discontinued due to ineffective cognitive improvement or safety challenges. Recent studies investigating the involvement of BACE1 in metabolic, vascular, and immune functions have indicated a role in aging, diabetes, hypertension, and cancer. These novel BACE1 functions have helped to identify new 'druggable' targets for BACE1 against aging comorbidities. In this review, we discuss BACE1 regulation during aging, and then provide recent insights into its enzymatic and nonenzymatic involvement in aging and age-related diseases. Our study not only proposes the perspective of BACE1's actions in various systems, but also provides new directions for using BACE1 inhibitors and modulators to delay aging and to treat age-related diseases.

Discovery of potent and selective dual cholinesterases and ß-secretase inhibitors in pomegranate as a treatment for Alzheimer's disease.

Pomegranate (Punica granatum L.) extract has been reported to inhibit cholinesterase and the ß-site amyloid precursor protein cleaving enzyme 1 (BACE1); however, most of its constituents' potential inhibition of these enzymes remains unknown. Thus, we investigated the anti-Alzheimer's disease (anti-AD) potential of 16 ellagitannin and gallotannin, and nine anthocyanin derivatives' inhibition of BACE1, AChE, and BChE, and gallagic acid inhibited both the best. Further, a kinetic study identified different modes of inhibition, and a molecular docking simulation revealed that active compounds inhibited these three enzymes with low binding energy through hydrophilic and hydrophobic interactions in the active site cavities. Gallagic acid and castalagin decreased Aß peptides secretion from neuroblastoma cells that overexpressed human ß-amyloid precursor protein significantly by 10 µM. Further, treatment with gallagic acid and castalagin reduced BACE1 and APPsß expression levels significantly without affecting amyloid precursor protein (APP) levels in the amyloidogenic pathway. Co-incubation of Aß42 with gallagic acid reduced Aß42-induced intracellular reactive oxygen species (ROS) production significantly. Our results suggest that pomegranate constituents, specifically gallagic acid, may be useful in developing therapeutic treatment modalities for AD.

Regulatory Effect of Adipose-Derived Mesenchymal Stem Cells and/ or Acitretin on Adam10 Gene in Alzheimer's Disease Rat Model.

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by progressive cognitive deterioration. All recent therapeutic strategies tend to inhibit the generation of the Aß peptide. These approaches tend to mediate both α - and γ -secretases to undergo the nonamyloidogenic pathway. ADAM10 is the main α-secretase that cleaves APP, and it is regulated by the metabolic product of vitamin A (retinoic acid), which is being widely used recently in AD research as a target for treatment. Mesenchymal stem cells (MSCs) are also used recently as a promising regenerative therapy for AD. OBJECTIVES: The present study aimed to: (1) study the effect of MSCs with/without acitretin on the regulation of Adam10 gene expression in AlCl3-induced AD rat model, and (2) validate the hypothesis that AD is a time-dependent progressive disease that spreads spontaneously even after the stopping of exposure to AlCl3. METHODS: The experimental work has been designed to include three successive phases; AlCl3 induction phase (I), AlCl3 withdrawal phase (W), and therapeutic phase (T). Forty-five male albino Wistar rats were randomly divided into 2 main groups: the control (C) group (15 rats) and AD group (30 rats). The therapeutic potential of MSCs with/without acitretin has been evaluated at behavioral, physiological, molecular, and histopathological levels. RESULTS: Among the three therapeutic groups, combined administration of both MSC and acitretin showed the best compensatory effects on most of the measured parameters. CONCLUSION: The present study approved that AD is a time-dependent progressive disease which spreads spontaneously without more AlCl3 exposure.

Improved Lysosomal Trafficking Can Modulate the Potency of Antibody Drug Conjugates.

Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.

The ß-amyloid precursor protein analog P165 improves impaired insulin signal transduction in type 2 diabetic rats.

This study was performed to understand whether P165 improves learning and memory by restoring insulin action using a diabetes mellitus (DM) rat model. A total of 34 male Sprague-Dawley rats were randomly divided into four groups: control group (n = 8), DM group (n = 8), DM group treated with a low dose of P165 (n = 9), and DM group treated with a high dose of P165 (n = 9). After 8 weeks of treatment, the animals were killed and the expression of insulin signaling-related proteins was examined in the hippocampus by Western blot and immunohistochemical staining. Administration of P165 in diabetic rats did not induce a significant effect on the fasting blood glucose level. The expression of IR, IRS-1, AKT, p-CREB, and Bcl-2 proteins was significantly enhanced in the hippocampus in diabetic rats. Treatment of diabetic rats with P165 at both low and high doses significantly attenuated the expression levels of these proteins. Moreover, immunohistochemistry staining showed that IR, IRS-1, AKT, p-CREB, and Bcl-2 were abundantly expressed in the CA1 region of the hippocampus. The number of cells positively stained for the above proteins was significantly higher in diabetic tissues compared to control tissues, whereas P165 treatments induced a significant reduction in the expression of these proteins. The expression of IR, IRS-1, AKT, p-CREB, and Bcl-2 was enhanced in DM rats, and administration of P165 normalized the expression of these molecules, suggesting that P165 can improve impaired insulin signal transduction.

Effects of amyloid precursor protein 17 peptide on the protection of diabetic encephalopathy and improvement of glycol metabolism in the diabetic rat.

Researchers have proposed that amyloid precursor protein 17 peptide (APP17 peptide), an active fragment of amyloid precursor protein (APP) in the nervous system, has therapeutic effects on neurodegeneration. Diabetic encephalopathy (DE) is a neurological disease caused by diabetes. Here we use multiple experimental approaches to investigate the effect of APP17 peptide on changes in learning behavior and glycol metabolism in rats. It was found that rats with DE treated by APP17 peptide showed reversed behavioral alternation. The [(18)F]-FDG-PET images and other results all showed that the APP17 peptide could promote glucose metabolism in the brain of the DE rat model. Meanwhile, the insulin signaling was markedly increased as shown by increased phosphorylation of Akt and enhanced GLUT4 activation. Compared with the DE group, the activities of SOD, GSH-Px, and CAT in the rat hippocampal gyrus were increased, while MDA decreased markedly in the DE + APP17 peptide group. No amyloid plaques in the cortex and the hippocampus were detected in either group, indicating that the experimental animals in the current study were not suffering from Alzheimer's disease. These results indicate that APP17 peptide could be used to treat DE effectively.

The n-terminal 5-MER peptide analogue P165 of amyloid precursor protein exerts protective effects on SH-SY5Y cells and rat hippocampus neuronal synapses.

The disturbance of the insulin-signaling pathway plays an important role in Alzheimer's disease. Resistance to insulin signaling renders neurons energy-deficient and vulnerable to oxidization or other metabolic insults and impairs synaptic plasticity. In search of neuroprotective drugs, we synthesized a peptide analogue, P165, an active domain of the soluble amyloid precursor protein, which is resistant to degradation and is suitable for oral administration in a clinical setting. Initially, we confirmed that P165 can protect cells from streptozotocin-caused damage and stimulate cell outgrowth using cultured SH-SY5Y cell lines treated with streptozotocin. P165 significantly reduced lactate dehydrogenase leakage from damaged cells, thereby rescuing cell energy production. Insulin signaling such as insulin receptor substrate-1 (IRS-1) and phosphoinositide 3-kinase (PI3K) proteins were upregulated to stimulate cell survival and growth. We proceeded to investigate the effect of P165 on streptozotocin-treated Alzheimer's disease (AD) rats. The data showed that P165 protected synaptic loss and dysfunction by increasing synaptophysin and PSD-95 (post synaptic density-95), while simultaneously decreasing α-synuclein expression. Moreover, animal behavior testing clearly showed that P165 increased rats' learning and memory activity. Overall, these results constitute evidence that peptide analogue 165 may protect synapse and improve learning and memory ability in AD.

Engineered proteolytic nanobodies reduce Abeta burden and ameliorate Abeta-induced cytotoxicity.

Deposition of beta-amyloid (Abeta) is considered an important early event in the pathogenesis of Alzheimer's disease (AD), and reduction of Abeta levels in the brain could be a viable therapeutic approach. A potentially noninflammatory route to facilitate clearance and reduce toxicity of Abeta is to degrade the peptide using proteolytic nanobodies. Here we show that a proteolytic nanobody engineered to cleave Abeta at its alpha-secretase site has potential therapeutic value. The Asec-1A proteolytic nanobody, derived from a parent catalytic light chain antibody, prevents aggregation of monomeric Abeta, inhibits further aggregation of preformed Abeta aggregates, and reduces Abeta-induced cytotoxicity toward a human neuroblastoma cell line. The nanobody also reduces toxicity induced by overexpression of the human amyloid precursor protein (APP) in a Chinese hamster ovary (CHO) cell line by cleaving APP at the alpha-secretase site which precludes formation of Abeta. Targeted proteolysis of APP and Abeta with catalytic nanobodies represents a novel therapeutic approach for treating AD where potentially harmful side effects can be minimized.

Amyloid precursor protein 17-mer peptide ameliorates hippocampal neurodegeneration in ovariectomized rats.

Amyloid precursor protein 17-mer peptide (APP 17-mer peptide) is an active fragment of amyloid precursor protein (APP) in the nervous system that mediates various neuronal activities and functions. Estrogen deprivation during menopause disproportionately increases the risk of many neurodegenerative diseases, including Alzheimer's disease (AD). Currently, therapeutic approaches to treat Alzheimer's disease are less than effective. We have previously shown that APP 17-mer peptide participates in neuronal function in aged-hippocampal neurons. In this study, we investigate the effects of estrogen and APP 17-mer peptide on hippocampal neurodegeneration in ovariectomized rats. The results showed that decreases in learning and memory function in ovariectomized rats were associated with degenerative changes in hippocampal neurons. Estrogen deprivation also enhances apoptotic cell death and decreases expression of the anti-apoptotic protein Bcl-2. Administration of APP 17-mer peptide ameliorates changes associated with estrogen deprivation without affecting estrogen levels. These results indicate that APP 17-mer peptide may prevent neurodegeneration caused by estrogen deficiency. Our findings also suggest that estrogen deficiency-induced neurodegeneration is regulated by activation of an intracellular "cross talk" signaling pathway, connecting neurotrophins with APP 17-mer peptide.

A novel neurotrophic peptide: APP63-73.

The objective of this study was to find out which N-terminal segment/s of amyloid precursor protein (APP) has any neurotrophic properties, since soluble APP-alpha (sAPP-alpha) has neurotrophic effects. We investigated neurotrophic properties of eight peptide segments of N-terminal APP. The APP63-73 was able to enhance neuronal growth; augment axonal and cell body growth in human neuroblastoma cell line, SH-SY5Y. Neurotrophic effects of chronic APP63-73 treatment were assessed in vivo using streptozotocin-induced diabetes and ovariectomized rats. Thus, this study demonstrated that APP63-73 peptide has neurotrophic effects both in vivo and in vitro.

The N-terminal copper-binding domain of the amyloid precursor protein protects against Cu2+ neurotoxicity in vivo.

The amyloid precursor protein (APP) contains a Cu binding domain (CuBD) localized between amino acids 135 and 156 (APP135-156), which can reduce Cu2+ to Cu1+ in vitro. The physiological function of this APP domain has not yet being established; nevertheless several studies support the notion that the CuBD of APP is involved in Cu homeostasis. We used APP synthetic peptides to evaluate their protective properties against Cu2+ neurotoxicity in a bilateral intra-hippocampal injection model. We found that human APP135-156 protects against Cu2+-induced neurotoxic effects, such as, impairment of spatial memory, neuronal cell loss, and astrogliosis. APP135-156 lacking two histidine residues showed protection against Cu2+; however, APP135-156 mutated in cysteine 144, a key residue in the reduction of Cu2+ to Cu1+, did not protect against Cu2+ neurotoxicity. In accordance with recent reports, the CuBD of the Caenorhabditis elegans, APL-1, protected against Cu2+ neurotoxicity in vivo. We also found that Cu2+ neurotoxicity is associated with an increase in nitrotyrosine immunofluorescence as well as with a decrease in Cu2+ uptake. The CuBD of APP therefore may play a role in the detoxification of brain Cu.