Synthesis and Pharmacological Characterization of Visabron, a Backbone Cyclic Peptide Dual Antagonist of α4ß1 (VLA-4)/α9ß1 Integrin for Therapy of Multiple Sclerosis.

Integrins α4ß1/ α9ß1 are important in the pathogenesis and progression of inflammatory and autoimmune diseases by their roles in leukocyte activation and trafficking. Natalizumab, a monoclonal antibody selectively targeting α4ß1 integrin and blocking leukocyte trafficking to the central nervous system, is an immunotherapy for multiple sclerosis (MS). However, due to its adverse effects associated with chronic treatment, alternative strategies using small peptide mimetic inhibitors are being sought. In the present study, we synthesized and characterized visabron c (4-4), a backbone cyclic octapeptide based on the sequence TMLD, a non-RGD unique α4ß1 integrin recognition sequence motif derived from visabres, a proteinous disintegrin from the viper venom. Visabron c (4-4) was selected from a minilibrary with conformational diversity based on its potency and selectivity in functional adhesion cellular assays. Visabron c (4-4)'s serum stability, pharmacokinetics, and therapeutic effects following ip injection were assessed in an experimental autoimmune encephalomyelitis (EAE) animal model. Furthermore, visabron c (4-4)'s lack of toxic effects in mice was verified by blood analysis, tissue pathology, immunogenicity, and "off-target" effects, indicating its significant tolerability and lack of immunogenicity. Visabron c (4-4) can be delivered systemically. The in vitro and in vivo data justify visabron c (4-4) as a safe alternative peptidomimetic lead compound/drug to monoclonal anti-α4 integrin antibodies, steroids, and other immunosuppressant drugs. Moreover, visabron c (4-4) design may pave the way for developing new therapies for a variety of other inflammatory and/or autoimmune diseases.

Novel humanin analogs confer neuroprotection and myoprotection to neuronal and myoblast cell cultures exposed to ischemia-like and doxorubicin-induced cell death insults.

Humanin (HN) is a 24-amino acid mitochondrial-derived peptide, best known for its ability to protect neurons from damage caused by ischemic stroke and neurodegenerative insults and cardiomyocytes from myocardial infarction or doxorubicin (Dox)-induced cardiotoxicity. This study examines the neuroprotective and myoprotective effects of HN novel synthetic analogs HUJInin and c(D-Ser14-HN), prepared by solid-phase peptide synthesis. The cellular models employed were oxygen-glucose-deprivation (OGD) followed by reoxygenation (R)-induced neurotoxicity in PC12 and SH-SY5Y neuronal cell cultures and Dox-induced cardiotoxicity in H9c2 and C2C12 myoblast cell cultures, respectively. Necrotic and apoptotic cell death was measured by LDH release and caspase-3 activity. Erk 1/2 and AKT phosphorylations were examined by western blotting. Mitochondrial calcium and mitochondrial membrane potential were measured using the fluorescent dye tetramethylrhodamine-methyl ester. It was found that HUJInin and c(D-Ser14-HN) conferred significant dose-dependent neuroprotection, a phenomenon related to attenuation of OGD insult-induced Erk 1/2 phosphorylation, stimulation of AKT phosphorylation and improvement of mitochondrial functions. These peptides also conferred myoprotective effect towards Dox-induced apo-necrotic cell death insults. HUJInin and c(D-Ser14-HN) synthetic analogs may provide new lead compounds for the development of a potential candidate drug for stroke treatment and/or Dox-induced cardiotoxicity therapy in cancer patients.

Neurotropic activity and safety of methylene-cycloalkylacetate (MCA) derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid.

Polyneuropathy is a disease involving multiple peripheral nerves injuries. Axon regrowth remains the major prerequisite for plasticity, regeneration, circuit formation, and eventually functional recovery and therefore, regulation of neurite outgrowth might be a candidate for treating polyneuropathies. In a recent study, we synthesized and established the methylene-cycloalkylacetate (MCAs) pharmacophore as a lead for the development of a neurotropic drug (inducing neurite/axonal outgrowth) using the PC12 neuronal model. In the present study we extended the characterizations of the in vitro neurotropic effect of the derivative 3-(3-allyl-2-methylenecyclohexyl) propanoic acid (MCA-13) on dorsal root ganglia and spinal cord neuronal cultures and analyzed its safety properties using blood biochemistry and cell counting, acute toxicity evaluation in mice and different in vitro "off-target" pharmacological evaluations. This MCA derivative deserves further preclinical mechanistic pharmacological characterizations including therapeutic efficacy in in vivo animal models of polyneuropathies, toward development of a clinically relevant neurotropic drug.

Integrin α2ß1-Targeted Self-Assembled Nanocarriers for Tumor Bioimaging.

Recent developments in near-infrared (NIR) dyes and imaging modalities enable tumor fluorescent images in preclinical and clinical settings. However, NIR dyes have several drawbacks, and therefore, there is an unmet diagnostic need for NIR dye encapsulation in appropriate pharmaceutical nanocarriers with targeting abilities for the purpose of achieving effective diagnosis and image-guided surgeries. Because integrin receptors are established diagnostic targets, the cyclic Arg-Gly-Asp (RGD) peptides, recognizing the αVß3 integrin, have been extensively investigated for radiology and bioimaging of tumors. However, the Lys(Arg)-Thr-Ser [K(R)TS] cyclic peptides, selective for collagen receptors α1ß1/α2ß1 integrins, which are overexpressed in many tumors, were not yet investigated and therefore used here for tumor bioimaging with a unique α2ß1-integrin-targeted nanocarrier, encapsulating the indocyanine green NIR dye. We synthesized three kinds of peptides: two cyclic RTS peptides functional only in the cyclic conformation and a linear peptide lacking the cyclic cysteine constrained RTS loop. We used them for the preparation of integrin-targeted self-assembled nanocarriers (ITNCs), referred to as OF5 and OF27, and a nontargeted control nanocarrier, referred to as OF70. Their selective association was demonstrated with α2ß1 integrin expressing cell cultures and three-dimensional tumor spheroids and by competition with a α2ß1 selective disintegrin. Cytotoxicity experiments in vitro demonstrated the safety of the ITNCs. The targeting potential and the biodistribution of the ITNCs, applied intravenously in A431 tumor-bearing nude mice, were evaluated in vivo using NIR bioimaging. Time-dependent biodistributions indicated that the ITNC OF27 showed higher fluorescent signals in main tissues, with no cytotoxic effects to major organs, and presented higher accumulation in tumors. Cumulatively, these results highlight the potential of the ITNC OF27 as an optical and innovative pharmaceutical bioimaging system, suitable for integrin α2ß1 receptor in vivo tumor targeting and visualization in the NIR region.

Neuroprotective Effects of Bioactive Compounds and MAPK Pathway Modulation in "Ischemia"-Stressed PC12 Pheochromocytoma Cells.

This review surveys the efforts taken to investigate in vitro neuroprotective features of synthetic compounds and cell-released growth factors on PC12 clonal cell line temporarily deprived of oxygen and glucose followed by reoxygenation (OGD/R). These cells have been used previously to mimic some of the properties of in vivo brain ischemia-reperfusion-injury (IRI) and have been instrumental in identifying common mechanisms such as calcium overload, redox potential, lipid peroxidation and MAPKs modulation. In addition, they were useful for establishing the role of certain membrane penetrable cocktails of antioxidants as well as potential growth factors which may act in neuroprotection. Pharmacological mechanisms of neuroprotection addressing modulation of the MAPK cascade and increased redox potential by natural products, drugs and growth factors secreted by stem cells, in either undifferentiated or nerve growth factor-differentiated PC12 cells exposed to ischemic conditions are discussed for future prospects in neuroprotection studies.

Methylene-Cycloalkylacetate (MCA) Scaffold-Based Compounds as Novel Neurotropic Agents.

One of the main symptoms in degenerative diseases is death of neuronal cell followed by the loss of neuronal pathways. In neuronal cultures, neurite outgrowths are cell sprouts capable of transforming into either axons or dendrites, to further form functional neuronal synaptic connections. Such connections have an important role in brain cognition, neuronal plasticity, neuronal survival, and regeneration. Therefore, drugs that stimulate neurite outgrowth may be found beneficial in ameliorating neural degeneration. Here, we establish the existence of a unique family of methylene-cycloalkylacetate-based molecules (MCAs) that interface with neuronal cell properties and operate as acceptable pharmacophores for a novel neurotropic (neurite outgrowth inducing) lead compounds. Using an established PC12 cell bioassay, we investigated the neurotropic effect of methylene-cycloalkylacetate compounds by comparison to NGF, a known neurotropic factor. Micrographs of the cells were collected by using a light microscope camera, and digitized photographs were analyzed for compound-induced neurotropic activity using an NIH image protocol. The results indicate that the alkene element, integrated within the cycloalkylacetate core, is indispensable for neurotropic activity. The discovered lead compounds need further mechanistic investigation and may be improved toward development of a neurotropic drug.

Nerve Growth Factor-Induced Angiogenesis: 1. Endothelial Cell Tube Formation Assay.

Nerve growth factor (NGF) is a neurotrophin promoting survival, proliferation, differentiation, and neuroprotection in the embryonal and adult nervous system. NGF also induces angiogenic effects in the cardiovascular system, which may be beneficial in engineering new blood vessels and for developing novel anti-angiogenesis therapies for cancer. Angiogenesis is a cellular process characterized by a number of events, including endothelial cell migration, invasion, and assembly into capillaries. In vitro endothelial tube formation assays are performed using primary human umbilical vein endothelial cells, human aortic endothelial cells, and other human or rodent primary endothelial cells isolated from the vasculature of both tumors and normal tissues. Immortalized endothelial cell lines are also used for these assays. When seeded onto Matrigel, these cells reorganize to create tubelike structure, which may be used as models for studying some aspects of in vitro angiogenesis. Image acquisition by light and fluorescence microscopy and/or quantification of fluorescently labeled cells can be carried out manually or digitally, using commercial software and automated image processing. Here we detail materials, procedure, assay conditions, and cell labeling for quantification of endothelial cell tube formation. This model can be applied to study cellular and molecular mechanisms by which NGF or other neurotrophins promote angiogenesis. This model may also be useful for the development of potential angiogenic and/or anti-angiogenic drugs targeting NGF receptors.

Nerve Growth Factor-Induced Angiogenesis: 2. The Quail Chorioallantoic Membrane Assay.

The avian chorioallantoic membrane (CAM) is a simple, highly vascularized extraembryonic membrane, which performs multiple functions during embryonic development. Therefore, the models of chicken and quail assays represent robust experimental platforms to study angiogenesis, which reflects perturbation of the entire vascular tree. This experimental approach, when combined with fractal morphometry, is sensitive to changes in vascular branching pattern and density. Nerve growth factor is a neurotrophin promoting angiogenesis in CAM models. Here, we provide a detailed protocol of the quail CAM, shell-less model, to study nerve growth factor effects on blood capillary sprouting. The quail CAM assay may be beneficial in investigations of cellular and molecular aspects of neurotrophin-induced angiogenesis and for developing novel anti-angiogenesis and anticancer therapies.

The Molecular Basis of Toxins' Interactions with Intracellular Signaling via Discrete Portals.

An understanding of the molecular mechanisms by which microbial, plant or animal-secreted toxins exert their action provides the most important element for assessment of human health risks and opens new insights into therapies addressing a plethora of pathologies, ranging from neurological disorders to cancer, using toxinomimetic agents. Recently, molecular and cellular biology dissecting tools have provided a wealth of information on the action of these diverse toxins, yet, an integrated framework to explain their selective toxicity is still lacking. In this review, specific examples of different toxins are emphasized to illustrate the fundamental mechanisms of toxicity at different biochemical, molecular and cellular- levels with particular consideration for the nervous system. The target of primary action has been highlighted and operationally classified into 13 sub-categories. Selected examples of toxins were assigned to each target category, denominated as portal, and the modulation of the different portal's signaling was featured. The first portal encompasses the plasma membrane lipid domains, which give rise to pores when challenged for example with pardaxin, a fish toxin, or is subject to degradation when enzymes of lipid metabolism such as phospholipases A2 (PLA2) or phospholipase C (PLC) act upon it. Several major portals consist of ion channels, pumps, transporters and ligand gated ionotropic receptors which many toxins act on, disturbing the intracellular ion homeostasis. Another group of portals consists of G-protein-coupled and tyrosine kinase receptors that, upon interaction with discrete toxins, alter second messengers towards pathological levels. Lastly, subcellular organelles such as mitochondria, nucleus, protein- and RNA-synthesis machineries, cytoskeletal networks and exocytic vesicles are also portals targeted and deregulated by other diverse group of toxins. A fundamental concept can be drawn from these seemingly different toxins with respect to the site of action and the secondary messengers and signaling cascades they trigger in the host. While the interaction with the initial portal is largely determined by the chemical nature of the toxin, once inside the cell, several ubiquitous second messengers and protein kinases/ phosphatases pathways are impaired, to attain toxicity. Therefore, toxins represent one of the most promising natural molecules for developing novel therapeutics that selectively target the major cellular portals involved in human physiology and diseases.

Protein toxins of the Echis coloratus viper venom directly activate TRPV1.

BACKGROUND: Peptide and protein toxins are essential tools to dissect and probe the biology of their target receptors. Venoms target vital physiological processes to evoke pain. Snake venoms contain various factors with the ability to evoke, enhance and sustain pain sensation. While a number of venom-derived toxins were shown to directly target TRPV1 channels expressed on somatosensory nerve terminals to evoke pain response, such toxins were yet to be identified in snake venoms. METHODS: We screened Echis coloratus saw-scaled viper venom's protein fractions isolated by reversed phase HPLC for their ability to activate TRPV1 channels. To this end, we employed heterologous systems to analyze TRPV1 and NGF pathways by imaging and electrophysiology, combined with molecular biology, biochemical, and pharmacological tools. RESULTS: We identified TRPV1 activating proteins in the venom of Echis coloratus that produce a channel-dependent increase in intracellular calcium and outwardly rectifying currents in neurons and heterologous systems. Interestingly, channel activation was not mediated by any of its known toxin binding sites. Moreover, although NGF neurotropic activity was detected in this venom, TRPV1 activation was independent of NGF receptors. CONCLUSIONS: Echis coloratus venom contains proteins with the ability to directly activate TRPV1. This activity is independent of the NGF pathway and is not mediated by known TRPV1 toxins' binding sites. GENERAL SIGNIFICANCE: Our results could facilitate the discovery of new toxins targeting TRPV1 to enhance current understanding of this receptor activation mechanism. Furthermore, the findings of this study provide insight into the mechanism through which snakes' venom elicit pain.

Novel Synthetic PEGylated Conjugate of α-Lipoic Acid and Tempol Reduces Cell Death in a Neuronal PC12 Clonal Line Subjected to Ischemia.

α-Lipoic acid (α-LA), a natural thiol antioxidant, and Tempol, a synthetic free radical scavenger, are known to confer neuroprotection following ischemic insults in both in vivo and in vitro models. The aim of this study was to synthesize and characterize a conjugate of α-LA and Tempol linked by polyethylene glycol (PEG) in order to generate a more efficacious neuroprotectant molecule. AD3 (α-Tempol ester-ω-lipo ester PEG) was synthesized, purified, and characterized by flash chromatography and reverse phase high pressure liquid chromatography and by 1H nuclear magnetic resonance, infrared spectroscopy, and mass spectrometry. AD3 conferred neuroprotection in a PC12 pheochromocytoma cell line of dopaminergic origin, exposed to oxygen and glucose deprivation (OGD) insult measured by LDH release. AD3 exhibited EC50 at 10 µM and showed a 2-3-fold higher efficacy compared to the precursor moieties, indicating an intrinsic potent neuroprotective activity. AD3 attenuated by 25% the intracellular redox potential, by 54% lipid peroxidation and prevented phosphorylation of ERK, JNK, and p38 by 57%, 22%, and 21%, respectively. Cumulatively, these findings indicate that AD3 is a novel conjugate that confers neuroprotection by attenuation of MAPK phosphorylation and by modulation of the redox potential of the cells.

Human placental eXpanded (PLX) mesenchymal-like adherent stromal cells confer neuroprotection to nerve growth factor (NGF)-differentiated PC12 cells exposed to ischemia by secretion of IL-6 and VEGF.

Mesenchymal stem cells are potent candidates in stroke therapy due to their ability to secrete protective anti-inflammatory cytokines and growth factors. We investigated the neuroprotective effects of human placental mesenchymal-like adherent stromal cells (PLX) using an established ischemic model of nerve growth factor (NGF)-differentiated pheochromocytoma PC12 cells exposed to oxygen and glucose deprivation (OGD) followed by reperfusion. Under optimal conditions, 2 × 105 PLX cells, added in a trans-well system, conferred 30-60% neuroprotection to PC12 cells subjected to ischemic insult. PC12 cell death, measured by LDH release, was reduced by PLX cells or by conditioned medium derived from PLX cells exposed to ischemia, suggesting the active release of factorial components. Since neuroprotection is a prominent function of the cytokine IL-6 and the angiogenic factor VEGF165, we measured their secretion using selective ELISA of the cells under ischemic or normoxic conditions. IL-6 and VEGF165 secretion by co-culture of PC12 and PLX cells was significantly higher under ischemic compared to normoxic conditions. Exogenous supplementation of 10 ng/ml each of IL-6 and VEGF165 to insulted PC12 cells conferred neuroprotection, reminiscent of the neuroprotective effect of PLX cells or their conditioned medium. Growth factors as well as co-culture conditioned medium effects were reduced by 70% and 20% upon pretreatment with 240 ng/ml Semaxanib (anti VEGF165) and/or 400 ng/ml neutralizing anti IL-6 antibody, respectively. Therefore, PLX-induced neuroprotection in ischemic PC12 cells may be partially explained by IL-6 and VEGF165 secretion. These findings may also account for the therapeutic effects seen in clinical trials after treatment with these cells.

Vimocin and vidapin, cyclic KTS peptides, are dual antagonists of α1ß1/α2ß1 integrins with antiangiogenic activity.

Obtustatin and viperistatin, members of the disintegrin protein family, served as lead compounds for the synthesis of linear and cyclic peptides containing the KTS binding motif. The most active linear peptide, a viperistatin analog, indicated the importance of Cys(19) and Cys(29), as well as the presence of Arg at position 24 for their biologic activity, and was used as the basic sequence for the synthesis of cyclic peptides. Vimocin (compound 6) and vidapin (compound 10) showed a high potency (IC50 = 0.17 nM) and intermediate efficacy (20 and 40%) in inhibition of adhesion of α1/α2 integrin overexpressor cells to respective collagens. Vimocin was more active in inhibition of the wound healing (53%) and corneal micropocket (17%) vascularization, whereas vidapin was more potent in inhibition of migration in the Matrigel tube formation assay (90%). Both compounds similarly inhibited proliferation (50-90%) of endothelial cells, and angiogenesis induced by vascular endothelial growth factor (80%) and glioma (55%) in the chorioallantoic membrane assay. These peptides were not toxic to endothelial cell cultures and caused no acute toxicity upon intravenous injection in mice, and were stable for 10-30 hours in human serum. The in vitro and in vivo potency of the peptides are consistent with conformational ensembles and "bioactive" space shared by obtustatin and viperistatin. These findings suggest that vimocin and vidapin can serve as dual α1ß1/α2ß1 integrin antagonists in antiangiogenesis and cancer therapy.