Peptide LIQ Promotes Cell Protection against Zinc-Induced Cytotoxicity through Microtubule Stabilization.

Stability of the microtubule protein (MTP) network required for its physiological functions is disrupted in the course of neurodegenerative disorders. Thus, the design of novel therapeutic approaches for microtubule stabilization is a focus of intensive study. Dynamin-related protein-1 (Drp1) is a guanosine triphosphatase (GTPase), which plays a prevailing role in mitochondrial fission. Several isoforms of Drp1 have been identified, of which one of these isoforms (Drp1-x01) has been previously described with MTP stabilizing activity. Here, we synthesized peptide LIQ, an 11-amino-acid peptide derived from the Drp1-x01 isoform, and reported that LIQ could induce tubulin assembly in vitro. Using a Stern-Volmer plot and continuous variation method, we proposed one binding site on tubulin for this peptide. Interestingly, FRET experiment and docking studies showed that LIQ binds the taxol-binding site on ß-tubulin. Furthermore, circular dichroism (CD) spectroscopy and 8-anilino-1-naphthalenesulfonic acid (ANS) assay provided data on tubulin structural changes upon LIQ binding that result in formation of more stable tubulin dimers. Flow cytometry analysis and fluorescence microscopy displayed that cellular internalization of 5-FAM-labeled LIQ is attributed to a mechanism that mostly involves endocytosis. In addition, LIQ promoted polymerization of tubulin and stabilized MTP in primary astroglia cells and also protected these cells against zinc toxicity. This excellent feature of cellular neuroprotection by LIQ provides a promising therapeutic approach for neurodegenerative diseases.

Site Directed Disulfide PEGylation of Interferon-ß-1b with Fork Peptide Linker.

The attachment of PEG to biopharmaceuticals has been applied for enhancement of bioavailability and improved stability. The PEG polymer is highly hydrated; thus effective attachment to inaccessible sites could be hindered. We have devised a scheme to address this issue by introducing a considerable distance between PEG and protein by addition of a linear peptide, appended to long chained reactive linkers. Second, the position of PEG conjugation directly affects biological activity. Accordingly, a disulfide bond could be considered as an ideal choice for site directed PEGylation; but reactivity of both thiol moieties to bridging reagent is critical for maintenance of protein structure. In our design, a forked structure with two arms provides essential flexibility to account for dissociation of reduced cysteines. An efficient yield for disulfide PEGylation of IFN-ß1b was attained and specificity, biophysical characterization, biological activity, and pharmacokinetics were surveyed.

Ac-SDKP ameliorates the progression of experimental autoimmune encephalomyelitis via inhibition of ER stress and oxidative stress in the hippocampus of C57BL/6 mice.

Despite the attention given to the treatment of multiple sclerosis (MS), still no certain cure is available. The main purpose of MS drugs is acting against neuroinflammation which underlies the pathology of MS. Neuroinflammation is associated with endoplasmic reticulum (ER) stress that mediates neural apoptosis. In the present study, we hypothesized that the tetrapeptide N-acetyl-ser-asp-lys-pro (Ac-SDKP) with the previously described anti-fibrotic effects might have anti-inflammatory, anti-oxidative and anti-ER stress roles in the hippocampus. We used myelin oligodendrocyte glycoprotein (MOG) to induce experimental autoimmune encephalomyelitis (EAE), a widely-accepted animal model of MS, in C57BL/6 mice. The protein levels of ER stress-related molecules including caspase-12, C/EBP homologous protein (CHOP), and protein disulfide isomerase (PDI) in the hippocampus were examined by immunoblotting. Hence, reactive oxygen species (ROS) production, lipid peroxidation and antioxidant capacity of the hippocampus were studied. Moreover, hippocampal morphology changes, leukocytes infiltration, and the levels of IL-6 and IL-1ß pro-inflammatory cytokines were evaluated. Our results displayed that Ac-SDKP down regulates caspase-12 and CHOP expression in the hippocampus-resident oligodendrocytes of EAE mice. Further, treatment with Ac-SDKP decreased oxidative stress markers and caspase-3 activation in the hippocampus of EAE mice. According to our findings, Ac-SDKP showed beneficial effects against ER stress and oxidative stress in addition to inflammation in the hippocampus of EAE mice. The present study provides the basis for further research on the therapeutic applications of Ac-SDKP to reduce ER stress and oxidative stress-induced apoptosis in neurodegenerative disorders.

Comparative evaluation of adolescent repeated psychological or physical stress effects on adult cognitive performance, oxidative stress, and heart rate in female rats.

Multiple adult health problems are associated with adolescent stress. As the brain discriminates physical and psychological stressors by activation of different neural networks, we hypothesized that behavioral and physiological performance would be modulated differently based on the nature of the stressors. Thus, we studied the comparative effects of adolescent repeated physical and psychological stresses on adult cognitive performance, pro-oxidant-antioxidant balance (PAB) and heart rate in female rats. The aim was to differentiate disparate potency of chronic psychological and physical stresses leading to long-term behavioral and physiological alterations. Twenty-one female rats were divided randomly into three groups of seven rats each; control, physical, and psychological stress. Experimental rats were exposed to the stressors for five consecutive days (10 min daily) via a two-communication box. After verifying stress induction by serum corticosterone measurement, the rats were returned to their home cage for 6 weeks, until adulthood, elevated plus maze (EPM), forced swimming test (FST), Y-maze, object recognition task (ORT), and passive avoidance test (PAT) were used as five different behavioral tests to evaluate cognitive performance of each group. Serum PAB and heart rate were measured to assess long-term stress-induced physiological disorders. The results showed exposure to adolescent psychological stress resulted in a larger set of significant changes (in behavioral variation, oxidative stress, and elevated heart rate) 6 weeks post-stress compared to adolescent physical stress. Hence, mental health care in adolescence and therapies targeting PAB and heart rate could be prevention and treatment approaches to confront persistent adolescent stress-induced disorders. Lay summaryThe aim of our study on female laboratory rats was to differentiate disparate potency of chronic psychological and physical stresses in adolescence leading to long-term behavioral and physiological alterations. The results suggest that psychological stresses result in a greater extent of changes compared to physical stress. Adolescent chronic psychological stress may reveal itself in the form of certain behavioral and physiological variations in adulthood. Therefore, mental health care in adolescence could be a valuable prevention approach to confront a variety of adult stress-induced disorders.

α-Cyperone of Cyperus rotundus is an effective candidate for reduction of inflammation by destabilization of microtubule fibers in brain.

ETHNOPHARMACOLOGICAL RELEVANCE: Cyperus rotundus L. (Cyperaceae), commonly known as purple nutsedge or nut grass is one of the most invasive and endemic weeds in tropical, subtropical and temperate regions. This plant has been extensively used in traditional medicine for anti-arthritic, antidiarrheal and antiplatelet properties as well as treatment for several CNS disorders such as epilepsy, depression and inflammatory disorders. Inflammation is evidently occurring in pathologically susceptible regions of the Alzheimer's disease (AD) brain as well as other disorders. Many cellular processes are responsible in chronic inflammation. Microtubule-based inflammatory cell chemotaxis is a well-recognized process that influences production of cytokines and phagocytosis. The effect of α-Cyperone, one of main ingredients of Cyperus rotundus on microtubule assembly and dynamics has not been examined and is the purpose of this investigation. MATERIALS AND METHODS: Microtubules and tubulin were extracted in order to explore their interaction with α-Cyperone by utilization of turbidimetric examinations, intrinsic fluorescence and circular dichroism spectroscopy (CD) studies. The molecular docking analysis was executed in order to facilitate a more detail and stronger evidence of this interaction. The BINding ANAlyzer (BINANA) algorithm was used to evaluate and further substantiate the binding site of α-Cyperone. RESULTS: It was demonstrated that α-Cyperone had a pronounced influence on the tubulin structure, decreased polymerization rate and reduced concentration of polymerized tubulin in vitro. The CD deconvolution analysis concluded that significant conformational changes occurred, demonstrated by a drastic increase in content of ß-strands upon binding of α-Cyperone. The fluorescence spectroscopy revealed that a static type of quenching mechanism is responsible for binding of α-Cyperone to tubulin. Upon characterization of various biophysical parameters, it was further deduced that ligand binding was spontaneous and a single site of binding was confirmed. Transmission electron microscopy revealed that upon binding of α-Cyperone to microtubule the number and complexity of fibers were noticeably decreased. The computational analysis of docking suggested that α-Cyperone binds preferably to ß-tubulin at a distinct location with close proximity to the GTP binding and hydrolysis site. The ligand interaction with ß-tubulin is mostly hydrophobic and occurs at amino acid residues that are exclusively on random coil. The BINANA 1.2.0 algorithm which counts and tallies close molecular interaction by performing defined set of simulations revealed that amino acid residues Arg 48 and Val 62 have registered the highest scores and are possibly crucial in ligand-protein interaction. CONCLUSION: α-Cyperone binds and interacts with tubulin and is capable of distinctly destabilizing microtubule polymerization. The effect of this interaction could result in reduction of inflammation which would be highly beneficial for treatment of inflammatory diseases such as AD.

Trichloroacetic acid treatment as a tricky way for rapid purification of 1N/4R tau protein.

Tau protein consists of six different isoforms and each one has particular physiological roles. In order to analyze the specific function of each single isoforms, large quantity of highly purified tau isoforms is essential. Many studies have been done to purify tau isoforms by heat treatment, followed by perchloric acid and glycerol precipitation. We found out that 1N/4R tau is soluble in glycerol, that is why mentioned methods do not work for purifying this isoform. In this study, large amounts of active and highly purified (97%) 1N/4R tau protein has been prepared by utilization of trichloroacetic acid as precipitating agent.

Multimeric peptide-based PEG nanocarriers with programmable elimination properties.

In the current study, the design, synthetic feasibility and biochemical characterization of biodegradable peptidic PEG-based nanocarriers are described. The components were selected to influence the body elimination pathway upon nanocarrier biodegradation. Two prototypical nanocarriers were prepared using non-PEGylated and PEGylated peptidic cores [CH(3)CO-(Lys-betaAla-betaAla)(X)-Cys-CONH(2) (X=2, 4)]. A homodimeric nanocarrier with 4 copies of fluorescein-PEG5kDa was synthesized by linking two PEGylated peptidic cores (X=2) using a disulfide bond. A dual labeled heterodimeric nanocarrier with 2 copies of fluorescein-PEG5kDa and 4 copies of Texas Red was also synthesized. Optimum conditions for linking imaging agents, PEG, or a peptidic core to a peptidic core were determined. Significantly higher yields (69% versus 30%) of the PEGylated peptidic core were obtained by using 2 copies of beta-alanine as a spacer along with increasing DMSO concentrations, which resulted in reduced steric hindrance. Stoichiometric addition of the components was also demonstrated and found to be important for reducing polydispersity. Nanocarrier biodegradation was evaluated in simulated intracellular and extracellular/blood environments using 3 mm and 10 microm glutathione in buffer, respectively. The nanocarrier was 9-fold more stable in the extracellular environment. The results suggest selective intracellular degradation of the nanocarrier into components with known body elimination pathways.

Endocytosis and membrane potential are required for HeLa cell uptake of R.I.-CKTat9, a retro-inverso Tat cell penetrating peptide.

Cell-penetrating peptides (CPPs) can enter many types of cells and have become useful tools for introducing a variety of cargo such as exogenous peptides, proteins, and nucleic acids into cultured cells in vitro. Tat CPPs derived from the HIV-1 Tat protein are the most widely used among the arginine-rich CPPs. Even though CPPs hold considerable promise for drug delivery, poor biological stability and high in vivo clearance may limit their effectiveness for delivering cargo. Therefore, we utilize a retro-inverso form of a Tat peptide, R.I.-CKTat9, which is proteolytically stable. In the current study, the cellular entry mechanism of this arginine-rich CPP is investigated. Fluorescently labeled R.I.-CKTat9 entered HeLa cells in a concentration- and energy-dependent manner demonstrating both diffuse and punctate (vesicular) appearance inside the cells. The labeled R.I.-CKTat9 colocalized with labeled transferrin in the punctate structure, suggesting that the peptide enters HeLa cells by clathrin-dependent endocytosis. Incubation of cells with an isotonic/high K(+) buffer (KPBS) or an NH(4)Cl solution abolished the diffuse but not the punctate fluorescence, suggesting that membrane potential plays a critical role. This result also suggests that the flux originates from the endosome, not the extracellular space, and relies on the acidity of the endosome. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain function and endocytosis inhibitors greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a single route of endocytosis and subsequent endosomal escape. Since cells in the mitotic (M) phase shut down endocytosis but maintain plasma membrane potential, this property was used to further confirm the endocytic mechanism. Direct measurement of plasma membrane potential confirmed its persistence in M phase arrested HeLa cells. Consistent with our working hypothesis, these cells did not show any vesicular nor diffuse fluorescence of labeled R.I.-CKTat9, providing compelling evidence for the sequential steps of endocytosis and endosomal escape. Binding of labeled R.I.-CKTat9 to the surface of HeLa cells at 0 degrees C was reduced under the mildly acidic conditions of early endosomes, suggesting an acidity-dependent endosomal escape mechanism. Overall, these results indicate that both endocytosis and membrane potential are required for R.I.-CKTat9 entry into HeLa cells and suggest that translocation occurs at the endosomal membrane.

Optimizing size and copy number for PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarrier uptake by macrophages.

Curing HIV-1 infection has remained elusive because of low and fluctuating drug levels arising from poor absorption, the development of viral reservoirs and sanctuary sites, toxicity, and patient nonadherence. The present study addresses the issue of insufficient drug exposure in macrophages. Viral reservoir sites such as macrophages are believed to be responsible for the viral rebound effect observed upon the discontinuation of anti-HIV drug therapy. In our proposed model, a drug can be covalently attached to a nanocarrier in order to facilitate the delivery of therapeutic agents to the site(s) of infection. As an initial step, we propose the covalent attachment of several copies of N-formyl-Met-Leu-Phe (fMLF), a known chemo-attractant for macrophages. In this article, one or more copies of fMLF were conjugated to multifunctional commercially available or novel, peptide-based PEG nanocarriers in which the structure was varied by appending PEGs with average molecular weights of 5, 20, and 40 kDa. U937 cell-specific binding and cellular uptake were analyzed. The results of uptake studies indicate that (i) uptake is energy dependent and mediated by a fMLF receptor, (ii) appending only 2 copies of the targeting ligand to the multifunctional nanocarrier appears sufficient for binding in vitro, and (iii) of the three configurations studied, the nanocarrier with a molecular weight of about 20 kDa, corresponding to a size of 20-60 nm, demonstrated the highest uptake. The results of the current studies demonstrate the feasibility of targeting macrophages and the suitability of using these synthetically versatile peptide--backbone PEG nanocarriers. The convenience, flexibility and possible limitations of this nanocarrier approach are discussed.

Peritoneal macrophage uptake, pharmacokinetics and biodistribution of macrophage-targeted PEG-fMLF (N-formyl-methionyl-leucyl-phenylalanine) nanocarriers for improving HIV drug delivery.

PURPOSE: To assess in vivo macrophage targeting potential of PEG-fMLF nanocarriers and to investigate their biodistribution, peritoneal macrophage uptake, and pharmacokinetics. METHODS: Multiple copies of fMLF were conjugated to purchased and novel (branched, peptide-based) PEG nanocarriers. Peritoneal macrophage uptake was evaluated in mice 4 hours after IP administration of fluorescence-labeled PEG-fMLF nanocarriers. Pharmacokinetics and biodistribution were determined in rats after IV administration of tritiated PEG-fMLF nanocarriers. RESULTS: Attachment of one, two, or four fMLF copies increased uptake in macrophages by 3.8-, 11.3-, and 23.6-fold compared to PEG without fMLF. Pharmacokinetic properties and tissue distribution also differed between nanocarriers with and without fMLF. Attachment of fMLF residues increased the t(1/2) of PEG(5K) by threefold but decreased the t(1/2) of PEG(20K) by 40%. Attachment of fMLF increased accumulation of nanocarriers into macrophages of liver, kidneys and spleen. However, on a molar basis, penetration was equivalent suggesting nanocarrier size and targeting moieties are important determinants. CONCLUSIONS: These results demonstrate the feasibility for targeting macrophages, a primary HIV reservoir site. However, these studies also suggest that balancing peripheral tissue penetration (a size-dependent phenomenon) versus target cell uptake specificity remains a challenge to overcome.

Novel multi-component nanopharmaceuticals derived from poly(ethylene) glycol, retro-inverso-Tat nonapeptide and saquinavir demonstrate combined anti-HIV effects.

BACKGROUND: Current anti-AIDS therapeutic agents and treatment regimens can provide a dramatically improved quality of life for HIV-positive people, many of whom have no detectable viral load for prolonged periods of time. Despite this, curing AIDS remains an elusive goal, partially due to the occurrence of drug resistance. Since the development of resistance is linked to, among other things, fluctuating drug levels, our long-term goal has been to develop nanotechnology-based drug delivery systems that can improve therapy by more precisely controlling drug concentrations in target cells. The theme of the current study is to investigate the value of combining AIDS drugs and modifiers of cellular uptake into macromolecular conjugates having novel pharmacological properties. RESULTS: Bioconjugates were prepared from different combinations of the approved drug, saquinavir, the antiviral agent, R.I.CK-Tat9, the polymeric carrier, poly(ethylene) glycol and the cell uptake enhancer, biotin. Anti-HIV activities were measured in MT-2 cells, an HTLV-1-transformed human lymphoid cell line, infected with HIV-1 strain Vbu 3, while parallel studies were performed in uninfected cells to determine cellular toxicity. For example, R.I.CK-Tat9 was 60 times more potent than L-Tat9 while the addition of biotin resulted in a prodrug that was 2850 times more potent than L-Tat9. Flow cytometry and confocal microscopy studies suggest that variations in intracellular uptake and intracellular localization, as well as synergistic inhibitory effects of SQV and Tat peptides, contributed to the unexpected and substantial differences in antiviral activity. CONCLUSION: Our results demonstrate that highly potent nanoscale multi-drug conjugates with low non-specific toxicity can be produced by combining moieties with anti-HIV agents for different targets onto macromolecules having improved delivery properties.

Quantitative assessment of the cell penetrating properties of RI-Tat-9: evidence for a cell type-specific barrier at the plasma membrane of epithelial cells.

Penetration of epithelial cells represents the rate-determining step for the absorption of many drugs and pharmaceutical macromolecules such as proteins and nucleic acid therapeutics. While the potential of using cell-penetrating peptides (CPPs) to facilitate absorption has been increasingly recognized, the mechanism of cell penetration and the uptake into certain cells have recently been called into question due to methodological artifacts. Therefore, the objective of this study was to quantitatively assess the ability of RI-Tat-9, a proteolytically stable CPP, to penetrate epithelial cell monolayers. The permeability of RI-Tat-9 with two epithelial cell lines, Madin-Darby canine kidney (MDCK) and Caco-2 cells, was comparable to the leakiness of the respective intact monolayers. Microscopic imaging showed that fluorescence-tagged RI-Tat-9 did not enter these cells, further supporting a paracellular transport mechanism. Although insufficient data were generated in these studies to generalize the observed phenomenon, the entry of RI-Tat-9 into nonepithelial T lymphocytic MT2 cells, possibly by endocytosis, suggested that a cell type-specific barrier might exist that controlled uptake of RI-Tat-9 by cells. Compared to that in MT2 and HeLa cells, the active uptake of the peptide into MDCK monolayers was much slower and showed no dependence of cell energy. Furthermore, the equilibrium binding of RI-Tat-9 to MDCK cells at 0 degrees C was indicative of an interaction with a nonspecific receptor. A correlation between binding density and concentration difference across a leaky separation barrier suggested that repulsion of free peptide molecules by bound peptide molecules at the MDCK monolayer surface may be significant at micromolar concentrations. The results of this study quantitatively show that Tat CPP uptake into two commonly used epithelial cell types is minimal and possibly cell type-specific. Implications for Tat CPP-assisted drug delivery are discussed.

Conjugates bearing multiple formyl-methionyl peptides display enhanced binding to but not activation of phagocytic cells.

N-Formyl-methionyl peptides can specifically bind to surface receptors on phagocytic cells. A single copy of N-formyl-methionine-leucine-phenylalanine (fMLF) covalently linked to a poly(ethylene glycol)-based polymer displayed reduced binding avidity (K(d) = 190 nM) for differentiated HL-60 cells relative to free fMLF (K(d) = 28 nM). Increasing the number of fMLF residues (up to eight) attached to a single polymer results in enhanced avidity for these cells (K(d) = 0.18 nM), which appears to be independent of whether the polymer backbone is linear or branched. However, no conjugate showed enhanced ability to activate phagocytic cells, relative to the free peptide (EC(50) = 5 nM), as measured by transient stimulation of release of calcium ions from intracellular stores into the cytoplasm. A polymer bearing four fMLF and four digoxigenin residues showed specific enhancement in binding to differentiated HL-60 cells and mouse peritoneal macrophages in situ relative to a polymer lacking fMLF; no such enhancement was seen in binding to receptor-negative lymphocytic Jurkat cells. These results suggest that multiple fMLF residues linked to a drug-delivery polymer can be used to target appended drugs to phagocytic cells with relatively little toxicity due to cellular activation.