Development of a receptor-based inhibitory penta-unit-conjugated peptide to enhance anthrax toxin neutralization.

Anthrax toxin is a key virulence factor for Bacillus anthracis. The cell-binding component of anthrax toxin, protective antigen (PA), mediates the entry of the toxin into cells by first binding to the extracellular von Willebrand factor A (VWA) domain of the cellular anthrax toxin receptor (ATR). Herein, we targeted the VWA domain of the cellular receptor to develop a more effective antitoxin agent for neutralization of anthrax toxin. We selected ATR-binding peptides by using a phage display: among these, we identified two novel peptides binding to the ATR with high affinity and specificity, and that neutralized anthrax toxicity in cells. Furthermore, to enhance the functional efficiency of the probes, the peptides were modified and conjugated to three polyvalent probe backbones: a 17 amino-acid-based cyclic form penta-unit, poly-d-lysine (PDL), or the M13 bacteriophage. One of the functionally modified polyvalent peptide probes, the penta-unit-conjugated probe (PUCP) produced the most potent neutralization of anthrax toxin, with half-maximal inhibitory concentration (IC50) of 20 nM. The PUCP disrupted anthrax toxin binding to its receptor and reduced endocytosis of anthrax toxin. This peptide-based approach may, therefore, represent a promising strategy to combat anthrax toxicosis and other bacterial diseases and may be efficient for disease treatment.

Advances in dermatology using DNA aptamer "Aptamin C" innovation: Oxidative stress prevention and effect maximization of vitamin C through antioxidation.

BACKGROUND: Vitamin C (also known as L-ascorbic acid) plays a critical role in reactive oxygen species (ROS) reduction and cell regeneration by protecting cell from oxidative stress. Although vitamin C is widely used in cosmetic and therapeutic markets, there is considerable evidence that vitamin C easily undergoes oxidation by air, pH, temperature, and UV light upon storage. This deficiency of vitamin C decreases its potency as an antioxidant and reduces the shelf-life of products containing vitamin C as its ingredient. To overcome the deficiency of vitamin C, we have developed Aptamin C, an innovative DNA aptamer maximizing the antioxidant efficacy of vitamin C by binding to the reduced form of vitamin C and delaying its oxidation. METHODS: Binding of Aptamin C with vitamin C was determined using ITC analysis. ITC experiment was performed 0.2 mmol/L vitamin C that was injected 25 times in 2 µL aliquots into the 1.8 mL sample cell containing the Aptamin C at a concentration of 0.02 mmol/L. The data were fitted to a one-site binding isotherm using with origin program for ITC v.5.0. RESULTS: To investigate the effect of Aptamin C and vitamin C complex in human skins, both in vitro and clinical tests were performed. We observed that the complex of Aptamin C and vitamin C was significantly effective in wrinkle improvement, whitening effect, and hydration increase. In the clinical test, subjects treated with the complex showed dramatic improvement in skin irritation and itching. No adverse reaction was presented by Aptamin C complex in the test. CONCLUSION: Taken together, these results showed that Aptamin C, an innovative novel compound, should potentially be served as a key cosmeceutical ingredient for a range of skin conditions.

Novel Peptide-Based Inhibitors for Microtubule Polymerization in Phytophthora capsici.

The plant disease Phytophthora blight, caused by the oomycete pathogen Phytophthora capsici, is responsible for major economic losses in pepper production. Microtubules have been an attractive target for many antifungal agents as they are involved in key cellular events such as cell proliferation, signaling, and migration in eukaryotic cells. In order to design a novel biocompatible inhibitor, we screened and identified inhibitory peptides against alpha- and beta-tubulin of P. capsici using a phage display method. The identified peptides displayed a higher binding affinity (nanomolar range) and improved specificity toward P. capsici alpha- and beta-tubulin in comparison to Homo sapiens tubulin as evaluated by fluorometric analysis. One peptide demonstrated the high inhibitory effect on microtubule formation with a nanomolar range of IC50 values, which were much lower than a well-known chemical inhibitor-benomyl (IC50 = 500 µM). Based on these results, this peptide can be employed to further develop promising candidates for novel antifungal agents against Phytophthora blight.

Ultrasensitive Fluorescence Detection of Alzheimer's Disease Based on Polyvalent Directed Peptide Polymer Coupled to a Nanoporous ZnO Nanoplatform.

Amyloid-beta 42 (Aß42), the key biomarker of Alzheimer's disease (AD), aggregates to form neurotoxic amyloid plaques. In this work, we modified two fluorescein isothiocyanate-labeled Aß42-targeting peptides and designed an Aß42-specific ultrasensitive polyvalent-directed peptide polymer (PDPP) to enhance AD diagnosis sensitivity. The dissociation constant of Aß42 by PDPP was 103-fold higher than the single-site-directed peptide. The improved binding was due to the ability of PDPP to detect multiple receptors on the target. The power of the PDPP diagnostic probe was verified in its application to detect Aß42 in cerebrospinal fluid (CSF), which showed a lower limit of detection (LOD) in the fg mL-1 range that is more sensitive than detection by antibodies or single peptides. In addition, we present a novel ultrasensitive diagnostic system using an array of nanoporous ZnO nanoparticles, which play a role in fluorescence signal amplification, to further improve AD diagnosis sensitivity. We enhanced the signal on the basis of the properties of nanoporous ZnO nanoparticles and measured and quantified an ultralow concentration (ag mL-1 range) of Aß42. This PDPP coupled to the nanoporous ZnO-based system is a novel approach to AD diagnosis that might also be useful for the detection of other target biomarkers and clinical applications.

Detection of Nonylphenol with a Gold-Nanoparticle-Based Small-Molecule Sensing System Using an ssDNA Aptamer.

Endocrine-disrupting chemicals (EDCs) threaten many kinds of life throughout the world. These compounds function the same as sexual hormones, inducing precocious puberty, gynecomastia, etc., in the human body. To prevent excess exposure to nonylphenol (NP), a simple and rapid detection system is needed. In this study, we develop a nonylphenol-specific aptamer from a random single-stranded DNA library and test a rapid sensor system based on the aptamer and gold nanoparticles (AuNPs). The aptamer was screened by a methodology involving reduced graphene oxide (rGO). As a result of screening and sequencing, a DNA aptamer was developed that recognizes the target with high binding affinity (Kd = 194.2 ± 65.9 nM) and specificity. The sensor system developed using the aptamer and gold nanoparticles is sensitive (LOD = 2.239 nM). Circular dichroism (CD) spectrometry results show that the free aptamer binds to the target molecule. The aptamer was characterized using gold nanoparticles to measure UV absorbance. Our results suggest that the sensor system developed using this aptamer is useful for field diagnosis of small molecules.

Development of a ssDNA aptamer system with reduced graphene oxide (rGO) to detect nonylphenol ethoxylate in domestic detergent.

Endocrine-disrupting chemicals are a major public health problem throughout the world. In the human body, these compounds functionalize the same as sexual hormones, inducing precocious puberty, gynecomastia, etc. To help prevent this occurrence, a simple detection system is needed. In this study, a nonylphenol ethoxylate (NPE)-specific aptamer was selected by reduced graphene oxide-systematic evolution of ligands by exponential enrichment. A random ssDNA library was incubated with rGO for adsorption, followed by elution with the target molecule. As a result of screening, a DNA aptamer was found that specifically bounds to the target with high binding affinity (Kd  = 100.9 ± 13.2 nM) and had a low limit of detection (LOD = 696 pM). Furthermore, this NPE-binding aptamer bounds selectively to the target. Characterization of the aptamer was confirmed by measuring the fluorescence signal recovery from rGO. In addition, detection of NPE was performed with several water samples, and the detection accuracy was 100 ± 10%. From these results, we expect that this aptamer could be applied to an on-site detection system for NPE in industrial sites or domestic fields.

Development of a novel imaging agent using peptide-coated gold nanoparticles toward brain glioma stem cell marker CD133.

CD133 is known as biomarker for glioblastoma (GBM) and also serves as a marker for cancer stem cells (CSCs), which carry out tumorigenesis and resist conventional therapeutics. The presence of CD133-presenting CSC is a one of the factors in maintenance of the tumorigenic potential of GBM. Thus, CD133 is a potential target for accurate diagnosis of GBM, which could improve its poor prognosis for patients when CSCs are present. Herein we designed a small peptide-based imaging agent with stimulus-responsive properties. A novel small peptide, CBP4, was screened by a phage display technique, and demonstrated binding to the target CD133 (ECD) comparable to that of an antibody. As a quencher, we used gold nanoparticles (GNPs); the targeting peptide was conjugated to GNPs with high efficiency. By means of a quenching effect, the peptide-coated GNP showed 'signal on-off' properties depending upon the presence of the target. In addition, the particles exhibited biocompatibility when localized in the cytosol. Thus, this study demonstrated that the peptide-coated GNPs can be utilized as an imaging agent for accurate diagnosis of GBM, and further as a drug carrier for therapeutic approaches. STATEMENT OF SIGNIFICANCE: The diagnosis and determination of prognosis made by cancer stem cell markers could be a key strategy to eradicate cancer stem cells and cure the cancer. The significance of this study is the characterization of quenching-based signal on-off mechanism and showed that the active targeting via peptide can contribute to the design of a stimulus-responsive cellular imaging agent. Moreover, small peptide based nano complexation showed specific recognition of the target stem cell and internalized on cellular cyotosol with stimulus responsive fluorescence. With its novel biocompatibility, the strategy might be a promising tool for drug carrier systems able to measure and visualize the delivered efficiency at intracellular sites.

Use of Multiple Peptide-Based SERS Probes Binding to Different Epitopes on a Protein Biomarker To Improve Detection Sensitivity.

We propose an analytical strategy to improve the sensitivity for detecting a protein biomarker through signal multiplication by manipulating multiple peptide-based surface-enhanced Raman scattering (SERS) probes to bind the biomarker. Protective antigen (PA) was used as an Anthrax biomarker in this study. For this purpose, five small peptides selective to various PA epitopes with different binding affinities were chosen and peptide-conjugated Au nanoparticle (AuNP) SERS probes were individually prepared using each peptide. Initially, five different SERS probes were separately used to detect PA and the sensitivities were compared. Next, the possibility of enhancing sensitivity by employing multiple SERS probes was examined. Rather than applying the probes simultaneously, which would induce competitive binding, each probe was added sequentially and an optimal probe-addition sequence was determined to provide maximal sensitivity. Finally, PA samples at seven different concentrations were measured with the optimal sequence. The limit of detection (LOD) was 0.1 aM, and the enhancement was more effective at lower PA concentrations. The proposed scheme can be further applicable to detect other protein biomarkers to diagnose various diseases.

Design of a PKCδ-specific small peptide as a theragnostic agent for glioblastoma.

Glioblastoma is an aggressive malignant brain tumor that starts in the brain or spine and frequently recurs after anticancer treatment. The development of an accurate diagnostic system combined with effective cancer therapy is essential to improve prognosis of glioma patients. Peptides, produced from phage display, are attractive biomolecules for glioma treatment because of their biostability, nontoxicity, and small size. In this study, we employed phage display methodology to screen for peptides that specifically recognize the target PKCδ as a novel biomarker for glioma. The phage library screening yielded four different peptides displayed on phages with a 20- to 200-pM Kd value for the recombinant PKCδ catalytic domain. Among these four phage peptides, we selected one to synthesize and tagged it with fluorescein isothiocyanate (FITC) based on the sequence of the PKCδ-binding phage clone. The synthetic peptide showed a relative binding affinity for antibody and localization in the U373 glioma cell. The kinase activity of PKCδ was inhibited by FITC-labeled peptide with an IC50 of 1.4 µM in vitro. Consequently, the peptide found in this study might be a promising therapeutic agent against malignant brain tumor.

Neuroprotective Effects of Acetyl-L-Carnitine Against Oxygen-Glucose Deprivation-Induced Neural Stem Cell Death.

Deprivation of oxygen and glucose is the main cause of neuronal cell death during cerebral infarction and can result in severe morbidity and mortality. In general, the neuroprotective therapies that are applied after ischemic stroke have been unsuccessful, despite many investigations. Acetyl-L-carnitine (ALCAR) plays an important role in mitochondrial metabolism and in modulating the coenzyme A (CoA)/acyl-CoA ratio. We investigated the protective effects of ALCAR against oxygen-glucose deprivation (OGD) in neural stem cells (NSCs). We measured cell viability, proliferation, apoptosis, and intracellular signaling protein levels after treatment with varying concentrations of ALCAR under OGD for 8 h. ALCAR protected NSCs against OGD by reducing apoptosis and restoring proliferation. Its protective effects are associated with increases in the expression of survival-related proteins, such as phosphorylated Akt (pAkt), phosphorylated glycogen synthase kinase 3b (pGSK3b), B cell lymphoma 2 (Bcl-2), and Ki-67 in NSCs that were injured by OGD. ALCAR also reduced the expression of death-related proteins, such as Bax, cytosolic cytochrome C, cleaved caspase-9, and cleaved caspase-3. We concluded that ALCAR exhibits neuroprotective effects against OGD-induced damage to NSCs by enhancing the expression of survival signals and decreasing that of death signals.

A novel peptide-based recognition probe for the sensitive detection of CD44 on breast cancer stem cells.

Metastasis and recurrence of breast cancer remain significant clinical problems. The expression level of CD44 protein is higher in breast cancer-initiating cancer stem cells; therefore, the early detection of CD44 using a sensitive diagnostic probe is important for breast cancer diagnosis and therapeutic purposes. In this study, we fabricated a polyvalent directed peptide polymer (PDPP) that specifically recognized the CD44 biomarker, as confirmed by immunocytochemistry tests and fluorescence-activated cell sorting assessment. Our results indicate that PDPP is useful as a novel tool for the sensitive detection of breast cancer stem cells.

Development of ssDNA aptamers as potent inhibitors of Mycobacterium tuberculosis acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS) from Mycobacterium tuberculosis (Mtb) is a promising potential drug target for an emerging class of new anti-tuberculosis agents. In this study, we identify short (30-mer) single-stranded DNA aptamers as a novel class of potent inhibitors of Mtb-AHAS through an in vitro DNA-SELEX method. Among all tested aptamers, two candidate aptamers (Mtb-Apt1 and Mtb-Apt6) demonstrated the greatest inhibitory potential against Mtb-AHAS activity with IC50 values in the low nanomolar range (28.94±0.002 and 22.35±0.001 nM respectively). Interestingly, inhibition kinetics analysis of these aptamers showed different modes of enzyme inhibition (competitive and mixed type of inhibition respectively). Secondary structure-guided mutational modification analysis of Mtb-Apt1 and Mtb-Apt6 identified the minimal region responsible for their inhibitory action and consequently led to 17-mer and 20-mer shortened aptamers that retained equivalent or greater inhibitory potential. Notably, a modeling and docking exercise investigated the binding site of these two potent inhibitory aptamers on the target protein and showed possible involvement of some key catalytic dimer interface residues of AHAS in the DNA-protein interactions that lead to its potent inhibition. Importantly, these two short candidate aptamers, Mtb-Apt1 (17-mer) and Mtb-Apt6 (20-mer), also demonstrated significant growth inhibition against multidrug-resistant (MDR-TB) and extensively drug-resistant (XDR-TB) strains of tuberculosis with very low MIC of 5.36 µg/ml and 6.24 µg/ml, respectively and no significant cytotoxicity against mammalian cell line. This is the first report of functional inhibitory aptamers against Mtb-AHAS and provides the basis for development of these aptamers as novel and strong anti-tuberculosis agents.

Development of receptor-based inhibitory RNA aptamers for anthrax toxin neutralization.

Anthrax toxin excreted by Bacillus anthracis is the key causative agent of infectious anthrax disease. In the present study, we targeted the binding of PA to the ATR/TEM8 Von Willebrand factor type A (VWA) domain, which we cloned into Escherichia coli and purified to homogeneity under denaturing conditions. To develop an anthrax toxin inhibitor, we selected and identified short single strand RNA aptamers (approximately 30mer) consisting of different sequences of nucleic acids with a high binding affinity in the 100 nanomolar range against the recombinant ATR/TEM8 VWA domain using systematic evolution of ligands by exponential enrichment (SELEX). Five candidate aptamers were further characterized by several techniques including secondary structural analysis. The inhibitor efficiency (IC50) of one of the aptamers toward anthrax toxin was approximately 5µM in macrophage RAW 264.7 cells, as determined from cytotoxicity analysis by MTT assay. We believe that the candidate aptamers should be useful for blocking the binding of PA to its receptor in order to neutralize anthrax toxin.

Proteolytic assay-based screening identifies a potent inhibitor of anthrax lethal factor.

Anthrax lethal factor (LF), a Zn(2+)-dependent metalloprotease, is a key virulence component of anthrax toxin. Here, we used proteolytic assay-based screening to identify novel LF inhibitors from a naturally extracted chemical library. The screening identified four compounds that inhibited in vitro proteolytic activity of LF with an IC(50) of low micromolar range (11-20 µM). Three of these compounds were toxic to the mouse macrophage-like cell line, RAW 264.7. Compound 200 was non-toxic, however, and successfully protected Raw 264.7 cells from a lethal toxin challenge with an IC(50) of 39.2 µM. We also identified possible binding modes of compound 200 by molecular docking.

Screening of peptides bound to breast cancer stem cell specific surface marker CD44 by phage display.

CD44, a cancer-associated membrane glycoprotein involved in cell adhesion and tumor progression, has been implicated as a cancer stem cell antigen in several cancers including breast cancer. If the detection sensitivity of CD44 as an early marker for cancer could be improved, this would have important clinical applications. As compared with early stage treatments of other kinds of cancer, treatment of breast cancer is more likely to results in positive outcomes, so this early detection is crucial. Therefore, CD44 is a potential diagnostic target for cancer detection. Herein, we have used a peptide library to screen novel diverse peptides that bind to CD44 with high affinity and characterized the specific binding of these peptides. Our work provides a basis to develop novel diagnostic peptides which may replace antibodies as CD44 detection probes.

Ultrasensitive diagnosis for an anthrax-protective antigen based on a polyvalent directed peptide polymer coupled to zinc oxide nanorods.

A flexible poly-D-lysine polymer conjugated with different target-binding peptides is demonstrated with an ultralow concentration detection limit compared to those of other conventional detection systems. This polyvalent directed peptide polymer (PDPP) exhibits increased binding affinity and detects anthrax protective antigen at low levels using a well-known zinc oxide nanorod detection system.

Phage display screen for peptides that bind Bcl-2 protein.

Bcl-2 family proteins are key regulators of apoptosis associated with human disease, including cancer. Bcl-2 protein has been found to be overexpressed in many cancer cells. Therefore, Bcl-2 protein is a potential diagnostic target for cancer detection. In the present study, the authors have identified several Bcl-2 binding peptides with high affinity (picomolar range) from a 5-round M13 phage display library screening. These peptides can be used to develop novel diagnostic probes or potent inhibitors with diverse polyvalencies.

Yeast-hybrid based high-throughput assay for identification of anthrax lethal factor inhibitors.

Inhibitors of anthrax lethal factor (LF) are currently being sought as effective therapeutics for the treatment of anthrax. Here we report a novel screening approach for inhibitors of LF, a yeast-hybrid-based assay system in which the expression of reporter genes from a Gal4 promoter is repressed by LF proteolytic activity. Yeast cells were co-transformed with LF and a chimeric transcription factor that contains an LF substrate sequence inserted between the DNA-binding and activation domains of Gal4. In the resulting yeast cells, LF cleaves the substrate, thus inactivating the chimeric Gal4 and resulting in lack of expression of reporter genes. Compounds that inhibit LF cleavage of its substrate are identified by changes in reporter gene activity. Relative to in vitro screens for inhibitors of LF proteolytic activity, this screen has the advantage of excluding compounds that are toxic or non-permeable to eukaryotic cells. Additionally, the screen has the advantage of being fast, easy and cheap because exogenous LF and substrate are not needed. An initial chemical library screen with this system has identified four candidate inhibitors which were confirmed to inhibit LF protease activity in an in vitro assay. Furthermore, FBS-00831, one of the compounds identified, protects Raw 264.7 macrophages from anthrax lethal toxin and the possible binding site on LF was also evaluated by molecular docking.

Recent advances in rapid and ultrasensitive biosensors for infectious agents: lesson from Bacillus anthracis diagnostic sensors.

Here, we review the cumulative efforts to develop rapid and ultrasensitive diagnostic systems, especially for the infectious agent, Bacillus anthracis, as a model system. This Minireview focuses on demonstrating the features of various probes for target molecule detection and recent methods of signal generation within the biosensors. Also, we discuss the possibility of using peptides as next-generation probe molecules.

Characterization of Capsicum annuum recombinant alpha- and beta-tubulin.

There are several conditions which might modulate polymerization to produce polymers having normal lattice structure. In the absence of 1 mM MgCl(2) the assembly was reduced by 36% in Capsicum annuum tubulin (CAnm tubulin). There was no significant difference in the final assembly formation in the presence of 5% to 10% glycerol. However, nucleation rate was slow and apparent study state was achieved lately in the presence of 10% glycerol. Taxol at 100 microM concentration increased 23% tubulin assembly. One millimolar CaCl(2), >or=1% dimethyl sulfoxide (DMSO) and physiologically low temperature reduced CAnm tubulin assembly. A value of 0.089 mg/ml was obtained as critical concentration for polymerization. Benomyl significantly reduced the number of cysteine residues accessible to 5,5'-dithiobis-(2-nitrobenzoic acid); there were 4.77 +/- 0.21 and 3.49 +/- 0.35 residues accessible per tubulin dimer in the presence of 50 and 100 microM benomyl respectively.