Development of peptide aptamers as alternatives for antibody in the detection of amyloid-beta 42 aggregates.

Alzheimer's disease (AD) causes cognitive impairment and serious social isolation. However, there are no effective treatments and even no established confirmatory diagnostic tools for the disease. Amyloid beta (Aß) aggregation in the brain is the best-known pathognomonic mechanism of AD, so various methods for Aß detection have been developed for the diagnosis of this disease. We synthesized two novel, ultra-sensitive peptide probes specialized in detecting Aß aggregates, and examined their potential for future diagnostic application. The peptides are produced through phage high-throughput screening (HTS) and amplified through a serial process called biopanning, which is a repeating method of elution and amplification of probes. We picked phages specific for amyloid from two kinds of phage display. The synthesized peptides were confirmed to have excellent binding affinity to Aß aggregates, by immunohistochemical staining and western blotting using the brains of 3X transgenic (Tg) AD mice at different stages (5-7, 12-17 months old) of AD severity. In the present study, it was confirmed that newly developed amyloid-binding peptides could be used as novel probes for the detection of Aß aggregates, which can be used for clinical diagnosis of AD in the future.

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.

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.

Optical Sensing Properties of ZnO Nanoparticles Prepared by Spray Pyrolysis.

We studied the optical sensing properties of ZnO nanoparticles prepared by spray pyrolysis. To investigate their optical sensing performance, we incubated peptides on ZnO nanoparticles. The photoluminescence (PL) peak intensity of peptides on the ZnO nanoparticles was higher than that of peptides on the ZnO film or on the glass plate. This observed PL enhancement is attributed to the optical confinement of ZnO nanoparticles. The low-temperature spectra displayed a strong exciton emission peak with multiple sidebands, attributed to the bound exciton and its longitudinal optical phonon sidebands. The strong exciton emission is thought to be the combined effect of optical confinement due to the nanoparticle geometry, reduction of defect emission by thermal annealing, and reduction of non-radiative relaxation at low temperatures.

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.

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.

Inhibition of anthrax lethal factor by ssDNA aptamers.

Anthrax is caused by Bacillus anthracis, a bacterium that is able to secrete the toxins protective antigen, edema factor and lethal factor. Due to the high level of secretion from the bacteria and its severe virulence, lethal factor (LF) has been sought as a biomarker for detecting bacterial infection and as an effective target to neutralize toxicity. In this study, we found three aptamers, and binding affinity was determined by fluorescently labeled aptamers. One of the aptamers exhibited high affinity, with a Kd value of 11.0 ±â€¯2.7 nM, along with low cross reactivity relative to bovine serum albumin and protective antigen. The therapeutic functionality of the aptamer was examined by assessing the inhibition of LF protease activity against a mitogen-activated protein kinase kinase. The aptamer appears to be an effective inhibitor of LF with an IC50 value of 15 ±â€¯1.5 µM and approximately 85% cell viability, suggesting that this aptamer provides a potential clue for not only development of a sensitive diagnostic device of B. anthracis infection but also the design of novel inhibitors of LF.

Development of a ssDNA aptamer for detection of residual benzylpenicillin.

Antibiotics are useful for improving the living conditions of livestock. However, residual antibiotics induce several human diseases such as food-borne illness and infection of carbapenem-resistant Enterobacteriaceae (CRE). In this study, the identification of a benzylpenicillin-specific aptamer was selected by rGO-SELEX (reduced Graphene Oxide-Systematic Evolution of Ligands by EXponential enrichment). A random ssDNA library was incubated with rGO for adsorption and eluted with benzylpenicillin. As a result of the selection process, a DNA aptamer was found that specifically bound to benzylpenicillin with high binding affinity, Kd = 383.4 nM, and had a low limit of detection (LOD) of 9.2 nM. The characterization of the aptamer was performed through the fluorescence recovery signal from rGO surface. In addition, detection of benzylpenicillin was performed in pretreated milk samples, and its detection accuracy was shown to be 100± 10%. This represented that BBA1 was used for fluorescence aptasensor system in real sample. Furthermore, this benzylpenicillin binding aptamer showed high specificity against other antibiotics except for ampicillin. With these advantageous characteristics, we expect that this aptamer could be applied to an on-site detection system for residual benzylpenicillin.

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.

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.

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.

Electrical graphene aptasensor for ultra-sensitive detection of anthrax toxin with amplified signal transduction.

Detection of the anthrax toxin, the protective antigen (PA), at the attomolar (aM) level is demonstrated by an electrical aptamer sensor based on a chemically derived graphene field-effect transistor (FET) platform. Higher affinity of the aptamer probes to PA in the aptamer-immobilized FET enables significant improvements in the limit of detection (LOD), dynamic range, and sensitivity compared to the antibody-immobilized FET. Transduction signal enhancement in the aptamer FET due to an increase in captured PA molecules results in a larger 30 mV/decade shift in the charge neutrality point (Vg,min ) as a sensitivity parameter, with the dynamic range of the PA concentration between 12 aM (LOD) and 120 fM. An additional signal enhancement is obtained by the secondary aptamer-conjugated gold nanoparticles (AuNPs-aptamer), which have a sandwich structure of aptamer/PA/aptamer-AuNPs, induce an increase in charge-doping in the graphene channel, resulting in a reduction of the LOD to 1.2 aM with a three-fold increase in the Vg,min shift.

Cloning, characterization and evaluation of potent inhibitors of Shigella sonnei acetohydroxyacid synthase catalytic subunit.

Acetohydroxyacid synthase (AHAS) is a thiamin diphosphate (ThDP)- and flavin adenine dinucleotide (FAD)-dependent plant and microbial enzyme that catalyzes the first common step in the biosynthesis of essential amino acids such as leucine, isoleucine and valine. To identify strong potent inhibitors against Shigella sonnei (S. sonnei) AHAS, we cloned and characterized the catalytic subunit of S. sonnei AHAS and found two potent chemicals (KHG20612, KHG25240) that inhibit 87-93% S. sonnei AHAS activity at an inhibitor concentration of 100uM. The purified S. sonnei AHAS had a size of 65kDa on SDS-PAGE. The enzyme kinetics revealed that the enzyme has a K(m) of 8.01mM and a specific activity of 0.117U/mg. The cofactor activation constant (K(s)) for ThDP and (K(c)) for Mg(++) were 0.01mM and 0.18mM, respectively. The dissociation constant (K(d)) for ThDP was found to be 0.14mM by tryptophan fluorescence quenching. The inhibition kinetics of inhibitor KHG20612 revealed an un-competitive inhibition mode with a K(ii) of 2.65mM and an IC(50) of 9.3µM, whereas KHG25240 was a non-competitive inhibitor with a K(ii of) 5.2mM, K(is) of 1.62mM and an IC(50) of 12.1µM. Based on the S. sonnei AHAS homology model structure, the docking of inhibitor KHG20612 is predicted to occur through hydrogen bonding with Met 257 at a 1.7Å distance with a low negative binding energy of -9.8kcal/mol. This current study provides an impetus for the development of a novel strong antibacterial agent targeting AHAS based on these potent inhibitor scaffolds.

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.

Evaluation of substituted triazol-1-yl-pyrimidines as inhibitors of Bacillus anthracis acetohydroxyacid synthase.

Acetohydroxyacid synthase (AHAS), a potential target for antimicrobial agents, catalyzes the first common step in the biosynthesis of the branched-chain amino acids. The genes of both catalytic and regulatory subunits of AHAS from Bacillus anthracis (Bantx), a causative agent of anthrax, were cloned, overexpressed in Escherichia coli, and purified to homogeneity. To develop novel anti-anthracis drugs that inhibit AHAS, a chemical library was screened, and four chemicals, AVS2087, AVS2093, AVS2387, and AVS2236, were identified as potent inhibitors of catalytic subunit with IC(50) values of 1.0 +/- 0.02, 1.0 +/- 0.04, 2.1 +/- 0.12, and 2.0 +/- 0.08 microM, respectively. Further, these four chemicals also showed strong inhibition against reconstituted AHAS with IC(50) values of 0.05 +/- 0.002, 0.153 +/- 0.004, 1.30 +/- 0.10, and 1.29 +/- 0.40 microM, respectively. The basic scaffold of the AVS group consists of 1-pyrimidine-2-yl-1H-[1,2,4]triazole-3-sulfonamide. The potent inhibitor, AVS2093 showed the lowest binding energy, -8.52 kcal/mol and formed a single hydrogen bond with a distance of 1.973 A. As the need for novel antibiotic classes to combat bacterial drug resistance increases, the screening of new compounds that act against Bantx-AHAS shows that AHAS is a good target for new anti-anthracis drugs.

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.

C-terminal amidation of PMAP-23: translocation to the inner membrane of Gram-negative bacteria.

PMAP-23 is a member of the cathelicidin family derived from pig myeloid cells and has potent antimicrobial activity. Amidation of the carboxyl terminus (C-terminus) of an antimicrobial peptide generally enhances its structural stability and antimicrobial activity or decreases its cytotoxicity. The aim of the present study was to investigate the effect of amidation on the mode of action in PMAP-23. Irrespective of amidation, PMAP-23 adopts a helix-hinge-helix structure in a membrane-mimetic environment. The antibacterial activities of PMAP-23C, which had a free C-terminus, and PMAP-23N, which had an amidated C-terminus, were similar against Gram-negative bacteria, reflecting a similar ability to neutralize lipopolysaccharide. However, PMAP-23N assumed a perpendicular orientation across the outer to the inner leaflet of the bacterial inner membrane, while PMAP-23C was orientated parallel to the lipid bilayer, as determined by following the blue shift in tryptophan fluorescence, as well as calcein release from liposomes and SYTOX Green uptake assays. These results suggest that N-terminal amidation of PMAP-23 provides structural stability and increases the peptide's cationic charge, facilitating translocation into the bacterial inner membrane.

Sensitive fluorescence assay of anthrax protective antigen with two new DNA aptamers and their binding properties.

A homogeneous assay of the protective antigen in anthrax toxin is reported using two new PA-specific aptamers for selective and sensitive detection, based on reduction in the fluorescence emission according to the formation of the aptamer-PA ternary complex. PA at 1 nM was readily detected using OliGreen as a fluorophore in HEPES buffer. We also demonstrated that the PA detection could be performed in blood serum. The binding interaction between the aptamer and PA was strong enough to dehybridize double-stranded DNA paired completely with 12 bases at room temperature. Moreover, this fluorescence study revealed that the binding sites of the two aptamers were located differently on the PA protein. We believe our approach may lay the groundwork for the real-time detection of PA.

Development of a Low-Molecular-Weight Aß42 Detection System Using a Enzyme-Linked Peptide Assay.

Alzheimer's disease (AD) is a degenerative brain disease that is the most common cause of dementia. The incidence of AD is rapidly rising because of the aging of the world population. Because AD is presently incurable, early diagnosis is very important. The disease is characterized by pathological changes such as deposition of senile plaques and decreased concentration of the amyloid-beta 42 (Aß42) peptide in the cerebrospinal fluid (CSF). The concentration of Aß42 in the CSF is a well-studied AD biomarker. The specific peptide probe was screened through four rounds of biopanning, which included the phage display process. The screened peptide showed strong binding affinity in the micromolar range, and the enzyme-linked peptide assay was optimized using the peptide we developed. This diagnostic method showed specificity toward Aß42 in the presence of other proteins. The peptide-binding site was also estimated using molecular docking analysis. Finally, the diagnostic method we developed could significantly distinguish patients who were classified based on amyloid PET images.