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Insect control traits are a key component of improving the efficacy of insect pest management and maximizing crop yields for growers. Insect traits based on proteins expressed by the bacteria Bacillus thuringiensis (Bt) have proven to be very effective tools in achieving this goal. Unfortunately, the adaptability of insects has led to resistance to certain proteins in current commercial products. Therefore, new insecticidal traits representing a different mode of action (MoA) than those currently in use are needed. Cry1Ja has good insecticidal activity against various lepidopteran species, and it provides robust protection against insect feeding with in planta expression. For Bt proteins, different MoAs are determined by their binding sites in the insect midgut. In this study, competitive binding assays are performed using brush border membrane vesicles (BBMVs) from Helicoverpa zea, Spodoptera frugiperda, and Chrysodeixis includens to evaluate the MoA of Cry1Ja relative to representatives of the various Bt proteins that are expressed in current commercial products for lepidopteran insect protection. This study highlights differences in the shared Cry protein binding sites in three insect species, Cry1Ja bioactivity against Cry1Fa resistant FAW, and in planta efficacy against target pests. These data illustrate the potential of Cry1Ja for new insect trait development. [ABSTRACT FROM AUTHOR]

The bean bug Riptortus pedestris is a notorious insect pest that can damage various crops, especially soybean, in East Asia. In insects, the olfactory system plays a crucial role in host finding and feeding behaviour in which the odorant‐binding proteins (OBPs) are believed to be involved in initial step in this system. In this study, we produced the R. pedestris adult antennae‐expressed RpedOBP4 protein using a recombinant expression system in E. coli. Fluorescence competitive binding confirmed that RpedOBP4 has binding affinities to 7 of 20 soybean volatiles (ligands), and that a neutral condition is the best environment for it. The binding property of RpedOBP4 to these ligands was further revealed by integrating data from molecular docking, site‐directed mutagenesis and ligand binding assays. This demonstrated that five amino acid residues (I30, L33, Y47, I57 and Y121) are involved in the binding process of RpedOBP4 to corresponding ligands. These findings will not only help us to more thoroughly explore the olfactory mechanism of R. pedestris during feeding on soybean, but also lead to the identification of key candidate targets for developing environmental and efficient behaviour inhibitors to prevent population expansion of R. pedestris in the future. [ABSTRACT FROM AUTHOR]

Dioryctria abietella is a major pest of conifer cones that can initiate specific behaviours (locating hosts, finding mates and selecting oviposition sites) through sensitive olfactory systems. General odorant‐binding proteins are involved in the first step in odorant perception in insects, but their specific functional roles in D. abietella chemoreception have not been fully elucidated. In this study, quantitative real‐time polymerase chain reaction (PCR) examinations showed that DabiGOBP2 is predominantly expressed in the antennae of female adults. Western blot experiments further confirmed that these proteins show biased expression in the tissues. Binding property analysis demonstrated that DabiGOBP2 exhibited ligand specificity to terpenes, especially myrcene (Ki = 8.19 ± 0.05). Electroantennogram (EAG) and Y‐tube assays showed that three plant volatiles elicited high EAG responses, but only dodecanal showed a strong attraction to females. However, myrcene and isobutyl benzoate were significantly repellent to males at high doses in the behaviour test. In conclusion, exploring the functions of DabiGOBP2 can help in better understanding the mechanism of the olfactory system of D. abietella and developing novel strategies to control this pest. [ABSTRACT FROM AUTHOR]

Peptide binding to major histocompatibility complexes (MHCs) is a central component of the immune system, and understanding the mechanism behind stable peptide-MHC binding will aid the development of immunotherapies. While MHC binding is mostly influenced by the identity of the so-called anchor positions of the peptide, secondary interactions from nonanchor positions are known to play a role in complex stability. However, current MHC-binding prediction methods lack an analysis of the major conformational states and might underestimate the impact of secondary interactions. In this work, we present an atomically detailed analysis of peptide-MHC binding that can reveal the contributions of any interaction toward stability. We propose a simulation framework that uses both umbrella sampling and adaptive sampling to generate a Markov state model (MSM) for a coronavirus-derived peptide (QFKDNVILL), bound to one of the most prevalent MHC receptors in humans (HLA-A24:02). While our model reaffirms the importance of the anchor positions of the peptide in establishing stable interactions, our model also reveals the underestimated importance of position 4 (p4), a nonanchor position. We confirmed our results by simulating the impact of specific peptide mutations and validated these predictions through competitive binding assays. By comparing the MSM of the wild-type system with those of the D4A and D4P mutations, our modeling reveals stark differences in unbinding pathways. The analysis presented here can be applied to any peptide-MHC complex of interest with a structural model as input, representing an important step toward comprehensive modeling of the MHC class I pathway. [ABSTRACT FROM AUTHOR]

Insects can exhibit flexible olfaction that is sensitive to complex natural chemical environments. Odorant-binding proteins (OBPs) in insects' antennal chemosensilla can act as transporters of plant volatiles and pheromones across the sensillar lymph. Although the physiological functions of OBPs have been widely reported, it is still unclear how OBP binds to ligands with various structures in detail. Here, we further investigated the ligand-binding modes and characteristics of AcerOBP2 from the Eastern honey bee (Apis cerana). The results showed that, as a specific protein distributed below the base of chemosensilla on the antennal surface, AcerOBP2 was strongly bound with the candidate floral volatiles and bee pheromones. By docking analysis and site-directed mutagenesis, four different binding modes were found in the five AcerOBP2 mutants between six ligands. Two key amino acids, Ser123 and Lys51, play a key role in AcerOBP2 binding to odors, depending on the presence or absence of hydrogen bonds. In addition, the binding modes depend on their chemical structures and the binding poses of the diverse ligands. These results not only further prompted the functional basis of the relationship between the chemical structures of odorants and bee OBPs, but also revealed the complexity of the flexible behavioral modes of odor binding in insect olfactory systems. • OBP2 is a specific protein distributed below the base of antennal chemosensilla. • OBP2 was strongly bound with the floral volatiles and bee pheromone components. • Four different binding modes were found in five OBP2 mutants between six ligands. • Ser123 and Lys51 play a key role in the binding of OBP2 to odors through H-bond. [ABSTRACT FROM AUTHOR]

The reference standards halo-GSK1482160 (F-, Br-, and I-) and their corresponding precursors desmethyl-halo-GSK1482160 (F-, Br-, and I-) were synthesized from (S)-1-methyl-5-oxopyrrolidine-2-carboxylic acid or (S)-5-oxopyrrolidine-2-carboxylic acid and 2-halo-3-(trifluoromethyl)benzylamine (F-, Br-, and I-) in one step with 45–93% yields. The target tracers [11C]halo-GSK1482160 (F-, Br-, and I-) were prepared from desmethyl-halo-GSK1482160 (F-, Br-, and I-) with [11C]CH 3 OTf under basic conditions (NaOH-Na 2 CO 3 , solid, w/w 1:2) through N -[11C]methylation and isolated by HPLC combined with SPE in 40–50% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity (A M) at end of bombardment (EOB) was 370–740 GBq/μmol. The potency of halo-GSK1482160 (F-, Br-, and I-) in comparison with GSK1482160 (Cl-) was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity K i values for halo-GSK1482160 (F-, Br-, and I-) and GSK1482160 (Cl-) are 54.2, 2.5, 1.9 and 3.1 nM, respectively. [ABSTRACT FROM AUTHOR]

Abstract The overexpression of P2X7R is associated with neuroinflammation and plays an important role in various neurodegenerative diseases. The [18F]fluoropropyl derivative of GSK1482160, [18F]IUR-1602, has been first prepared and examined as a new potential P2X7R radioligand. The reference standard IUR-1602 was synthesized from tert -butyl (S)-5-oxopyrrolidine-2-carboxylate, fluoropropylbromide, and 2-chloro-3-(trifluoromethyl)benzylamine with overall chemical yield 13% in three steps. The target tracer [18F]IUR-1602 was synthesized from desmethyl-GSK1482160 with 3-[18F]fluoropropyl tosylate, prepared from propane-1,3-diyl bis(4-methylbenzenesulfonate) and K[18F]F/Kryptofix2.2.2, in two steps and isolated by HPLC combined with SPE in 2–7% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity at end of bombardment (EOB) was 74–370 GBq/μmol. The potency of IUR-1602 in comparison with GSK1482160 was determined by a radioligand competitive binding assay using [11C]GSK1482160, and the binding affinity K i values for IUR-1602 and GSK1482160 are 23.6 and 3.07 nM, respectively. The initial in vitro evaluation results, 8-fold less potency of [18F]IUR-1602 compared to [11C]GSK1482160, prevent further in vivo evaluation of [18F]IUR-1602 in animals and human. Highlights • A new P2X7R radioligand [18F]IUR-1602 was synthesized. • A 2-step 2-pot fully automated [18F]-radiosynthesis for [18F]IUR-1602 was developed. • A semi-preparative RP HPLC-SPE technique was employed in radiosynthesis. • K i value of IUR-1602 was 23.6 ± 0.96 nM. [ABSTRACT FROM AUTHOR]

The Japanese pine sawyer, Monochamus alternatus Hope (Coleoptera: Cerambycidae), is a major pest of pines and it is also the key vector of the exotic pinewood nematode in China. In the present study, we cloned, expressed, and purified a chemosensory protein (CSP) in M. alternatus. We surveyed its expression in various developmental stages of male and female adult tissues and determined its binding affinities for different pine volatiles using a competitive binding fluorescence assay. A CSP known as CSP5 in M. alternatus was obtained from an antennal cDNA library and expressed in Escherichia coli. Quantitative reverse transcription polymerase chain reaction results indicated that the CSP5 gene was mainly expressed in male and female antennae. Competitive binding assays were performed to test the binding affinity of recombinant CSP5 to 13 odour molecules of pine volatiles. The results showed that CSP5 showed very strong binding abilities to myrcene, (+)-ß-pinene, and (-)-isolongifolene, whereas the volatiles 2-methoxy-4-vinylphenol, p-cymene, and (+)-limonene oxide have relatively weak binding affinity at pH 5.0. Three volatiles myrcene, (+)-ß-pinene, and (-)-isolongifolene may play crucial roles in CSP5 binding with ligands but this needs further study for confirmation. The sensitivity of insect to host plant volatiles can effectively be used to control and monitor the population through mass trapping as part of integrated pest management programs. [ABSTRACT FROM AUTHOR]

The reference standard IUR-1601 (( S )- N -(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from tert -butyl ( S )-5-oxopyrrolidine-2-carboxylate, fluoroethylbromide, and 2-chloro-3-(trifluoromethyl)benzylamine with overall chemical yield 12% in three steps. The target tracer [ 18 F]IUR-1601 (( S )- N -(2-chloro-3-(trifluoromethyl)benzyl)-1-(2-[ 18 F]fluoroethyl)-5-oxopyrrolidine-2-carboxamide) was synthesized from desmethyl-GSK1482160 with 2-[ 18 F]fluoroethyl tosylate, prepared from 1,2-ethylene glycol-bis-tosylate and K[ 18 F ]F/Kryptofix2.2.2, in two steps and isolated by HPLC combined with SPE in 1–3% decay corrected radiochemical yield. The radiochemical purity was >99%, and the molar activity at end of bombardment (EOB) was 74–370 GBq/μmol. The potency of IUR-1601 in comparison with GSK1482160 was determined by a radioligand competitive binding assay using [ 11 C]GSK1482160, and the binding affinity K i values for IUR-1601 and GSK1482160 are 4.31 and 5.14 nM, respectively. [ABSTRACT FROM AUTHOR]

Brominated phenolic compounds (BPCs) are found in the environment, and in human and wildlife tissues, and some are considered to have endocrine disrupting activities. The goal of this study was to determine how structural differences of 3 BPC classes impact binding affinities for the thyroid receptor beta (TRß) in humans and zebrafish. BPC classes included halogenated bisphenol A derivatives, halogenated oxidative transformation products of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) and brominated phenols. Affinities were assessed using recombinant TRb protein in competitive binding assays with 125I-triiodothyronine (125I-T3) as the radioligand. Zebrafish and human TRß displayed similar binding affinities for T3 (Ki=0.40 and 0.49 nM) and thyroxine (T4, Ki=6.7 and 6.8 nM). TRß affinity increased with increasing halogen mass and atomic radius for both species, with the iodinated compounds having the highest affinity within their compound classes. Increasing halogen mass and radius increases the molecular weight, volume, and hydrophobicity of a compound, which are all highly correlated with increasing affinity. TRb affinity also increased with the degree of halogenation for both species. Human TRb displayed higher binding affinities for the halogenate bisphenol A compounds, whereas zebrafish TRb displayed higher affinities for 2,4,6-trichlorophenol and 2,4,6-trifluorophenol. Observed species differences may be related to amino acid differences within the ligand binding domains. Overall, structural variations impact TRb affinities in a similar manner, supporting the use of zebrafish as a model for TRb disruption. Further studies are necessary to investigate how the identified structural modifications impact downstreamreceptor activities and potential in vivo effects. [ABSTRACT FROM AUTHOR]

Aims: To confirm the different characteristics of genotype-specific and common neutralizing epitopes of hepatitis E virus (HEV). Methods: A competitive binding assay was established with known genotype-common neutralizing monoclonal antibodies (mAbs) 3G1 and 5G5 as well as genotype-specific neutralizing mAbs 2B1 and 4C5. HEV ORF2 recombinant p166W01 derived from genotype 1 and p166Chn derived from genotype 4 were used as coated antigens, to determine whether the mAbs recognize independent, similar, or overlapping epitopes. mAbs were produced, purified, and conjugated with horseradish peroxidase (HRP). HRP-conjugated 2B1 could react only with p166W01 but not p166Chn, HRP-conjugated 4C5 could react only with p166Chn but not p166W01, while HRP-conjugated 3G1 and 5G5 could react both with p166W01 and p166Chn. Thus, competitive binding assays were performed successively using p166W01 and p166Chn antigen. Results and Conclusion: The results of competitive binding assays revealed that the binding of HRP-conjugated 2B1 to p166W01 could not be inhibited by 5G5 or 3G1. Similarly, the binding of HRP-conjugated 4C5 to p166Chn could not be inhibited by 5G5 or 3G1. However, the mAbs 5G5 and 3G1 blocked each other’s binding to p166W01 and p166Chn, suggesting that common and genotype-specific neutralizing mAbs recognize independent epitopes. [ABSTRACT FROM AUTHOR]

Contaminants of emerging concern are continuously increasing, which makes it important to develop high throughput screening techniques for the evaluation of their potential biological effects, especially endocrine disrupting effects, which would directly influence the population dynamics in environment. A novel competitive binding assay based on enzyme fragmentation complementation technology was established to screen the binding affinities of emerging chemicals for estrogen receptor (ER) α or β isoforms. Exogenous compounds could compete with the fragment (ED-ES) of genetically engineered β-galactosidase enzyme (β-gal) for the binding to ERα or β, thus quantitatively altering the formation of enzymatically active β-gal and the hydrolysis of luminescent substrate. According to the monitoring of luminescence curves and the optimization of ERα or β concentrations, it was found that luminescent signals were sustainably emitted for 9 h, and 40 nM ERα or β in the system would lead to the most sensitive luminescence response. Using 17β-estrodiol (E 2 ) and genistein as the representative estrogenic hormones, their binding affinities for ERα and β were evaluated. The results were consistent with those determined by traditional methods, which confirmed the reliability of this competitive binding assay based on β-gal. Four polyfluorinated iodine alkanes (PFIs) with specific structural characteristics in iodine substitution and carbon chain length were screened, and the results showed diverse binding affinities and different preferences of these chemicals to ERα or β isoforms. The binding affinities of PFIs for ERα were consistent with the result from MVLN transcriptional reporter assay. Overall, the competitive binding assay presented in this study provided a promising alternative to high throughput screening of emerging chemicals with estrogenic effects, which would be important in explanation of their potential toxicological effects and human exposure risks. [ABSTRACT FROM AUTHOR]

P2X4 receptor has become an interesting molecular target for treatment and PET imaging of neuroinflammation and associated brain diseases such as Alzheimer’s disease. This study reports the first design, synthesis, radiolabeling and biological evaluation of new candidate PET P2X4 receptor radioligands using 5-BDBD, a specific P2X4 receptor antagonist, as a scaffold. 5-(3-Hydroxyphenyl)-1-[ 11 C]methyl-1,3-dihydro-2 H -benzofuro[3,2- e ][1,4]diazepin-2-one ( N- [ 11 C]Me-5-BDBD analog, [ 11 C] 9 ) and 5-(3-Bromophenyl)-1-[ 11 C]methyl-1,3-dihydro-2 H -benzofuro[3,2- e ][1,4]diazepin-2-one ( N- [ 11 C]Me-5-BDBD, [ 11 C] 8c ) were prepared from their corresponding desmethylated precursors with [ 11 C]CH 3 OTf through N -[ 11 C]methylation and isolated by HPLC combined with SPE in 30–50% decay corrected radiochemical yields with 370–1110 GBq/µmol specific activity at EOB. 5-(3-[ 18 F]Fluorophenyl)-1,3-dihydro-2 H -benzofuro[3,2- e ][1,4]diazepin-2-one ([ 18 F]F-5-BDBD, [ 18 F] 5a ) and 5-(3-(2-[ 18 F]fluoroethoxy)phenyl)-1,3-dihydro-2 H -benzofuro[3,2- e ][1,4]diazepin-2-one ([ 18 F]FE-5-BDBD, [ 18 F] 11 ) were prepared from their corresponding nitro- and tosylated precursors by nucleophilic substitution with K[ 18 F]F/Kryptofix 2.2.2 and isolated by HPLC-SPE in 5–25% decay corrected radiochemical yields with 111–740 GBq/µmol specific activity at EOB. The preliminary biological evaluation of radiolabeled 5-BDBD analogs indicated these new radioligands have similar biological activity with their parent compound 5-BDBD. [ABSTRACT FROM AUTHOR]

The reference standard methyl (2-amino-5-(benzylthio)thiazolo[4,5- d ]pyrimidin-7-yl)- d -leucinate ( 5 ) and its precursor 2-amino-5-(benzylthio)thiazolo[4,5- d ]pyrimidin-7-yl)- d -leucine ( 6 ) were synthesized from 6-amino-2-mercaptopyrimidin-4-ol and BnBr with overall chemical yield 7% in five steps and 4% in six steps, respectively. The target tracer [ 11 C]methyl (2-amino-5-(benzylthio)thiazolo[4,5- d ]pyrimidin-7-yl)- d -leucinate ([ 11 C] 5 ) was prepared from the acid precursor with [ 11 C]CH 3 OTf through O -[ 11 C]methylation and isolated by HPLC combined with SPE in 40–50% radiochemical yield, based on [ 11 C]CO 2 and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the specific activity (SA) at EOB was 370–1110 GBq/μmol with a total synthesis time of ∼40-min from EOB. The radioligand depletion experiment of [ 11 C] 5 did not display specific binding to CX 3 CR1, and the competitive binding assay of ligand 5 found much lower CX 3 CR1 binding affinity. [ABSTRACT FROM AUTHOR]

Thyroid hormone (TH) disrupting compounds are potentially important environmental contaminants due to their possible adverse neurological and developmental effects on both humans and wildlife. Currently, the most successful bio-analytical method to detect and evaluate TH disruptors, which target the plasma transport of TH in environmental samples, is the radio-ligand thyroxine-transthyretin (T 4 -TTR) binding assay. Yet, costly materials and tedious handling procedures prevent the use of this assay in high throughput analysis that is nowadays urgently demanded in environmental quality assessment. For the first time a miniaturized fluorescence T 4 -TTR binding assay was developed in a 96 well microplate and tested with eight TH disrupting compounds. For most of the compounds, the sensitivity of the newly developed assay was slightly lower than the radio-ligand binding assay, however, throughput was enhanced at least 100-fold, while using much cheaper materials. The TH disrupting potency of 22 herring gull ( Larus argentatus ) egg extracts, collected from two different locations (Musvær and Reiaren) in Norway, was evaluated to demonstrate the applicability of the assay for environmental samples. [ABSTRACT FROM AUTHOR]

Conjugation of aptamers and their corresponding analytes onto plasmonic nanoparticles mediates the formation of nanoparticle assemblies: molecularly bound nanoclusters that cause a measurable change in the colloid's optical properties. The optimization of a surface-enhanced Raman spectroscopy (SERS) competitive binding assay utilizing plasmonic "target" and magnetic "probe" nanoparticles for the detection of the toxin bisphenol-A (BPA) is presented. These assay nanoclusters were housed inside three types of optofluidic chips patterned with magnetically activated nickel pads, in either a straight or array pattern. Both Fe2O3 and Fe2CoO4 were compared as potential magnetic cores for the silver-coated probe nanoparticles. We found that the Ag@Fe2O3 particles were, on average, more uniform in size and more stable than Ag@Fe2CoO4, whereas the addition of cobalt significantly improved the collection time of particles. Using Raman mapping of the assay housed within the magnetofluidic chips, it was determined that a 1 × 5 array of 50 μm square nickel pads provided the most uniform SERS enhancement of the assay (coefficient of variation ~25%) within the magnetofluidic chip. Additionally, the packaged assay demonstrated the desired response to BPA, verifying the technology's potential to translate magnetic nanoparticle assays into a user-free optical analysis platform. [ABSTRACT FROM AUTHOR]

A full-length cDNA encoding Cydia pomonella pheromone binding protein 1 (CpomPBP1) was cloned and characterized. CpomPBP1, possessing the typical characteristics of lepidopteran odorant binding proteins, was detected to be specifically expressed in the antennae of male and female moths at the mRNA and protein level. Soluble recombinant CpomPBP1 was subjected to in vitro binding to analyse its binding properties and to search for potentially active semiochemicals. A competitive binding assay showed that three 12-carbon ligands, codlemone, 1-dodecanol and E,E-2,4-dodecadienal, were able to bind to CpomPBP1 in decreasing order of affinity. Moreover, unlike the wild-type CpomPBP1, the C-terminus truncated CpomPBP1 exhibited high affinity to ligands even in an acidic environment, suggesting that the C-terminus plays a role in preventing ligands from binding to CpomPBP1 in a lower pH environment. [ABSTRACT FROM AUTHOR]

Two chromium(III) complexes containing derivatives of 1,10-phenanthroline were synthesized and characterized using different spectroscopic methods. The interaction of the synthesized complexes with DNA was performed. From the absorption titration data, the binding constant for the complexes [Cr(imiphen) 2 Cl 2 ] + ( 1 ) and [Cr(furphen) 2 Cl 2 ] + ( 2 ) with DNA were found to be 5.69 ± 0.3 × 10 4 M − 1 for 1 and 3.7 ± 0.2 × 10 4 M − 1 for 2 , respectively. The viscosity studies show that both the complexes bind to DNA in a partial intercalative mode. The results of competitive binding assay reveal that both complexes displace EB from DNA marginally. Gel electrophoresis data show that both the complexes 1 and 2 requires co-reagent to induce DNA cleavage but complex 2 exhibit better nuclease activity compared to complex 1 . The results from this study describe the role of ligand structure in the binding and cleavage of DNA as furan based ligand exhibit better cleaving ability compared to imidazole moiety. [ABSTRACT FROM AUTHOR]

Extracellular signal-regulated kinase 2 (ERK2) is a drug target for type 2 diabetes mellitus. A peptide-type ERK2 inhibitor (PEP) was discovered in the previous study through the knowledge-based method and showed physiological effects on the db / db mice model of type 2 diabetes. Here, the crystal structure showed that PEP bound to the allosteric site without the interruption of the ATP competitive inhibitor binding to ERK2. An in silico biased-screening using the focused library rendered three compounds with inhibitory activity of IC 50 <100 μM. Among them, two compounds revealed the concentration-dependent competition with PEP and could be lead compounds for antidiabetic medicine. [ABSTRACT FROM AUTHOR]

Sphingosine-1-phosphate receptors (S1PRs) are important regulators of vascular permeability, inflammation, angiogenesis and vascular maturation. Identifying a specific S1PR PET radioligand is imperative, but it is hindered by the complexity and variability of current for binding affinity measurement procedures. Herein, we report a streamlined protocol for radiosynthesis of [ 32 P]S1P with good radiochemical yield (36–50%) and high radiochemical purity (>99%). We also report a reproducible procedure for determining the binding affinity for compounds targeting S1PRs in vitro . [ABSTRACT FROM AUTHOR]

Oligomers incorporating the tetrapeptide MSH4, the minimum active sequence of melanocyte stimulating hormone, were synthesized by an A 2 + B 2 strategy involving microwave-assisted copper-catalyzed azide-alkyne cycloaddition. A 2 contained an MSH4 core while B 2 contained a (Pro-Gly) 3 spacer. Soluble mixtures containing compounds with up to eight MSH4 units were obtained from oligomerizations at high monomer concentrations. The avidities of several oligomeric mixtures were evaluated by means of a competitive binding assay using HEK293 cells engineered to overexpress the melanocortin 4 receptor. When based on total MSH4 concentrations, avidities were only minimally enhanced compared with a monovalent control. The lack of variation in the effect of ligands on probe binding is consistent with high off rates for MSH4 in both monovalent and oligomeric constructs relative to that of the competing probe. [ABSTRACT FROM AUTHOR]

Abstract: The development of high-throughput screening (HTS) assays with increased sensitivity for the identification of potent and selective inhibitors of galectins has been hampered by the weak binding affinities between galectins and their carbohydrate ligands. To circumvent this obstacle, we have developed an AlphaScreen assay for a 384-well plate format in a competitive binding configuration for discovery of new inhibitors of galectin-3. His-tagged galectin-3 was bound to nickel chelate acceptor beads, whereas biotinylated asialofetuin (biotin–ASF), a galectin-3 nanomolar binding partner, was bound to streptavidin-coated donor beads. Inhibitors of the carbohydrate–galectin interaction lead to a reduction of the AlphaScreen signal by competing with the biotin–ASF. The obtained IC50 values for known carbohydrate ligands of galectin-3 are in good agreement with the K d values reported and measured for galectin-3 by isothermal titration calorimetry (ITC). Thus, the developed AlphaScreen assay in a competitive binding configuration offers several advantages over the existing screening assays for inhibitors of glycan–lectin interactions. In addition, the assay format for the galectin-3/ASF pair could be easily applied in screening for glycan- and/or small molecule-based inhibitors of other members of the galectin family. [Copyright &y& Elsevier]

We have developed competitive and direct binding methods to examine small-molecule inhibitors of protein tyrosine phosphatase activity. Focusing on the Yersinia pestis outer protein H, a potent bacterial protein tyrosine phosphatase, we describe how an understanding of the kinetic interactions involving Yersinia pestis outer protein H, peptide substrates, and small-molecule inhibitors of protein tyrosine phosphatase activity can be beneficial for inhibitor screening, and we further translate these results into a microarray assay for high-throughput screening. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: Novel technetium-labeled ligands, 99mTc-NCAM and 99mTc-NHAM were developed from the N-methyl-d-aspartate (NMDA) receptor agonist memantine as a lead compound by coupling with N2S2. This study evaluated the binding affinity and specificity of the ligands for the NMDA receptor. Methods: Ligand biodistribution and uptake specificity in the brain were investigated in mice. Binding affinity and specificity were determined by radioligand receptor binding assay. Three antagonists were used for competitive binding analysis. In addition, uptake of the complexes into SH-SY5Y nerve cells was evaluated. Results: The radiochemical purity of 99mTc-labeled ligands was more than 95%. Analysis of brain regional uptake showed higher concentration in the frontal lobe and specific uptake in the hippocampus. 99mTc-NCAM reached a higher target to nontarget ratio than 99mTc-NHAM. The results indicated that 99mTc-NCAM bound to a single site on the NMDA receptor with a K d of 701.21 nmol/l and a B max of 62.47 nmol/mg. Specific inhibitors of the NMDA receptor, ketamine and dizocilpine, but not the dopamine D2 and 5HT1A receptor partial agonist aripiprazole, inhibited specific binding of 99mTc-NCAM to the NMDA receptor. Cell physiology experiments showed that NCAM can increase the viability of SH-SY5Y cells after glutamate-induced injury. Conclusions: The new radioligand 99mTc-NCAM has good affinity for and specific binding to the NMDA receptor, and easily crosses the blood–brain barrier; suggesting that it might be a potentially useful tracer for NMDA receptor expression. [Copyright &y& Elsevier]

. We investigated the virus-host interaction for hepatitis E virus (HEV) by performing competitive binding assays using in vitro assembled virus-like particles (VLPs). We used Escherichia coli expressed native capsid protein (pORF2) and its mutants with an attached Gly (5) -Ala (linker) reporter [enhanced green fluorescent protein (EGFP)/firefly luciferase (Fluc)]. Transmission electron microscopy and nanoparticle tracking showed near uniform particles of approximately 30-35 nm in diameter for pORF2 VLPs and 60-100 nm for reporter-linked VLPs. Binding of reporter-linked full-length (1-660aa) and N-terminal truncated (Δ1-112aa) pORF2 VLPs to Huh7 cell surfaces was found to be specific with 1.92 ± 0.065 × 105 sites per cell. Saturation binding indicated an equilibrium dissociation constant ( Kd) of 121.1 ± 23.83 and 123.8 ± 16.15 n m for pORF2-linker-EGFP and pORF2-linker-Fluc VLPs respectively. A similar binding pattern was observed for Δ1-112aa pORF2-linker-EGFP and Δ1-112aa pORF2-linker-Fluc VLPs with Kd values of 123.6 ± 10.60 and 135.6 ± 16.19 n m respectively. The affinity (log Ki) of pORF2 binding on Huh7 cells in the presence of EGFP-tagged and Fluc-tagged pORF2 VLPs was found to be approximately 2.0. However, no VLP formation or binding was observed with refolded C-terminal truncated (Δ458-660aa) pORF2. We investigated HEV internalization using fluorescent VLPs (EGFP-VLPs), which showed vesicle-mediated uptake starting at 5 min post-incubation. The uptake of VLPs could be stopped by inhibitors for clathrin-dependent endocytosis, but not by caveosome inhibitors. No binding and uptake of EGFP-VLPs were observed on non-hepatic cell lines (HeLa and SiHa). These findings suggest that HEV attaches to the host cell via a specific high affinity receptor and enters the cytoplasm by clathrin-mediated endocytosis. [ABSTRACT FROM AUTHOR]

Abstract: Urinary tract infection (UTI) caused by uropathogenic Escherichia coli (UPEC) is one of the most prevalent infectious diseases. Particularly affected are women, who have a 40–50% risk to experience at least one symptomatic UTI episode at some time during their life. In the initial step of the infection, the lectin FimH, located at the tip of bacterial pili, interacts with the high-mannosylated uroplakin Ia glycoprotein on the urinary bladder mucosa. This interaction is critical for the ability of UPEC to colonize and invade the bladder epithelium. X-ray structures of FimH co-crystallized with two different ligands, the physiological binding epitope oligomannose-3 and the antagonist biphenyl α-d-mannoside 4a revealed different binding modes, an in-docking-mode and an out-docking-mode, respectively. To accomplish the in-docking-mode, that is the docking mode where the ligand is hosted by the so-called tyrosine gate, FimH antagonists with increased flexibility were designed and synthesized. All derivatives 5–8 showed nanomolar affinities, but only one representative, the 4-pyridiyl derivative 5j, was as potent as the reference compound n-heptyl α-d-mannoside (1b). Furthermore, a loss of affinity was observed for C-glycosides and derivatives where the triazole aglycone is directly N-linked to the anomeric center. A conformational analysis by NMR revealed that the triazolyl-methyl-C-mannosides 8 adopt an unusual 1 C 4 chair conformation, explaining the comparably lower affinity of these compounds. Furthermore, to address the druglikeness of this new class of FimH antagonists, selected pharmacokinetic parameters, which are critical for oral bioavailability (lipophilicity, solubility, and membrane permeation), were determined. [Copyright &y& Elsevier]

Abstract: Uropathogenic Escherichia coli (UPEC) is the primary cause of urinary tract infections (UTIs). In the first step of this infective process, the virulence factor FimH located on type 1 pili allows UPEC to specifically adhere to oligosaccharides, which are part of glycoproteins on the urinary bladder mucosa. This initial step prevents the clearance of E. coli from the urinary tract and enables the invasion of the host cells. Because FimH antagonists can block this interaction, they exhibit a promising therapeutic potential as anti-infectives. For the evaluation of their binding properties, a reliable, target-based affinity assay is required. Here, we describe the expression and purification of the carbohydrate recognition domain of FimH (FimH-CRD) as well as the development of a competitive binding assay. FimH-CRD linked with a thrombin cleavage site to a 6His-tag is recombinantly expressed and purified by affinity chromatography. For the evaluation of FimH antagonists, a cell-free binding assay based on the interaction of a biotinylated polyacrylamide glycopolymer with the FimH-CRD was developed. Complexation of the biotinylated glycopolymer with streptavidin coupled to horseradish peroxidase allows the quantification of the binding properties of FimH antagonists. The assay format was optimized and validated by a comparison with affinity data from reported assays. [ABSTRACT FROM AUTHOR]

Abstract: A series of novel aryl-substituted triazolyl d-galactosamine derivatives was synthesized as ligands for the carbohydrate recognition domain of the major subunit H1 (H1-CRD) of the human asialoglycoprotein receptor (ASGP-R). The compounds were biologically evaluated with a newly developed competitive binding assay, surface plasmon resonance and by a competitive NMR binding experiment. With compound 1b, a new ligand with a twofold improved affinity to the best so far known d-GalNAc was identified. This small, drug-like ligand can be used as targeting device for drug delivery to hepatocytes. [Copyright &y& Elsevier]

Abstract: 8-anilino-1-naphthalenesulfonic acid (ANS) is believed to strongly bind cationic groups of proteins and polyamino acids through ion pair formation. A paucity of data exists on the fluorescent properties of ANS in these interactions. ANS binding to arginine and lysine derivatives was studied by fluorescence and circular dichroism spectroscopies to augment published information attained by isothermal titration calorimetry (ITC). Fluorescence enhancement with a hypsochromic shift results from the interaction of the charged group of lysine and arginine with the sulfonate group of ANS. Ion pairing between Arg (or Lys) and the sulfonate group of ANS reduce the intermolecular charge transfer (CT) rate constant that leads to enhancement of fluorescence. A positive charge near the –NH group of ANS changes the intramolecular CT process producing a blue shift of fluorescence. The Arg side chain compared to that of Lys more effectively interacts with both the –NH and sulfonate groups of ANS. ANS binding also induces a random coil-alpha helix transition in poly-Arg. Our data, in contrast to ITC results, indicate that electrostatic interactions between ANS derivatives and positively charged side chains do not account for binding affinity in the micromolar range. In addition to ion pairing complementary interactions, such as van der Waals, should be considered for high affinity (K d <1 mM) external binding sites of proteins. [Copyright &y& Elsevier]

Simple Summary: Bradysia odoriphaga (Diptera: Sciaridae) is an important underground pest in Chinese chives. Chemical pesticides are the main prevention and control method, however, but this method not only leads to the increase of the insect's resistance, but also causes pesticide residues and pollutes the environment. Previous studies have shown that olfaction plays a crucial role in the recognition of plant volatiles and sex pheromones, but the mechanism of olfactory action is still unclear. In the present study, Real-time PCR (qRT-PCR) analysis revealed that BodoOBP8 was highly expressed in the antennae of both sexes, and speculated that it is very likely to participate in the olfactory process. Then we used prokaryotic expression, fluorescence competitive binding, homology modeling, and molecular docking to prove its olfactory function. The results of this study increase our understanding of the binding of BodoOBP8 with plant volatiles and sex pheromone, facilitating the development of novel ways to control B. odoriphaga. The belowground pest Bradysia odoriphaga (Diptera: Sciaridae) has a sophisticated and sensitive olfactory system to detect semiochemical signals from the surrounding environment. In particular, odorant-binding proteins (OBPs) are crucial in capturing and transporting these semiochemical signals across the sensilla lymph to the corresponding odorant receptors. In this study, we cloned a full-length cDNA sequence of BodoOBP8 from B. odoriphaga. Real-time PCR (qRT-PCR) analysis revealed that BodoOBP8 has the highest expression levels in males, with more pronounced expression in the male antennae than in other tissues. In this study, the recombinant protein BodoOBP8 was successfully expressed by a bacterial system to explore its function. Competitive binding assays with 33 host plant volatiles and one putative sex pheromone (n-heptadecane) revealed that purified BodoOBP8 strongly bound to two sulfur compounds (methyl allyl disulfide and diallyl disulfide) and to n-heptadecane; the corresponding dissolution constants (Ki) were 4.04, 6.73, and 4.04 μM, respectively. Molecular docking indicated that Ile96, Ile103, Ala107, and Leu111, located in the hydrophobic cavity of BodoOBP8, are the key residues mediating the interaction of BodoOBP8 with two sulfur compounds (methyl allyl disulfide and diallyl disulfide) and n-heptadecane. These results show that BodoOBP8 plays a role in the recognition of plant volatiles and sex pheromones, suggesting its application as a molecular target for the screening of B. odoriphaga attractants and repellents and facilitating a new mechanism of B. odoriphaga control. [ABSTRACT FROM AUTHOR]

Odorant-binding proteins (OBP) in the mucus of the olfactory epithelium are thought to transfer the hydrophobic odorous compounds through the aqueous barrier towards the chemo-sensory cells. To evaluate their binding properties, two distinct OBP subtypes of the rat were expressed as N-terminal His-tagged fusion proteins in Escherichia coli, thus allowing an efficient purification. Based on gel chromatography and CD spectroscopy analysis the recombinant OBP subtypes seem to share several structural features with other members of the lipocalin family. Approaches to elucidate whether heterologous expressed OBPs interact with odorous compounds revealed that OBP1 specifically binds 2-[3H]-isobutyl-3-methoxypyrazine whereas OBP2 did not shown any specific binding to this compound. In contrast, the chromophore 1-anilinonaphthalene 8-sulfonic acid (1,8-ANS) specifically interacted with OBP2 but not with OBP1. Displacement experiments monitored by the relative fluorescence intensity revealed that fatty acids with appropriate chain length act as efficient competitors. Some odorous compounds, notably lilial (p-tert-butyl-α-methyl dihydrocinnamic aldehyde) and citralva (3,7-dimethyl-2,6-octadienenitrile), also displaced efficiently the chromophore, whereas pyrazine derivatives including 2-isobutyl-3-methoxypyrazine and other odorants did not. These results indicate that rat OBPs have distinct ligand specificities. [ABSTRACT FROM AUTHOR]

• Eosin Y interacts with DNA with a binding constant of about 10−4 M−1. • Binding of eosin Y to DNA is not accompanied by the changes of the visible spectrum. • Competitive binding assay was used to calculate the eosin Y – DNA binding parameters. • Eosin Y forms heteroassociates with methylene blue, but not with proflavine. The interaction between xanthene dye eosin Y and double stranded DNA has been studied by spectrophotometry. The conventional titration study does not show the interaction in the eosin Y – DNA system. Therefore, the competitive binding assay was carried out. The DNA-targeted ligands proflavine and methylene blue were used as competitors. Multivariate curve resolution – alternative least squares method (MCR-ALS) was applied to analyze the spectrophotometric titration data. The experimental binding isotherms were fitted by Scatchard and McGee equations. The binding constant of eosin Y with DNA was found to be 1.7·104 M−1. It is shown that the competitive binding assay requires consideration of heteroassociation for the correct determination of ligand-DNA binding parameters. [ABSTRACT FROM AUTHOR]

Abstract: A series of 17α-(heteroaryl)vinyl estradiols was prepared to evaluate the influence of heteroatom on the affinity and efficacy of estrogenic ligands for the estrogen receptor-alpha ligand binding domain (ERα-LBD). The products demonstrated reduced binding affinity compared to the parent 17α-E-phenylvinyl estradiol, but the binding was relatively independent of the heteroatom. The greatest influence of the heteroatom was evident in the efficacy of the compounds as the thienyl derivatives 2f,g were more potent than either the pyridyl 2b–d or pyrimidinyl 2e analogs. The results suggest that a subtle interplay of interactions between the ligands and the receptor influences the biological response. [Copyright &y& Elsevier]

Some thyroid-disrupting chemicals (TDCs) affect thyroid function by activating the pathways mediated by a typical thyroid hormone (TH) membrane receptor, integrin α v β 3. The present study introduces improved competitive binding assays for the rapid and sensitive evaluation of the binding affinities of TDCs for integrin α v β 3. Based on different probes, two assays were modified: a fluorescence competitive binding assay and a radioligand competitive binding assay. The chemicals tested included the known TH, 3,3′,5,5′-tetraiodo- l -thyronine (T4); a deaminated analog of T4, tetraiodothyroacetic acid (tetrac); and phthalate esters (PAEs). The relative binding potency of T4 was studied, and the concentration required to displace 50% of the ligands from their receptors (RIC50) of T4 was 4.9 × 105 and 9.7 × 104 nM for the fluorescence and radioligand competitive binding assays, respectively, suggesting that the radioligand competitive binding assay might be more sensitive for the evaluation of the binding affinity for integrin α v β 3. The three PAEs, including diethyl hexyl phthalate (DEHP), benzyl butyl phthalate (BBP) and dibutyl phthalate (DnBP), demonstrated binding affinities for integrin α v β 3 in the following order of potency: DnBP > DEHP > BBP tested by the radioligand competitive binding assay. A docking simulation of each of the three PAEs with integrin α v β 3 confirmed the calculated binding energies, which had a strong positive relationship with the log RIC20 values of the 3 PAEs (R = 0.99, p < 0.001). The present study shows that the established radioligand competitive binding assay could be used as a valuable tool for quantifying the affinity of TDCs for integrin α v β 3. Image 1 • A fluorescence competitive binding assay using the T4-BSA-F probe was developed. • A radioligand competitive binding assay using the 99mTc-3PRGD 2 probe was established. • PAEs had binding affinity for integrin α v β 3 in the following order: DnBP > DEHP > BBP [ABSTRACT FROM AUTHOR]