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Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4′-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins.

The elimination of ejaculates and males with low fertility despite good sperm motility and morphology is crucial to maintain high pregnancy rates after artificial insemination (AI) in farm animals. The ability of sperm to survive in the female tract is particularly crucial in pigs due to the large variation in the timing between AI and ovulation and the high number of oocytes to fertilise. The objective of this study was to characterise a new in vitro model of oviduct sperm reservoir using porcine oviduct epithelial spheroids (OES) and to assess the variability in sperm binding to OES among gilts, boars and their ejaculates. Isthmic mucosa fragments were collected from gilt oviducts at a slaughterhouse, and after 48 h of culture, the OES that had spontaneously formed were sorted according to their vesicle shape and size (150–200 μm in diameter) for characterisation and sperm binding assays. The OES contained viable, cytokeratin-positive and vimentin-negative cells, of which 36.4 ± 2.0% were multiciliated. The average proportion of multiciliated cells per OES did not change among culture replicates. After co-incubation with boar fresh semen, only sperm of normal morphology were found to bind, by their head, to cilia of OES. The density of sperm bound to the OES surface increased linearly with sperm concentration. The bound sperm density on OES was used to assess the binding capacity of fresh ejaculates collected from Pietrain boars. For a given ejaculate, the bound sperm density did not vary among pools of OES female donors. The analysis of five successive ejaculates from nine boars indicated significant differences in bound sperm densities on the OES among individual boars and their ejaculates (P < 0.01). There was no correlation between the sperm bound density and sperm parameters measured by computer-assisted sperm analysis or the initial dilution of the ejaculate. In conclusion, the OES characterised in this study offered physiological conditions to study sperm binding to the isthmic reservoir and evidenced that sperm from different ejaculates and different boars vary in their ability to bind to these oviduct spheroids despite homogeneous motility and morphology. • Isthmus epithelial spheroids provided a standardised tool for sperm binding assays. • Binding capacity of boar fresh semen to spheroids did not change among gilt donors. • A considerable variation in sperm binding to spheroids was observed among boars. • A high variation in binding was also observed among ejaculates from individual boars. • This variability could not be explained by sperm parameters measured by CASA. [ABSTRACT FROM AUTHOR]

Nimotuzumab has been used for treating squamous cell carcinoma of the head and neck. Its efficacy is attributed to its mechanism of action, which involves the binding to the epidermal growth factor receptor. Ensuring the quality of nimotuzumab is crucial for its safety and efficacy. Utilizing a binding assay to measure its efficacy is a valid approach since it directly correlates with its potency. Here, we developed and validated the cell-based binding assay to determine the potency of nimotuzumab. A-431 cells and fluorescein isothiocyanate mouse anti-human immunoglobulin G antibody were used and the fluorescent intensity was measured to evaluate the binding activity. The binding assay conditions were optimized during the method development and the final method was fully validated. The binding method was established by optimizing the suitability and appropriate amount of the antibody, incubation time for binding assay, and the condition for cell washing. The results of specificity and the linearity range between 1.5 and 4.5 µg/ml of nimotuzumab were in the acceptable criteria. The recoveries of nimotuzumab at 75, 100, and 125% of the target concentration demonstrated that the method was accurate. The relative standard deviations for repeatability and intermediate precision were within the limit. The data revealed that the developed method was successfully validated. The developed and validated cellbased potency assay can be employed for the potency determination of nimotuzumab products marketed in Thailand and the method can later be adapted to evaluate biosimilar nimotuzumab that will be developed and registered in the foreseeable future. [ABSTRACT FROM AUTHOR]

Serotonin, or 5-hydroxytryptamine (5-HT), is a biogenic amine most noted as a neurotransmitter, an activator of the utmost subtype family of G-protein-coupled receptors (GPCR). Drugs targeting 5-HT1A and other 5-HT receptors treat central nervous system diseases such as schizophrenia and depression. Recent advances in serotonin receptor structure research gave us several crystal 5-HT1A receptor structures, most notably 5-HT1A bound to the antipsychotic drug aripiprazole (Abilify7#174;). This discovery prompted us to evaluate a series of newly synthesized ligands for serotonergic activity since those arylpiperazine derivatives share minimal general structure with aripiprazole. The results of molecular docking analysis of unsubstituted starting substances encouraged us to propound further modifications of the tail and head parts of the parent molecules to maximize receptor binding affinity. Intrigued by the results of molecular analysis, all foreseen derivatives were synthesized. The pharmacological activity of all nine (5a and 6a are synthesized previously) compounds was assessed by the in vitro tests and in silico pharmacokinetics predictions for the most promising candidates. All tested ligands have improved affinity compering to parent compounds (10a and 11a), 8b and 9b expressed the best pharmacological profile with an improved binding affinity toward serotonin 5-HT1A receptors (Ki 12.1 and 4.8 nM, respectively). [ABSTRACT FROM AUTHOR]

Simple Summary: The bamboo weevil beetle, Cyrtotrachelus buqueti (Coleoptera: Curculionidae) is the primary bamboo pest and seriously influences the development of the bamboo industry. Sensory neuron membrane proteins (SNMPs) play important roles in insect pheromone communication. However, SNMPs of C. buqueti are still uncharacterized. With the aim of developing control techniques to control this pest, one novel SNMP from C. buqueti, CbuqSNMP1b, was functionally characterized in this study. The results indicated that CbuqSNMP1b was predominantly expressed in the antennae of both sexes and showed significantly higher transcription levels in the adult stage, suggesting that CbuqSNMP1b is involved in the process of olfaction. Fluorescence binding assays revealed that CbuqSNMP1b could bind to three out of fourteen volatile compounds emitted from C. buqueti and showed the strongest binding affinity to dibutyl phthalate, demonstrating the role of SNMP1 in detecting odorants. Molecular docking was performed to further understand the binding mode between CbuqSNMP1b and these three ligands, and the results showed that hydrophobic interactions were the prevailing forces within the binding cavities of CbuqSNMP1b. The results of this study will be helpful to understand the function of SNMP1 in C. buqueti and lay a foundation for developing new methods to control this pest. Sensory neuron membrane proteins (SNMPs) play important roles in insect chemoreception and SNMP1s have been reported to be essential in detecting sex pheromones in Drosophila and some lepidopteran species. However, SNMPs for Cyrtotrachelus buqueti (Coleoptera: Curculionidae), a major insect pest of bamboo plantations, remain uncharacterized. In this study, a novel SNMP gene, CbuqSNMP1b, from C. buqueti was functionally characterized. The expression of CbuqSNMP1b was significantly higher in antennae than in other tissues of both sexes and the expression level was significantly male-biased. Additionally, CbuqSNMP1b showed significantly higher transcription levels in the adult stage and very low transcription levels in other stages, suggesting that CbuqSNMP1b is involved in the process of olfaction. Fluorescence binding assays indicated that CbuqSNMP1b displayed the strongest binding affinity to dibutyl phthalate (Ki = 9.03 μM) followed by benzothiazole (Ki = 11.59 μM) and phenol (Ki = 20.95 μM) among fourteen C. buqueti volatiles. Furthermore, molecular docking revealed key residues in CbuqSNMP1b that interact with dibutyl phthalate, benzothiazole, and phenol. In conclusion, these findings will lay a foundation to further understand the olfactory mechanisms of C. buqueti and promote the development of novel methods for controlling this pest. [ABSTRACT FROM AUTHOR]

Pheromone-binding proteins (PBPs) play important roles in binding and transporting sex pheromones. However, the PBP genes identified in coleopteran insects and their information sensing mechanism are largely unknown. Cyrtotrachelus buqueti (Coleoptera: Curculionidae) is a major insect pest of bamboo plantations. In this study, a novel PBP gene, CbuqPBP2, from C. buqueti was functionally characterized. CbuqPBP2 was more abundantly expressed in the antennae of both sexes than other body parts, and its expression level was significantly male-biased. Fluorescence competitive binding assays showed that CbuqPBP2 exhibited the strongest binding affinity to dibutyl phthalate (Ki = 6.32 μM), followed by styrene (Ki = 11.37 μM), among twelve C. buqueti volatiles. CbuqPBP2, on the other hand, showed high binding affinity to linalool (Ki = 10.55), the main volatile of host plant Neosinocalamus affinis. Furthermore, molecular docking also demonstrated the strong binding ability of CbuqPBP2 to dibutyl phthalate, styrene, and linalool, with binding energy values of −5.7, −6.6, and −6.0 kcal/mol, respectively, and hydrophobic interactions were the prevailing forces. The knockdown of CbuqPBP2 expression via RNA interference significantly reduced the electroantennography (EAG) responses of male adults to dibutyl phthalate and styrene. In conclusion, these results will be conducive to understanding the olfactory mechanisms of C. buqueti and promoting the development of novel strategies for controlling this insect pest. [ABSTRACT FROM AUTHOR]

Benzodiazepines (BZD) are widely used in neurological disorders. The use of classical benzodiazepines is limited due to side effects. In this study, based on the structure–activity relationship (SAR) of Benzodiazepine receptors, new derivatives of 4-amino-3,5-diphenyl-1,2,4-triazole as benzodiazepine agonists with selective effects were designed and synthesized. Docking studies showed that pharmacophore groups of the designed structures and zolpidem, a benzodiazepine receptor agonist, are properly matched and are located well in the GABA receptor. The triazole group of the compound 4j, N N-(3,5-diphenyl-4H-1,2,4-triazol-4-yl)-2-((4-fluorobenzyl)amino)acetamide, was near the nitrogen moiety of the imidazole ring of zolpidem providing the hydrogen bond acceptor in the suitable direction in the BDZ-binding site of GABAA receptor model (α1β2ϒ2). The compounds were synthesized with acceptable yield and in-vitro affinity for the BZD receptor was determined. Compound 4j had the best affinity for the BZD site of action on GABAA receptor complex (Ki = 2.56 nM and IC50 = 6.10 nM). In addition, the sedative-hypnotic effect, the locomotor activity, and evaluated memory of the novel compounds were assessed by pentobarbital-induced sleeping, open field, and passive avoidance tests respectively. Most of the novel compounds showed significant hypnotic activity with no impairment on learning and memory performance in the mouse. The pharmacological effects of the compounds were antagonized by flumazenil, a BZD antagonist, which confirms the involvement of BZD receptors in the biological effects.

The autophagy-related 12 (ATG12)–autophagy-related 5 (ATG5)–autophagy-related 16-like 1 (ATG16L1) ternary complex forms a dimer that facilitates the translocation of autophagy-related 8 (ATG8) proteins from autophagy-related 3 (ATG3) to phosphatidylethanolamine (PE). This event is fundamental for cargo sequestration and autophagy progression. Thus, one possible strategy for inhibiting autophagy is to disrupt the critical ATG5-ATG16L1 interaction during this process. So far very few known specific autophagy modulators can block autophagy effectively. We recently discovered a small-molecule compound, T1742, which is able to block the ATG5-ATG16L1 and ATG5-TECAIR interactions in vitro at the low-micromolar range (IC50 = 1~2 μM). Flow cytometry assay and western blot experiments indicated that T1742 can also effectively inhibit autophagy in living cells in a dose-dependent manner. To the best of our knowledge, T1742 represents the first small-molecule autophagy inhibitor that disrupts the protein-protein interactions involving ATG5. Such compounds may serve as a new chemical tool for deciphering the mechanism of autophagy or a potential candidate for therapeutic application.

Human exposure to dicarbonyls occurs via ingestion (e.g., food), inhalation (e.g., electronic cigarettes) and dysregulation of endogenous metabolic pathways (e.g., glycolysis). Dicarbonyls are electrophiles able to induce carbonylation of endogenous substrate. They have been associated with the onset and progression of several human diseases. Several studies have advocated the use of dicarbonyl binders as food preservatives or as drugs aimed at mitigating carbonylation. This study presents the setup of an easy and cheap assay for the screening of selective and potent dicarbonyl binders. The method is based on the incubation of the candidate molecules with a molecular probe. The activity is then determined by measuring the residual concentration of the molecular probe over time by liquid chromatography (LC). However, the naturally occurring dicarbonyls (e.g., glyoxal, methylglyoxal) are not appealing as probes since they are hard to separate and detect using the most popular LC variants. Benzylglyoxal (BGO) was therefore synthesized and tested, proving to be a convenient probe that allows a direct quantification of residual dicarbonyls by reversed phase LC without derivatization. The method was qualified by assessing the binding ability of some molecules known as binders of natural occurring dicarbonyls, obtaining results consistent with literature. [ABSTRACT FROM AUTHOR]

Receptor‐derived peptides have played an important role in elucidating chemokine‐receptor interactions. For the inflammatory chemokine CXC‐class chemokine ligand 8 (CXCL8), a site II‐mimetic peptide has been derived from parts of extracellular loops 2 and 3 and adjacent transmembrane helices of its receptor CXC‐class chemokine receptor 1 (Helmer et al., RSC Adv., 2015, 5, 25657). The peptide sequence with a C‐terminal glutamine did not bind to CXCL8, whereas one with a C‐terminal glutamate did but with low micromolar affinity. We sought to improve the affinity and protease stability of the latter peptide through cyclization while also cyclizing the former for control purposes. To identify a cyclization strategy that permits a receptor‐like interaction, we conducted a molecular dynamics simulation of CXCL8 in complex with full‐length CXC‐class chemokine receptor 1. We introduced a linker to provide an appropriate spacing between the termini and used an on‐resin side‐chain‐to‐tail cyclization strategy. Upon chemokine binding, the fluorescence intensity of the tetramethylrhodamine (TAMRA)‐labeled cyclic peptides increased whereas the fluorescence anisotropy decreased. Additional molecular dynamics simulations indicated that the fluorophore interacts with the peptide macrocycle so that chemokine binding leads to its displacement and observed changes in fluorescence. Macrocyclization of both 18‐amino acid‐long peptides led to the same low micromolar affinity for CXCL8. Likewise, both TAMRA‐labeled linear peptides interacted with CXCL8 with similar affinities. Interestingly, the linear TAMRA‐labeled peptides were more resistant to tryptic digestion than the unlabeled counterparts, whereas the cyclized peptides were not degraded at all. We conclude that the TAMRA fluorophore tends to interact with peptides altering their protease stability and behavior in fluorescence‐based assays. [ABSTRACT FROM AUTHOR]

Agonists of Benzodiazepine (BZD) receptor are exhaustively used in the control of muscle spasms, seizure, anxiety, and insomnia. BZDs have some unwanted effects; therefore, the development of new BZD receptor agonists with better efficacy and fewer unwanted effects is one of the subjects of interest. In this study, based on the pharmacophore/receptor model of the BZD binding site of GABAA receptors, a series of new 2-substituted-5-(4-chloro-2-phenoxy)phenyl-1,3,4-oxadiazole derivatives (6a-f) were designed. Energy minima conformers of the designed compounds and diazepam were well matched in conformational analysis and showed proper interaction with the BZD-binding site of the GABAA receptor model (α1β2ϒ2) in docking studies. The designed compounds were synthesized in acceptable yield and evaluated for their in vitro affinity to the benzodiazepine receptor of rat brains by radioligand receptor binding assay. The results demonstrated that the affinities of most of the novel compounds were even higher than diazepam. The novel compound 6a with the best affinity in radioligand receptor binding assay (Ki=0.44 nM and IC50= 0.73±0.17 nM) had considerable hypnotic activity and weak anticonvulsant and anxiolytic effects with no negative effect on memory in animal models. Flumazenil as a selective benzodiazepine receptor antagonist was able to prevent hypnotic and anticonvulsant effects of 6a indicating the role of BZD receptors in these effects.

Abstract Dopamine-deficient (DD) mice exhibit psychomotor hyperactivity that might be related to a decrease in muscarinic signaling. In the present study, muscarinic acetylcholine receptor M2 (CHRM2) density decreased in the cortex in DD mice. This is significant because cortical CHRM2 acts as an autoreceptor; therefore, changes in CHRM2 levels could alter acetylcholine in DD mice. We also found that the CHRM1/CHRM4 agonist xanomeline and CHRM2 agonist arecaidine propargyl ester tosylate inhibited hyperactivity in DD mice, suggesting that postsynaptic CHRM1 and CHRM2 and presynaptic CHRM2 may be involved in hyperactivity in DD mice.

Sensory neuron membrane proteins (SNMPs) play important roles in insect chemoreception and SNMP1s have been reported to be essential in detecting sex pheromones in Drosophila and some lepidopteran species. However, SNMPs for Cyrtotrachelus buqueti (Coleoptera: Curculionidae), a major insect pest of bamboo plantations, remain uncharacterized. In this study, a novel SNMP gene, CbuqSNMP1b, from C. buqueti was functionally characterized. The expression of CbuqSNMP1b was significantly higher in antennae than in other tissues of both sexes and the expression level was significantly male-biased. Additionally, CbuqSNMP1b showed significantly higher transcription levels in the adult stage and very low transcription levels in other stages, suggesting that CbuqSNMP1b is involved in the process of olfaction. Fluorescence binding assays indicated that CbuqSNMP1b displayed the strongest binding affinity to dibutyl phthalate (Ki = 9.03 μM) followed by benzothiazole (Ki = 11.59 μM) and phenol (Ki = 20.95 μM) among fourteen C. buqueti volatiles. Furthermore, molecular docking revealed key residues in CbuqSNMP1b that interact with dibutyl phthalate, benzothiazole, and phenol. In conclusion, these findings will lay a foundation to further understand the olfactory mechanisms of C. buqueti and promote the development of novel methods for controlling this pest.

Serotonin receptors modulate numerous behavioral and neuropsychological processes. Therefore, they are the target for the action of many drugs, such as antipsychotics, antidepressants, antiemetics, migraine remedies, and many others. The 5-HT1A receptors have been involved in the pathogenesis and treatment of anxiety and depression and represent a promising target for new drugs with reduced extrapyramidal side effects. In most antidepressants, a piperazine-based structural motif can be identified as a common moiety. Here we describe the synthesis, pharmacological, and in silico characterization of a novel arylpiperazines series with excellent 5-HT1A affinity. The final compounds, 4a, 8a, and 8b, were selected according to predictions of in silico pharmacokinetics, docking analysis, and molecular dynamics in conjunction with physical properties, and metabolic stability. The accentuated molecules could serve as a lead compound for developing 5-HT1A drug-like molecules for depression treatment.

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KDapp for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl- on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl-), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208 s-1 for D-glucose to 95 s-1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steadystate transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,Kapp = 210 mM). Affinity for D-glucose increases 14-fold (KD,Naapp = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9 mM) compared to KD,Naapp due to optimized kinetics. In contrast, KM and KDapp values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding. [ABSTRACT FROM AUTHOR]

Angiotensin II analogue and β-arrestin biased agonist TRV027 (Sarcosine¹, D-Alanine8-Angiotensin (Ang) II; SD Ang II), developed by Trevena, Inc. in the early 2010s, brought hopes of a novel treatment for cardiovascular diseases, due to its ability to simultaneously cause signaling through the β-arrestin signaling pathway, while antagonizing the pathophysiological effects of Ang II mediated by the AT1 receptor G protein signaling cascades. However, a phase II clinical trial of this agent revealed no significant benefit compared to placebo treatment. Using 125I-Sarcosine¹, Isoleucine8-Ang II (125I-SI Ang II) radioligand receptor competition binding assays, we assessed the relative affinity of TRV027 compared to SI Ang II for liver AT1 receptors. We also compared radioiodinated TRV027 (125I-SD Ang II) binding affinity for liver AT1 receptors with 125I-SI Ang II. We found that despite its anticipated gain in metabolic stability, TRV027 and 125I-SD Ang II had reduced affinity for the AT1 receptor compared with SI Ang II and 125I-SI Ang II. Additionally, male-female comparisons showed that females have a higher AT1 receptor density, potentially attributed to tissue-dependent estrogen and progesterone effects. Peptide drugs have become more popular over the years due to their increased bioavailability, fast onset of action, high specificity, and low toxicity. Even though Trevena®'s biased agonist peptide TRV027 offered greater stability and potency compared to earlier AT1R biased agonists, it failed its phase II clinical trial in 2016. Further refinements to AT1R biased agonist peptides to improve affinity, as seen with SI Ang II, with better stability and bioavailability, has the potential to achieve the anticipated biased agonism. [ABSTRACT FROM AUTHOR]

Agonists of Benzodiazepine (BZD) receptor are exhaustively used in the control of muscle spasms, seizure, anxiety, and insomnia. BZDs have some unwanted effects; therefore, the development of new BZD receptor agonists with better efficacy and fewer unwanted effects is one of the subjects of interest. In this study, based on the pharmacophore/receptor model of the BZD binding site of GABAA receptors, a series of new 2-substituted-5-(4-chloro2-phenoxy)phenyl-1,3,4-oxadiazole derivatives (6a-f) were designed. Energy minima conformers of the designed compounds and diazepam were well matched in conformational analysis and showed proper interaction with the BZD-binding site of the GABAA receptor model (α1β2ϒ2) in docking studies. The designed compounds were synthesized in acceptable yield and evaluated for their in vitro affinity to the benzodiazepine receptor of rat brains by radioligand receptor binding assay. The results demonstrated that the affinities of most of the novel compounds were even higher than diazepam. The novel compound 6a with the best affinity in radioligand receptor binding assay (Ki=0.44 nM and IC50= 0.73±0.17 nM) had considerable hypnotic activity and weak anticonvulsant and anxiolytic effects with no negative effect on memory in animal models. Flumazenil as a selective benzodiazepine receptor antagonist was able to prevent hypnotic and anticonvulsant effects of 6a indicating the role of BZD receptors in these effects. [ABSTRACT FROM AUTHOR]

Leukocyte immunoglobulin-like receptor B1 (LILRB1) is widely expressed on various immune cells and the engagement of LILRB1 to HLA class I and pathogen-derived proteins can modulate the immune response. In the current study, 108 LILRB1 alleles were identified by screening the LILRB1 locus from the 1000 Genomes Phase 3 database. Forty-six alleles that occurred in three or more individuals encode 28 LILRB1 allotypes, and the inferred LILRB1 allotypes were then grouped into 9 LILRB1 D1-D2 variants for further analysis. We found that variants 1, 2, and 3 represent the three most frequent LILRB1 D1-D2 variants and the nine variants show frequency differences in populations. The binding assay demonstrated that variant 1 bound to HLA class I with the highest avidity, and all tested LILRB1 D1-D2 variants bound to HLA-C with lower avidity than to HLA-A and -B. Locus-specific polymorphisms at positions 183, 189, and 268 in HLA class I and dimorphisms in HLA-A (positions 207 and 253) and in HLA-B (position 194) affect their binding to LILRB1. Notably, the electrostatic interaction plays a critical role in the binding of LILRB1 to HLA class I as revealed by electrostatic analysis and by comparison of different binding avidities caused by polymorphisms at positions 72 and 103 of LILRB1. In this paper, we present a comprehensive study of the population genetics and binding abilities of LILRB1. The data will help us better understand the LILRB1-related diversity of the immune system and lay a foundation for functional studies. [ABSTRACT FROM AUTHOR]

Pheromone-binding proteins (PBPs) play important roles in binding and transporting sex pheromones. However, the PBP genes identified in coleopteran insects and their information sensing mechanism are largely unknown. Cyrtotrachelus buqueti (Coleoptera: Curculionidae) is a major insect pest of bamboo plantations. In this study, a novel PBP gene, CbuqPBP2, from C. buqueti was functionally characterized. CbuqPBP2 was more abundantly expressed in the antennae of both sexes than other body parts, and its expression level was significantly male-biased. Fluorescence competitive binding assays showed that CbuqPBP2 exhibited the strongest binding affinity to dibutyl phthalate (Ki = 6.32 μM), followed by styrene (Ki = 11.37 μM), among twelve C. buqueti volatiles. CbuqPBP2, on the other hand, showed high binding affinity to linalool (Ki = 10.55), the main volatile of host plant Neosinocalamus affinis. Furthermore, molecular docking also demonstrated the strong binding ability of CbuqPBP2 to dibutyl phthalate, styrene, and linalool, with binding energy values of −5.7, −6.6, and −6.0 kcal/mol, respectively, and hydrophobic interactions were the prevailing forces. The knockdown of CbuqPBP2 expression via RNA interference significantly reduced the electroantennography (EAG) responses of male adults to dibutyl phthalate and styrene. In conclusion, these results will be conducive to understanding the olfactory mechanisms of C. buqueti and promoting the development of novel strategies for controlling this insect pest.

Abstract Angiotensin II analogue and β‐arrestin biased agonist TRV027 (Sarcosine1, d‐Alanine8‐Angiotensin (Ang) II; SD Ang II), developed by Trevena, Inc. in the early 2010s, brought hopes of a novel treatment for cardiovascular diseases, due to its ability to simultaneously cause signaling through the β‐arrestin signaling pathway, while antagonizing the pathophysiological effects of Ang II mediated by the AT1 receptor G protein signaling cascades. However, a phase II clinical trial of this agent revealed no significant benefit compared to placebo treatment. Using 125I‐Sarcosine1, Isoleucine8‐Ang II (125I‐SI Ang II) radioligand receptor competition binding assays, we assessed the relative affinity of TRV027 compared to SI Ang II for liver AT1 receptors. We also compared radioiodinated TRV027 (125I‐SD Ang II) binding affinity for liver AT1 receptors with 125I‐SI Ang II. We found that despite its anticipated gain in metabolic stability, TRV027 and 125I‐SD Ang II had reduced affinity for the AT1 receptor compared with SI Ang II and 125I‐SI Ang II. Additionally, male–female comparisons showed that females have a higher AT1 receptor density, potentially attributed to tissue‐dependent estrogen and progesterone effects. Peptide drugs have become more popular over the years due to their increased bioavailability, fast onset of action, high specificity, and low toxicity. Even though Trevena®'s biased agonist peptide TRV027 offered greater stability and potency compared to earlier AT1R biased agonists, it failed its phase II clinical trial in 2016. Further refinements to AT1R biased agonist peptides to improve affinity, as seen with SI Ang II, with better stability and bioavailability, has the potential to achieve the anticipated biased agonism.

Beside the ongoing efforts to determine structural information, detailed functional studies on transporters are essential to entirely understand the underlying transport mechanisms. We recently found that solid supported membrane-based electrophysiology (SSME) enables the measurement of both sugar binding and transport in the Na+/sugar cotransporter SGLT1 (Bazzone et al, 2022a). Here, we continued with a detailed kinetic characterization of SGLT1 using SSME, determining KM and KDapp for different sugars, kobs values for sugar-induced conformational transitions and the effects of Na+, Li+, H+ and Cl− on sugar binding and transport. We found that the sugar-induced pre-steady-state (PSS) charge translocation varies with the bound ion (Na+, Li+, H+ or Cl−), but not with the sugar species, indicating that the conformational state upon sugar binding depends on the ion. Rate constants for the sugar-induced conformational transitions upon binding to the Na+-bound carrier range from 208 s−1 for D-glucose to 95 s−1 for 3-OMG. In the absence of Na+, rate constants are decreased, but all sugars bind to the empty carrier. From the steady-state transport current, we found a sequence for sugar specificity (Vmax/KM): D-glucose > MDG > D-galactose > 3-OMG > D-xylose. While KM differs 160-fold across tested substrates and plays a major role in substrate specificity, Vmax only varies by a factor of 1.9. Interestingly, D-glucose has the lowest Vmax across all tested substrates, indicating a rate limiting step in the sugar translocation pathway following the fast sugar-induced electrogenic conformational transition. SGLT1 specificity for D-glucose is achieved by optimizing two ratios: the sugar affinity of the empty carrier for D-glucose is similarly low as for all tested sugars (KD,Kapp = 210 mM). Affinity for D-glucose increases 14-fold (KD,Naapp = 15 mM) in the presence of sodium as a result of cooperativity. Apparent affinity for D-glucose during transport increases 8-fold (KM = 1.9 mM) compared to KD,Naapp due to optimized kinetics. In contrast, KM and KDapp values for 3-OMG and D-xylose are of similar magnitude. Based on our findings we propose an 11-state kinetic model, introducing a random binding order and intermediate states corresponding to the electrogenic transitions detected via SSME upon substrate binding.

Dopamine-deficient (DD) mice exhibit psychomotor hyperactivity that might be related to a decrease in muscarinic signaling. In the present study, muscarinic acetylcholine receptor M2 (CHRM2) density decreased in the cortex in DD mice. This is significant because cortical CHRM2 acts as an autoreceptor; therefore, changes in CHRM2 levels could alter acetylcholine in DD mice. We also found that the CHRM1/CHRM4 agonist xanomeline and CHRM2 agonist arecaidine propargyl ester tosylate inhibited hyperactivity in DD mice, suggesting that postsynaptic CHRM1 and CHRM2 and presynaptic CHRM2 may be involved in hyperactivity in DD mice. [ABSTRACT FROM AUTHOR]

The olfactory system is an important component of insect behavior and is vital for survival and reproduction. However, the genomic characterization and molecular basis of the olfactory response of Megalurothrips usitatus remain relatively unknown. RNA sequencing-built developmental transcriptomes of nymphs, pupae, and adult M. usitatus were examined in order to establish the sequence-based background of M. usitatus olfactory responses. A total of 56,669 unigenes were annotated using GO, NR, Pfam, eggNOG, SwissProt, and KEGG. The number of differentially expressed genes between pupae and nymphs, males and nymphs, and females and nymphs were 10,498, 9,235, and 10,964, respectively. One odorant-binding protein (MusiOBP1) and one chemosensory protein (MusiCSP1) were selected from the transcriptome, and their full-length sequences were obtained using RACE PCR. The relative expression of MusiOBP1 was the highest in primordial females, whereas the relative expression of MusiCSP1 was the highest in primordial pupae. The strongest binding ability to the odor-binding protein MusiOBP1 was observed for β-citronellol. 3-Hydroxy-2-methyl-4-pyrone showed the strongest binding affinity to MusiCSP1. Our analysis suggests that MusiOBP1 and MusiCSP1 may play significant roles in mediating M. usitatus host recognition. This research will improve our knowledge of odorant-binding proteins and chemosensory proteins, which will in turn improve our understanding of insect olfactory systems. [ABSTRACT FROM AUTHOR]

Avidin-biotin binding, the most robust non-covalent protein-ligand interaction occurring in nature, has wide-ranging applications in biotechnology. A frequent challenge in these applications is accurately determining the number of unoccupied biotin binding sites in avidin-containing fusion proteins. We delineate a novel assay protocol in miniaturized format to quantify available biotin binding sites based on the affinity of the anionic dye 4'-hydroxyazobenzene-2-carboxylic acid for biotin binding sites within avidin. We apply this assay as a quality control assay to evaluate the number of available biotin binding sites in different fusion protein production batches. This method offers a streamlined alternative to fluorescence-based assays commonly employed to assess biotin binding, is less time-consuming than other methods and is applicable to diverse fusion proteins.

The green peach aphid, Myzus persicae , is a worldwide agricultural pest. Chlorpyrifos has been widely used to control M. persicae for decades, thus leading to a high resistance to chlorpyrifos. Recent studies have found that insect odorant binding proteins (OBPs) play essential roles in insecticide resistance. However, the potential resistance mechanism underlying the cross-link between aphid OBPs and chlorpyrifos remains unclear. In this study, two OBPs (MperOBP3 and MperOBP7) were found overexpressed in M. persicae chlorpyrifos-resistant strains (CRR) compared to chlorpyrifos-sensitive strains (CSS); furthermore, chlorpyrifos can significantly induce the expression of both OBPs. An in vitro binding assay indicated that both OBPs strongly bind with chlorpyrifos; an in vivo RNAi and toxicity bioassay confirmed silencing either of the two OBPs can increase the susceptibility of aphids to chlorpyrifos, suggesting that overexpression of MperOBP3 and MperOBP7 contributes to the development of resistance of M. persicae to chlorpyrifos. Our findings provide novel insights into insect OBPs-mediated resistance mechanisms.

Human exposure to dicarbonyls occurs via ingestion (e.g., food), inhalation (e.g., electronic cigarettes) and dysregulation of endogenous metabolic pathways (e.g., glycolysis). Dicarbonyls are electrophiles able to induce carbonylation of endogenous substrate. They have been associated with the onset and progression of several human diseases. Several studies have advocated the use of dicarbonyl binders as food preservatives or as drugs aimed at mitigating carbonylation. This study presents the setup of an easy and cheap assay for the screening of selective and potent dicarbonyl binders. The method is based on the incubation of the candidate molecules with a molecular probe. The activity is then determined by measuring the residual concentration of the molecular probe over time by liquid chromatography (LC). However, the naturally occurring dicarbonyls (e.g., glyoxal, methylglyoxal) are not appealing as probes since they are hard to separate and detect using the most popular LC variants. Benzylglyoxal (BGO) was therefore synthesized and tested, proving to be a convenient probe that allows a direct quantification of residual dicarbonyls by reversed phase LC without derivatization. The method was qualified by assessing the binding ability of some molecules known as binders of natural occurring dicarbonyls, obtaining results consistent with literature.

Adrenergic receptors are among the most studied G protein-coupled receptors. Activation or blockade of these receptors is a major therapeutic approach for the treatment of numerous disorders such as cardiac hypertrophy, congestive heart failure, hypertension, angina pectoris, cardiac arrhythmias, depression, benign prostate hyperplasia, anaphylaxis, asthma, and hyperthyroidism. Among all nine cloned adrenoceptor subtypes and the subsequent development of animal models, a significant target for various neurological conditions treatment is alpha1-adrenergic receptors. 2-{[4-(2-Methoxyphenyl)piperazin-1-yl]alkyl}-1H-benzo[d]imidazoles, their 5 substituted derivatives, and structurally similar, arylpiperazine based alpha1-adrenergic receptors antagonists (trazodone, naftopidil, and urapidil) have been subjects of comparative analysis. Most of the novel compounds showed alpha1-adrenergic affinity in the range from 22 nM to 250 nM. The in silico docking and molecular dynamics simulations, binding data together with absorption, distribution, metabolism, and excretion (ADME) calculations identified the promising lead compounds. The results brought out the conclusions which allowed us to propose a rationale for the activity of these molecules and to highlight six compounds (2–5, 8, and 12) that exhibited an acceptable pharmacokinetic profile to the advanced investigation as the potential alpha1-adrenergic receptor antagonists. [ABSTRACT FROM AUTHOR]

Antibody‐based therapeutic agents and other biopharmaceuticals are now used in the treatment of many diseases. However, when these biopharmaceuticals are administrated to patients, an immune reaction may occur that can reduce the drug's efficacy and lead to adverse side‐effects. The immunogenicity of biopharmaceuticals can be evaluated by detecting and measuring antibodies that have been produced against these drugs, or antidrug antibodies. Methods for antidrug antibody detection and analysis can be important during the selection of a therapeutic approach based on such drugs and is crucial when developing and testing new biopharmaceuticals. This review examines approaches that have been used for antidrug antibody detection, measurement, and characterization. Many of these approaches are based on immunoassays and antigen binding tests, including homogeneous mobility shift assays. Other techniques that have been used for the analysis of antidrug antibodies are capillary electrophoresis, reporter gene assays, surface plasmon resonance spectroscopy, and liquid chromatography‐mass spectrometry. The general principles of each approach will be discussed, along with their recent applications with regards to antidrug antibody analysis. [ABSTRACT FROM AUTHOR]

The anesthetic effect of Alpinia galanga oil (AGO) has been reported. However, knowledge of its pathway in mammals is limited. In the present study, the binding of AGO and its key compounds, methyl eugenol, 1,8-cineole, and 4-allylphenyl acetate, to gamma-aminobutyric acid type A (GABAA) receptors in rat cortical membranes, was investigated using a [3H]muscimol binding assay and an in silico modeling platform. The results showed that only AGO and methyl eugenol displayed a positive modulation at the highest concentrations, whereas 1,8-cineole and 4-allylphenyl acetate were inactive. The result of AGO correlated well to the amount of methyl eugenol in AGO. Computational docking and dynamics simulations into the GABAA receptor complex model (PDB: 6X3T) showed the stable structure of the GABAA receptor–methyl eugenol complex with the lowest binding energy of −22.16 kcal/mol. This result shows that the anesthetic activity of AGO and methyl eugenol in mammals is associated with GABAA receptor modulation. An oil-in-water nanoemulsion containing 20% w/w AGO (NE-AGO) was formulated. NE-AGO showed a significant increase in specific [3H]muscimol binding, to 179% of the control, with an EC50 of 391 µg/mL. Intracellular studies show that normal human cells are highly tolerant to AGO and the nanoemulsion, indicating that NE-AGO may be useful for human anesthesia. [ABSTRACT FROM AUTHOR]

The 3C-like main protease of SARS-CoV-2 (3CLPro) is responsible for the cleavage of the viral polyprotein. This process is essential for the viral life cycle. Therefore, 3CLPro is a promising target to develop antiviral drugs for COVID-19 prevention and treatment. Traditional enzymatic assays for the identification of 3CLPro inhibitors rely on peptide-based colorimetric or fluorogenic substrates. However, the COVID-19 pandemic has limit or delay access to these substrates, especially for researchers in developing countries attempting to screen natural product libraries. We explored the use of the fluorescent probe 8-anilinonaphthalene-1-sulfonate (ANS) as an alternative assay for inhibitor identification. Fluorescence enhancement upon binding of ANS to 3CLPro was observed, and this interaction was competitive with a peptide substrate. The utility of ANS-based competitive binding assay to identify 3CLPro inhibitors was demonstrated with the flavonoid natural products baicalein and rutin. The molecular nature of ANS and rutin interaction with 3CLPro was explored with molecular modeling. Our results suggested that ANS could be employed in a competitive binding assay to facilitate the identification of novel SARS-CoV-2 antiviral compounds.

Bacillus thuringiensis Cry1I insecticidal proteins are structurally similar to other three-domain Cry proteins, although their size, activity spectrum, and expression at the stationary phase are unique among other members of the Cry1 family. The mode of action of Cry1 proteins is not completely understood but the existence of an activation step prior to specific binding is widely accepted. In this study, we attempted to characterize and determine the importance of the activation process in the mode of action of Cry1I, as Cry1Ia protoxin or its partially processed form showed significantly higher toxicity to Ostrinia nubilalis than the fully processed protein either activated with trypsin or with O. nubilalis midgut juice. Oligomerization studies showed that Cry1Ia protoxin, in solution, formed dimers spontaneously, and the incubation of Cry1Ia protoxin with O. nubilalis brush border membrane vesicles (BBMV) promoted the formation of dimers of the partially processed form. While no oligomerization of fully activated proteins after incubation with BBMV was detected. The results of the in vitro competition assays showed that both the Cry1Ia protoxin and the approx. 50 kDa activated proteins bind specifically to the O. nubilalis BBMV and compete for the same binding sites. Accordingly, the in vivo binding competition assays show a decrease in toxicity following the addition of an excess of 50 kDa activated protein. Consequently, as full activation of Cry1I protein diminishes its toxicity against lepidopterans, preventing or decelerating proteolysis might increase the efficacy of this protein in Bt-based products. Key points: • Processing Cry1I to a 50 kDa stable core impairs its full toxicity to O. nubilalis • Partially processed Cry1Ia protoxin retains the toxicity of protoxin vs O. nubilalis • Protoxin and its final processed forms compete for the same functional binding sites [ABSTRACT FROM AUTHOR]

Olfaction is of great significance for insect mate-seeking and host-locating behaviors. Insect odorant-binding proteins (OBPs), especially those antenna-enriched OBPs, are thought to discriminate, capture and transport odorant molecules to olfactory receptors, but this has not been fully clarified in Bactrocera minax (Enderlein), an economically important pest of citrus crops. Our previous studies showed that seven OBP genes (BminOBP1 - 7) were identified from B. minax adults via a head transcriptome analysis, of which only BminOBP3 and 6 were highly expressed in antennae, suggesting an olfactory role. To confirm their functions, here, BminOBP3 and 6 were cloned, expressed in Escherichia coli cells. Binding properties of the recombinant BminOBPs with 13 volatiles, most of which can elicit a significant behavioral response from B. minax adults, were determined by fluorescent competitive binding assays. The results showed that Both BminOBP3 and 6 exhibited a remarkable selectivity towards the 13 ligands tested. BminOBP3 displayed strong binding affinity only with undecanol. BminOBP6 demonstrated strong binding affinity with undecanol and limonene among 13 ligands tested. Undecanol is believed to be main sex pheromone component of B. minax. Limonene is an important volatile compound enriched in citrus fruits. Taken together, we concluded that BminOBP3 and 6 may play a prominent role in the process of B. minax mate-seeking and host-locating behaviors through recognizing and transporting these volatiles. It is conceivable that this study will increase our molecular understanding of B. minax olfaction, facilitating the development of OBP-based behavioral interference that is potentially useful for the integrated management of B. minax. [ABSTRACT FROM AUTHOR]

Fatty acid-binding proteins (FABPs) are a family of lipid chaperones, which contribute to systemic metabolic regulation through diverse lipid signalings. In this study, a midgut-specific FABP gene (Slfabp2) was cloned from Spodoptera litura. RT-PCR and Western blot analysis indicated that RNA and protein levels of SlFABP2 gradually increased and reached a peak at the prepupal stage and maintained a high level during the pupal stage. The expression of SlFABP2 protein was induced by starvation treatment. In vitro binding assay revealed that the recombinant SlFABP2 had high affinities of binding long-chain fatty acids, such as palmitic acid, arachidonate and oleic acid. The results suggest that SlFABP2 may have a unique function that transports intracellular fatty acids and can regulate the metabolism of lipids in metamorphosis. This work provides experimental clues for understanding the potential function of SlFABP2 in fatty acid metabolism in S. litura.

Context: Bioassays provide information on the functionality of thyrotropin receptor antibodies (TSH-R-Ab) and thus may offer more clinical utility than binding assays., Objective: In this prospective, blinded, US-based study, the clinical performance of several TSH-R-Ab assays was compared., Setting: US endocrinology clinic., Subjects: One hundred sixty-two unselected, consecutive, well-documented patients with various thyroid diseases and healthy controls., Intervention(s): Blinded TSH-R-Ab measurements., Main Outcome Measure(s): Sensitivity and specificity of 4 TSH-R-Ab assays., Results: The 4 TSH-R-Ab assays were negative in all 42 patients without autoimmune thyroid disease (AITD). In 104 patients with Graves' disease (GD), irrespective of the disease duration, TSH-R-Ab positivity was present in 65 (63%), 67 (65%), and 87 (84%) for the Cobas and Immulite binding assays and stimulatory TSH-R-Ab [thyroid-stimulating immunoglobin (TSI)] bioassay, respectively (TSI vs Immulite P < .0025, TSI vs Cobas P < .0009). Fifteen newly diagnosed GD patients were all positive in the TSI bioassay, but only 11 (73%) were positive in the Cobas and Immulite binding assays. Nine GD patients with biochemical subclinical hyperthyroidism were TSI-positive but Immulite- and Cobas-negative. Two GD patients were blocking TSH-R-Ab [thyroid-blocking immunoglobin (TBI)]-positive and TSI-negative, and the Immulite and Cobas were positive in both. Additional serum samples from AITD patients that consisted of 30 TBI-positive and 10 TSI-positive samples were blindly tested in the binding assays. Only 6 of the 10 TSI-positive samples were positive in both binding assays, and 30 and 28 of the TBI-positive samples were positive in the Cobas and Immulite assays, respectively., Conclusion: Binding TSH-R-Ab assays are less sensitive than TSI bioassays and are not specific for stimulating antibodies. Measuring the function of TSH-R-Ab in a bioassay can provide useful information to clinicians., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com. See the journal About page for additional terms.)

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The loss of cholinergic neurons and synapses relates to the severity of dementia in several neurodegenerative pathologies; and the vesicular acetylcholine transporter (VAChT) provides a reliable biomarker of cholinergic function. We recently characterized and (11)C-labeled a new VAChT inhibitor, (-)-TZ659. Here we report the in vitro and ex vivo characterization of (-)-TZ659. A stably transfected PC12(A123.7) cell line which expresses human VAChT (hVAChT) was used for the in vitro binding characterization of (-)-[(3)H]TZ659. A saturated binding curve was obtained with Kd=1.97±0.30nM and Bmax=3240±145.9fmol/mg protein. In comparison, a PC12(A123.7) cell line that expresses mutant hVAChT showed decreased binding affinity (Kd=15.94±0.28nM). Competitive binding assays using a panel of other CNS ligands showed no inhibition of (-)-[(3)H]TZ659 binding. On the other hand, binding inhibitions were observed only using VAChT inhibitors (Ki=0.20-31.35nM). An in vitro assay using rat brain homogenates showed that (-)-[(3)H]TZ659 had higher binding in striatum than in cerebellum, with a target: non-target ratio>3.46. Even higher ex vivo striatum-to-cerebellum ratios (9.56±1.11) were observed using filtered homogenates of brain tissue after rats were injected intravenously with (-)-[(11)C]TZ659. Ex vivo autoradiography of (-)-[(11)C]TZ659 confirmed high striatal uptake, with a consistently high striatum-to-cerebellum ratio (2.99±0.44). In conclusion, (-)-TZ659 demonstrated high potency and good specificity for VAChT in vitro and in vivo. These data suggest that (-)-[(11)C]TZ659 may be a promising PET tracer to image VAChT in the brain.

The present study used a binding assay to identify novel target biomolecules of l-menthol ([−]-menthol) that promote mouse ambulation. Among 88 different ligands to specific biomolecules examined, 0.1 mM l-menthol inhibited the binding of 13 ligands with relatively high inhibition rates. The assays showed that l-menthol acts on calcium channels, sodium channels, γ-aminobutyric acid type A (GABAA) receptor, GABA transporter, dopamine transporter, dopamine D4 receptor, adenosine A2a receptor, α2A-adrenergic receptor, histamine H2 receptor, bombesin receptor, angiotensin AT1 receptor, vasopressin V2 receptor, and leukotriene B4 receptor over a similar concentration range. The inhibition constant (Ki) for l-menthol inhibition of binding of [3H]-WIN35,428 to the human recombinant dopamine transporter was 6.15 × 10−4 mol/L. The Ki for l-menthol inhibition of binding of [3H]-ethynylbicycloorthobenzoate (EBOB), a ligand of GABAA receptor picrotoxin site, was 2.88 × 10−4 mol/L. These results should aid future research by providing clues for investigating the mechanisms underlying l-menthol activities, including the ambulation-promoting effect. The present results suggest that the dopamine transporter, adenosine A2a receptor, dopamine D4 receptor, α2A-adrenergic receptor, and GABAA receptor are promising candidate molecules that are involved in the mechanisms underlying the psychostimulant-like effect of l-menthol.

We adapted a radioligand receptor binding assay for measuring insulin levels in unknown samples. The assay enables rapid and accurate determination of insulin concentrations in experimental samples, such as from insulin-secreting cells. The principle of the method is based on the binding competition of insulin in a measured sample with a radiolabeled insulin for insulin receptor (IR) in IM-9 cells. Both key components, radiolabeled insulin and IM-9 cells, are commercially available. The IR binding assay was used to determine unknown amounts of insulin secreted by MIN6 β cell line after stimulation with glucose, arginine, ornithine, dopamine, and serotonin. The experimental data obtained by the IR binding assay were compared to the results determined by RIA kits and both methods showed a very good agreement of results. We observed the stimulation of glucose-induced insulin secretion from MIN6 cells by arginine, weaker stimulation by ornithine, but inhibitory effects of dopamine. Serotonin effects were either stimulatory or inhibitory, depending on the concentration of serotonin used. The results will require further investigation. The study also clearly revealed advantages of the IR binding assay that allows the measuring of a higher throughput of measured samples, with a broader range of concentrations than in the case of RIA kits. The IR binding assay can provide an alternative to standard RIA and ELISA assays for the determination of insulin levels in experimental samples and can be especially useful in scientific laboratories studying insulin production and secretion by β cells and searching for new modulators of insulin secretion. [ABSTRACT FROM AUTHOR]

To explore thermofluorimetric analysis (TFA) in detail, we compared two related aptamers. The first, LINN2, is a DNA aptamer previously selected against EGFR recombinant protein. In this work we selected a second aptamer, KM4, against EGFR-overexpressing A549 cells. The two aptamers were derived from the same pool and bind the same target but behave differently in TFA. Our results suggest four overall conclusions about TFA of aptamers: 1. Some aptamers show reduced fluorescence upon target binding suggesting that target-bound aptamer is not always fluorescent. 2. Many aptamers do not obey the intuitive assumptions that aptamer–target interactions stabilize a folded conformation. 3. TFA may be most appropriate for aptamers with significant double-stranded structure. 4. Kinetic effects may be significant and the order of operations in preparing samples should be carefully optimized.

Lysophosphatidic acid (LPA) activates cognate G protein-coupled receptors (GPCRs) to initiate biological signaling cascades. Lysophospholipid (LP) receptor binding properties remain incompletely assessed because of difficulties with ligand lipophilicity and lipid “stickiness.” These inherent attributes produce high levels of nonspecific binding within cell-membrane preparations used to assess GPCRs, as has been shown in classical binding assays using radiolabeled ligands, making accurate measurements of lipid binding kinetics difficult to achieve. Backscattering interferometry (BSI) is an optical technology that measures molecular binding interactions by reporting changes in the refractive index of a solution after binding events. Here, we report the use of BSI to assess LPA1 for its ability to bind to naturally occurring lipids and a synthetic LPA1 antagonist (ONO-9780307), under both primary- and competition-binding conditions. Assessment of 12 different lipids demonstrated that the known LP ligand, 1-oleoyl-LPA, as well as an endocannabinoid metabolite, anandamide phosphate, are specific ligands for LPA1, whereas other LPs tested were not. Newly determined dissociation constants (Kd values) for orthosteric lipid ligands approximated 10−9 M, substantially lower (i.e., with higher affinity) than measured Kd values in classical binding or cell-based assays. These results demonstrate that BSI may have particular utility in assessing binding interactions between lipid receptors and their lipid ligands and could provide new screening approaches for lipid receptor identification and drug discovery.

Surface protein gp-120 of HIV-1 virus plays an important role in the infection of HIV-1, but detection of gp-120 during the early stage of infection is very difficult. Herein, we report a binding bioassay based on an RNA aptamer B40t77, which binds specifically to gp-120. The bioassay is built upon a hydrophobic glass slide with surface immobilized gp-120. When the glass surface is incubated in a solution containing B40t77, the aptamer is able to bind to gp-120 specifically and remove it from the surface after a short incubation time of 30 min. The result of the binding event can be amplified by using liquid crystal (LC) into optical signals in the final step. By using this bioassay, we are able to detect as low as 1 μg/ml of gp-120 with high specificity within 30 min. No response is obtained when gp-120 is replaced by other protein such as bovine serum albumin (BSA). This is the first qualitative bioassay which provides a simple way for the detection of gp-120 with the naked eye. The assay is robust, low-cost and does not require additional labeling. Thus, the bioassay is potentially useful for the early detection of HIV-1 in resources-limited regions.

The anesthetic effect of Alpinia galanga oil (AGO) has been reported. However, knowledge of its pathway in mammals is limited. In the present study, the binding of AGO and its key compounds, methyl eugenol, 1,8-cineole, and 4-allylphenyl acetate, to gamma-aminobutyric acid type A (GABAA) receptors in rat cortical membranes, was investigated using a [3H]muscimol binding assay and an in silico modeling platform. The results showed that only AGO and methyl eugenol displayed a positive modulation at the highest concentrations, whereas 1,8-cineole and 4-allylphenyl acetate were inactive. The result of AGO correlated well to the amount of methyl eugenol in AGO. Computational docking and dynamics simulations into the GABAA receptor complex model (PDB: 6X3T) showed the stable structure of the GABAA receptor–methyl eugenol complex with the lowest binding energy of −22.16 kcal/mol. This result shows that the anesthetic activity of AGO and methyl eugenol in mammals is associated with GABAA receptor modulation. An oil-in-water nanoemulsion containing 20% w/w AGO (NE-AGO) was formulated. NE-AGO showed a significant increase in specific [3H]muscimol binding, to 179% of the control, with an EC50 of 391 µg/mL. Intracellular studies show that normal human cells are highly tolerant to AGO and the nanoemulsion, indicating that NE-AGO may be useful for human anesthesia.

To identify drugs that could potentially be used to treat infection with SARS-CoV-2, a high throughput 384-well assay was developed to measure the binding of the receptor binding domain (RBD) of the viral S1 protein to its main receptor, angiotensin converting enzyme 2 (ACE2). The RBD was fused to both a HiBIT tag and an IL6 secretion signal to enable facile collection from the cell culture media. The addition of culture media containing this protein, termed HiBIT-RBD, to cells expressing ACE2 led to binding that was specific to ACE2 and both time and concentration dependant, Binding could be inhibited by both RBD expressed in E. coli and by a full length S1 - Fc fusion protein (Fc-fused S1) expressed in eukaryotic cells. The mutation of residues that are known to play a role in the interaction of RBD with ACE2 also reduced binding. This assay may be used to identify drugs which inhibit the viral uptake into cells mediated by binding to ACE2. Image 1 1.A high-throughput, 384-well plate assay was developed to measure the binding S1 RBD to ACE2; 2.HiBIT-RBD binds to cells expressing ACE2 specifically and in a time dependant fashion; 3.The binding of HiBIT-RBD to ACE2 can be inhibited using recombinantly expressed SARS-CoV-2 RBD and full-length, Spike S1; 4.Site specific mutations within the RBD demonstrate the specificity of this assay. [ABSTRACT FROM AUTHOR]

The key factor in successful development and marketing of biosimilar antibodies is a deep understanding of their critical quality attributes and the ability to control them. Comprehensive functional characterization is therefore at the heart of the process and is a crucial part of regulatory requirements. Establishment of a scientifically sound molecule-specific functional in vitro assay panel requires diligent planning and high flexibility in order to respond to both regulatory requirements and the ever-changing demands relevant to the different stages of the development and production process. Relevance of the chosen assays to the in vivo mechanism of action is of key importance to the stepwise evidence-based demonstration of biosimilarity. Use of a sound interdisciplinary approach and orthogonal state-of-the-art techniques is also unavoidable for gaining in-depth understanding of the biosimilar candidate. The aim of the present review is to give a snapshot on the methodic landscape as depicted by the available literature discussing the in vitro techniques used for the functional characterization of approved biosimilar therapeutic antibodies. Emerging hot topics of the field and relevant structure-function relationships are also highlighted. [ABSTRACT FROM AUTHOR]

Odorant binding proteins (OBPs) are critical components in insect olfactory systems where they bind, solubilize and transport odorant molecules to receptors. Here, we cloned three OBPs (PxylGOBP1, PxylGOBP2 and PxylOBP24) from the diamondback moth, Plutella xylostella, one of the most destructive pests of cruciferous crops. These three OBPs were expressed in Escherichia coli as recombinant proteins, purified and characterized by fluorescence binding assays with 39 ligands including sex pheromone and plant‐derived chemical compounds. PxylGOBP1 and PxylGOBP2 showed significantly different binding affinities to theses ligands, suggesting distinct binding preferences of these two general odorant binding proteins. PxylOBP24 showed no or extremely low binding activities to selected ligands, suggesting it may be involved in non‐olfactory functions. Circular dichroism spectral results demonstrated that PxylGOBP1 and PxylGOBP2 shared similar secondary structures while PxylOBP24 was significantly different. This study improves our knowledge of insect OBPs, which will assist in a better understanding of insect olfactory system and developing more environmentally friendly pest control strategies for P. xylostella. [ABSTRACT FROM AUTHOR]

Fluorescence (or Förster) resonance energy transfer (FRET) is a straightforward and sensitive technique to evaluate molecular interactions. However, most of the popular FRET pairs suffer cross-excitation of the acceptor, which could lead to false positives. To overcome this problem, we selected a large Stokes shift (LSS) fluorophore as a FRET donor. As a successful example, we employed a new FRET pair mAmetrine (an LSS yellow fluorescence protein)/DY-547 (a cyanine derivative) to substitute CFP/fluorescein that we previously employed to study molecular interactions between cyclic nucleotide-binding domains and cyclic nucleotides. The new FRET pair is practically free of cross-excitation of the acceptor. Namely, a change in the fluorescence spectral shape implies evidence of FRET without other control experiments.

Morphine‐6β‐D‐glucuronide (M6βG), an active metabolite of morphine, and its isomer morphine‐6α‐D‐glucuronide (M6αG) were synthesized from 3‐O‐protected morphinethrough glycosylation and alkaline hydrolysis. All structures were determined by spectroscopic analysis, and especially it is the first time to report the single crystal of compound M6αG. In vitro binding assay showed that M6βG bound to mu opioid receptor (MOR), kappa opioid receptor (KOR), and delta opioid receptor (DOR) with nanomolar affinity (Ki = 28.03, 116.88, and 375.13 nM) and M6αG bound to them with similar affinity (Ki = 1070.13, 20 637.93, and 677.36 nM). The selectivity of M6αG toward KOR is much higher. Hot‐plate test showed that the analgesic effect of M6βG is better than that of M6αG, that is maybe because the mechanism of M6αG is different. [ABSTRACT FROM AUTHOR]

Sitodiplosis mosellana, a periodic but devastating wheat pest, relies on wheat spike volatiles as a cue in selecting hosts for oviposition. Insect odorant‐binding proteins (OBPs) are thought to play essential roles in filtering, binding and transporting hydrophobic odorant molecules to specific receptors. To date, the molecular mechanisms underlying S. mosellana olfaction are poorly understood. Here, three S. mosellana antenna‐specific OBP genes, SmosOBP11, 16 and 21, were cloned and bacterially expressed. Binding properties of the recombinant proteins to 28 volatiles emitted from wheat spikes were investigated using fluorescence competitive binding assays. Sequence analysis suggested that these SmosOBPs belong to the Classic OBP subfamily. Ligand‐binding analysis showed that all three SmosOBPs preferentially bound alcohol, ester and ketone compounds, and SmosOBP11 and 16 also selectively bound terpenoid compounds. In particular, the three SmosOBPs had high binding affinities (Ki < 20 μmol/L) to 3‐hexanol and cis‐3‐hexenylacetate that elicited strong electroantennogram (EAG) response from female antennae. In addition, SmosOBP11 displayed significantly higher binding (Ki < 8 μmol/L) than SmosOBP16 and 21 to 1‐octen‐3‐ol, D‐panthenol, α‐pinene and heptyl acetate which elicited significant EAG response, suggesting that SmosOBP11 plays a major role in recognition and transportation of these volatiles. These findings have provided important insight into the molecular mechanism by which S. mosellana specifically recognizes plant volatiles for host selection, and have facilitated identification of effective volatile attractants that are potentially useful for pest monitoring and trapping. [ABSTRACT FROM AUTHOR]