Development of a high throughput cytochrome P450-ligand binding assay.

Human cytochrome P450 enzymes are membrane-embedded monooxygenases responsible for xenobiotic metabolism, steroidogenesis, fatty acid metabolism, and vitamin metabolism. Their active sites can accommodate diverse small molecules and understanding these interactions is key to decoding enzymatic functionality and designing drugs. The most common method for characterizing small molecule binding is quantifying absorbance changes that typically occur when substrates enter the active site near the heme iron. Traditionally such titrations are monitored by a spectrophotometer, requiring significant manual time, protein, and increasing solvents. This assay was adapted for semi-automated high throughput screening, increasing throughput 50-fold while requiring less protein and keeping solvent concentrations constant. This 384-well assay was validated for both type I and II shifts typically observed for substrates and heme-coordinating inhibitors, respectively. This assay was used to screen a library of ∼100 diverse imidazole-containing compounds which can coordinate with the heme iron if compatible with the overall active site. Three human cytochrome P450 enzymes were screened: drug-metabolizing CYP2A6 and CYP2D6 and sterol-metabolizing CYP8B1. Each bound different sets of imidazole compounds with varying Kd values, providing a unique binding fingerprint. As a final validation, the Kd values were used to generate pharmacophores to compare to experimental structures. Applications for the high-throughput assay include 1) facilitating generation of pharmacophores for enzymes where structures are not available, 2) screening to identify ligands for P450 orphans, 3) screening for inhibitors of P450s drug targets, 4) screening potential new drugs to avoid and/or control P450 metabolism, and 5) efficient validation of computational predictions.

Different binding properties of odorant-binding protein 8 to insecticides in Orius sauteri.

Chemical sensing systems are vital in the growth and development of insects. Orius sauteri (Poppius) (Hemiptera: Anthocoridae) is an important natural enemy of many pests. The molecular mechanism of odorant binding proteins (OBPs) binding with common insecticides is still unknow in O. sauteri. In this study, we expressed in vitro OsauOBP8 and conducted fluorescence competition binding assay to investigate the function of OsauOBP8 to insecticides. The results showed that OsauOBP8 could bind with four common insecticides (phoxim, fenitrothion, chlorpyrifos, deltamethrin). Subsequently, we used molecular docking to predict and obtained candidate six amino acid residues (K4, K6, K13, R31, K49, K55) and then mutated. The result showed that three key residues (K4, K6, R31) play important role in OsauOBP8 bound to insecticides. Our study identified the key binding sites of OsauOBP8 to insecticides and help to better understand the molecular mechanism of OBPs to insecticides in O. sauteri.

Development and Validation of an ADA-Tolerant Assay for Quantification of an Exatecan-Based ADC in Monkey Plasma.

BACKGROUND: The development of an anti-drug antibody (ADA)-tolerant pharmacokinetic (PK) assay is important when the drug exposure is irrelevant to toxicity in the presence of ADA. We aimed to develop and validate an ADA-tolerant assay for an exatecan-based antibody-drug conjugate (ADC) in monkey plasma. RESULTS: The assay tolerated 5.00 µg/mL of ADA at 12 µg/mL of ADC. Its accuracy and precision results satisfied the acceptance criteria. Furthermore, the assay was free from hook and matrix effects and exhibited good dilutional linearity. Additionally, the ADC in plasma samples was stable under different storage conditions. METHOD: An ADA-tolerant ADC assay was configured with an anti-payload antibody for capture, and a drug-target protein combined with a horseradish peroxidase (HRP)-labeled antibody against a drug-target-protein tag for detection. Samples were firstly acidified to dissociate drug and ADA complexes, and to convert the carboxylate form to the lactone form of exatecan molecules; then, the ADAs in the samples were removed with a naked antibody-coated microplate. The treated samples were further incubated with coated anti-payload antibody and captured ADC molecules were quantified by the detection reagent. The developed assay was optimized and validated against regulatory guidelines. CONCLUSIONS: The assay met both methodological and sample-related ADA tolerance requirements, and was applicable to a nonclinical study in cynomolgus monkeys.

Assessment of serum total 25-hydroxyvitamin D assays for Vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions.

The Vitamin D External Quality Assessment Scheme (DEQAS) distributes human serum samples four times per year to over 1000 participants worldwide for the determination of total serum 25-hydroxyvitamin D [25(OH)D)]. These samples are stored at -40 °C prior to distribution and the participants are instructed to store the samples frozen at -20 °C or lower after receipt; however, the samples are shipped to participants at ambient conditions (i.e., no temperature control). To address the question of whether shipment at ambient conditions is sufficient for reliable performance of various 25(OH)D assays, the equivalence of DEQAS human serum samples shipped under frozen and ambient conditions was assessed. As part of a Vitamin D Standardization Program (VDSP) commutability study, two sets of the same nine DEQAS samples were shipped to participants at ambient temperature and frozen on dry ice. Twenty-eight laboratories participated in this study and provided 34 sets of results for the measurement of 25(OH)D using 20 ligand binding assays and 14 liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods. Equivalence of the assay response for the frozen versus ambient DEQAS samples for each assay was evaluated using multi-level modeling, paired t-tests including a false discovery rate (FDR) approach, and ordinary least squares linear regression analysis of frozen versus ambient results. Using the paired t-test and confirmed by FDR testing, differences in the results for the ambient and frozen samples were found to be statistically significant at p < 0.05 for four assays (DiaSorin, DIAsource, Siemens, and SNIBE prototype). For all 14 LC-MS/MS assays, the differences in the results for the ambient- and frozen-shipped samples were not found to be significant at p < 0.05 indicating that these analytes were stable during shipment at ambient conditions. Even though assay results have been shown to vary considerably among different 25(OH)D assays in other studies, the results of this study also indicate that sample handling/transport conditions may influence 25(OH)D assay response for several assays.

Interlaboratory comparison of 25-hydroxyvitamin D assays: Vitamin D Standardization Program (VDSP) Intercomparison Study 2 - Part 2 ligand binding assays - impact of 25-hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance.

An interlaboratory comparison study was conducted by the Vitamin D Standardization Program (VDSP) to assess the performance of ligand binding assays (Part 2) for the determination of serum total 25-hydroxyvitamin D [25(OH)D]. Fifty single-donor samples were assigned target values for concentrations of 25-hydroxyvitamin D2 [25(OH)D2], 25-hydroxyvitamin D3 [25(OH)D3], 3-epi-25-hydroxyvitamin D3 [3-epi-25(OH)D3], and 24R,25-dihydroxyvitamin D3 [24R,25(OH)2D3] using isotope dilution liquid chromatography-tandem mass spectrometry (ID LC-MS/MS). VDSP Intercomparison Study 2 Part 2 includes results from 17 laboratories using 32 ligand binding assays. Assay performance was evaluated using mean % bias compared to the assigned target values and using linear regression analysis of the test assay mean results and the target values. Only 50% of the ligand binding assays achieved the VDSP criterion of mean % bias ≤ |± 5%|. For the 13 unique ligand binding assays evaluated in this study, only 4 assays were consistently within ± 5% mean bias and 4 assays were consistently outside ± 5% mean bias regardless of the laboratory performing the assay. Based on multivariable regression analysis using the concentrations of individual vitamin D metabolites in the 50 single-donor samples, most assays underestimate 25(OH)D2 and several assays (Abbott, bioMérieux, DiaSorin, IDS-EIA, and IDS-iSYS) may have cross-reactivity from 24R,25(OH)2D3. The results of this interlaboratory study represent the most comprehensive comparison of 25(OH)D ligand binding assays published to date and is the only study to assess the impact of 24R,25(OH)2D3 content using results from a reference measurement procedure.

Functional differentiation of three pheromone binding proteins in Orthaga achatina using mixed-type sex pheromones.

Pheromone-binding proteins (PBPs) play important roles in perception of insect sex pheromones, functioning to recognize and transport pheromone components onto the olfactory receptors of the odorant sensing neurons. Orthaga achatina, a serious pest of camphor trees, uses a mixture of three Type I (Z11-16:OAc, Z11-16:OH and Z11-16:Ald) and one Type II (Z3,Z6,Z9,Z12,Z15-23:H) sex pheromone components in its sex communication, in which Z11-16:OAc is the major component and others are minor components. In this study, we for the first time demonstrated that the three PBPs differentiated in recognition among pheromone components in a moth using mixed-type sex pheromones. First, tissue expression study showed that all three PBPs of O. achatina were expressed only in antennae and highly male-biased, suggesting their involvement in perception of the sex pheromones. Second, the three PBPs were expressed in Escherichia coli and the binding affinities of PBPs to four sex pheromone components and some pheromone analogs were determined by the fluorescence competition binding assays. The results showed that OachPBP1 bound all four sex pheromone components with high binding affinity, while OachPBP2 had high or moderate binding affinity only to three Type I components, and OachPBP3 had high binding affinity only to three minor pheromone components. Furthermore, key amino acid residues that bind to sex pheromone components were identified in three PBPs by 3-D structure modeling and ligand molecular docking, predicting the interactions between PBPs and pheromone components. Our study provides a fundamental insight into the olfactory mechanism in moths that use mixed-type sex pheromones.

Application of blood microsampling in cynomolgus monkey and demonstration of equivalent monoclonal antibody PK parameters compared to conventional sampling.

PURPOSE: The purpose of this study was to evaluate the suitability of whole blood microsampling procedures in non-human primate (NHP) to support toxicokinetic assessments of biotherapeutics in non-human primates. METHOD: A one-month single dose intravenous pharmacokinetic (PK) study was performed in male cynomolgus monkeys with a human IgG1 control monoclonal antibody (mAb) as a surrogate monoclonal antibody biotherapeutic. In this study, both serum samples (conventional sample collection) and microsampling samples were collected. Microsampling samples were collected from two sites on cynomolgus monkey, with each site using two different devices for the whole blood collection. The drug concentrations from all sample types were determined using a quantitative ligand binding assay (LBA). The PK parameters obtained from microsampling samples and serum samples were examined using a standard PK analysis method. The comparability of key PK parameters from both sample types were analyzed statistically. RESULTS: Similar profiles of drug concentrations versus timepoints from all sampling procedures were observed. The correlations of PK concentration data obtained from serum and microsampling samples were ≥ 0.97 using Brand Alman Plot analysis. The key PK parameters obtained from microsampling samples were comparable to those obtained from serum samples (the % differences of mean PK parameters obtained from both sample types were within ±25%). CONCLUSION: This study confirmed that PK parameters obtained from samples using microsampling were comparable to that of serum samples in cynomolgus monkeys. Therefore, the microsampling procedure described can be used as a substitute for conventional sampling procedure to support PK/TK studies of biotherapeutics in non-clinical product developments.

Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and Vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2.

An interlaboratory study was conducted through the Vitamin D Standardization Program (VDSP) to assess commutability of Standard Reference Materials® (SRMs) and proficiency testing/external quality assessment (PT/EQA) samples for determination of serum total 25-hydroxyvitamin D [25(OH)D] using ligand binding assays and liquid chromatography-tandem mass spectrometry (LC-MS/MS). A set of 50 single-donor serum samples were assigned target values for 25-hydroxyvitamin D2 [25(OH)D2] and 25-hydroxyvitamin D3 [25(OH)D3] using reference measurement procedures (RMPs). SRM and PT/EQA samples evaluated included SRM 972a (four levels), SRM 2973, six College of American Pathologists (CAP) Accuracy-Based Vitamin D (ABVD) samples, and nine Vitamin D External Quality Assessment Scheme (DEQAS) samples. Results were received from 28 different laboratories using 20 ligand binding assays and 14 LC-MS/MS methods. Using the test assay results for total serum 25(OH)D (i.e., the sum of 25(OH)D2 and 25(OH)D3) determined for the single-donor samples and the RMP target values, the linear regression and 95% prediction intervals (PIs) were calculated. Using a subset of 42 samples that had concentrations of 25(OH)D2 below 30 nmol/L, one or more of the SRM and PT/EQA samples with high concentrations of 25(OH)D2 were deemed non-commutable using 5 of 11 unique ligand binding assays. SRM 972a (level 4), which has high exogenous concentration of 3-epi-25(OH)D3, was deemed non-commutable for 50% of the LC-MS/MS assays.

Structural and Biomolecular Analyses of Borrelia burgdorferi BmpD Reveal a Substrate-Binding Protein of an ABC-Type Nucleoside Transporter Family.

Borrelia burgdorferisensu lato, the causative agent of tick-borne Lyme borreliosis (LB), has a limited metabolic capacity and needs to acquire nutrients, such as amino acids, fatty acids, and nucleic acids, from the host environment. Using X-ray crystallography, liquid chromatography-mass spectrometry, microscale thermophoresis, and cellular localization studies, we show that basic membrane protein D (BmpD) is a periplasmic substrate-binding protein of an ABC transporter system binding to purine nucleosides. Nucleosides are essential for bacterial survival in the host organism, and these studies suggest a key role for BmpD in the purine salvage pathway of B. burgdorferi sensu lato Because B. burgdorferisensu lato lacks the enzymes required for de novo purine synthesis, BmpD may play a vital role in ensuring access to the purines needed to sustain an infection in the host. Furthermore, we show that, although human LB patients develop anti-BmpD antibodies, immunization of mice with BmpD does not confer protection against B. burgdorferi sensu lato infection.

Nucleotide binding kinetics and conformational change analysis of tissue transglutaminase with switchSENSE.

Function, activity, and interactions of proteins crucially depend on their three-dimensional structure and are often regulated by effector binding and environmental changes. Tissue transglutaminase (Transglutaminase 2, TG2) is a multifunctional protein, allosterically regulated by nucleotides and Ca2+ ions, which trigger opposing conformational changes. Here we introduce switchSENSE as a versatile tool for TG2 characterization and provide novel insights into protein conformation as well as analyte binding kinetics. For the first time, we succeeded in measuring the kinetic rate constants and affinities (kon, koff, KD) for guanosine nucleotides (GMP, GDP, GTP, GTPγS). Further, the conformational changes induced by GDP, Ca2+ and the covalent inhibitor Z-DON were observed by changes in TG2's hydrodynamic diameter. We confirmed the well-known compaction by guanosine nucleotides and extension by Ca2+, and provide evidence for TG2 conformations so far not described by structural analysis. Moreover, we analyze the influence of the peptidic Z-DON inhibitor and the R580A mutation on the conformational responsiveness of TG2 to its natural effectors. In summary, this work shows how the combination of structural and kinetic information obtained by switchSENSE opens new perspectives for the characterization of conformationally active proteins and their interactions with ligands, e.g. potential drug candidates.

Probe-dependence of competitive fluorescent ligand binding assays to odorant-binding proteins.

Ligand binding experiments between small chemicals and proteins and the evaluation of dissociation constants of their complexes in competitive binding assays often rely on displacement of reporter probes by the tested ligand. The most widely adopted protocol uses a fluorescent ligand which changes its emission spectrum when bound to a protein. A decrease of fluorescence, caused by the addition of a second ligand to the complex is generally interpreted as displacement of the fluorescent probe by the ligand, and therefore as a measure of the affinity of the ligand for the protein. Working with an odorant-binding protein (OBP), we found drastic differences in the calculated affinities when using 1-aminoanthracene or N-phenyl-1-naphthylamine as the fluorescent reporter. This fact was quite unexpected, as OBPs are small compact proteins with a single binding pocket without allosteric sites. Such observation raises doubts on the reliability of the fluorescent binding assay, perhaps the most widely used approach to evaluate affinities of small organic compounds to OBPs and other binding proteins. We recommend that the results of fluorescent binding experiments with OBPs should be confirmed by using two different probes or alternative methods. The reliability of current protocols for ligand binding assays is rather limited, while we still wait for a label-free approach that could be simple, fast and free from the use of radioactive tracers.

Experimental design and measurement uncertainty in ligand binding studies by affinity capillary electrophoresis.

In all life sciences ligand binding assays (LBAs) play a crucial role. Unfortunately these are very error prone. One part of this uncertainty results from the unavoidable random measurement uncertainty, another part can be attributed to the experimental design. To investigate the latter, uncertainty propagation was evaluated as a function of the given experimental design. A design space including the normalized maximum response range (nMRR), the data point position (DPP), the data point range (DPR) and the number of data points (NoDP) was defined. Based on ten measured ms ACE source data sets 20 specific parameter sets were selected by Design of Experiments. Monte Carlo simulations using 100 000 repeats for every parameter set were employed. The resulting measurement uncertainty propagation factors (measurement uncertainty multiplier: MUM) were used to describe the whole design space by polynomial regression. The resulting 5-dimensional response surface was investigated to evaluate the design parameter's influence and to find the minimal uncertainty propagation. It could be shown, that the nMRR is of highest importance, followed by DPP and DPR. Interestingly, the NoDP is less relevant. However, the interactions of the four parameters need to be carefully considered during design optimization. Using at least five data points which cover over 40% of the upper part of the binding hyperbola (DPP > 0.57) the MUM will be minimized (MUM approximately 1.5) when the nMRR is appropriate. It is possible to reduce the measurement uncertainty propagation more than one order of magnitude.

Fit-for-Purpose Validation of a Ligand Binding Assay for Toxicokinetic Study Using Mouse Serial Sampling.

PURPOSE: The purpose of this study was to validate a ligand binding assay for the quantitation of a monoclonal antibody-based biotherapeutics (PF-57781346) in samples collected via capillary microsampling to support a regulated mouse toxicity study. METHOD: A quantitative ligand binding assay on the Gyrolab platform was developed to quantify PF-57781346 in blood samples derived from capillary mouse serial sampling. The method validation evaluated assay characteristics including accuracy and precision, influence of sample processing on drug quantitation, whole blood matrix selectivity, dilution linearity and the stability of the drug in the study sample matrix. RESULTS: The method validation demonstrated acceptable analytical characteristics. The whole blood selectivity testing demonstrated accuracy between -4.8% and 13.9% in 10 out of 10 individual whole blood samples, suggesting that drug quantitation from whole blood is not impacted by the serial sampling procedure. Short-term and long-term drug stability in study sample matrix were established to cover required stability for sample storage and analysis (accuracy between -7.3% and 6.1%). CONCLUSION: We reported a successful validation of a bioanalytical method that quantifies PF-55781346 in samples collected via capillary microsampling. The experience shared in this study could serve as a model process for bioanalytical method validation when capillary microsampling is used.

Quantification of biopharmaceuticals and biomarkers in complex biological matrices: a comparison of liquid chromatography coupled to tandem mass spectrometry and ligand binding assays.

The quantification of proteins (biopharmaceuticals or biomarkers) in complex biological samples such as blood plasma requires exquisite sensitivity and selectivity, as all biological matrices contain myriads of proteins that are all made of the same 20 proteinogenic amino acids, notwithstanding post-translational modifications. This review describes and compares the two main approaches, namely, ligand binding assays (LBAs) and liquid chromatography coupled to tandem mass spectrometry in the selected reaction monitoring (SRM) mode. While LBAs remain the most widely used approach, SRM assays are gaining interest due to their generally better analytical performance (precision and accuracy) and their capacity for multiplex analyses. This article focuses on the possible reasons for the discrepancies between results obtained by LBAs and SRM assays.

A multifactorial screening strategy to identify anti-idiotypic reagents for bioanalytical support of antibody therapeutics.

Antibodies are critical tools for protein bioanalysis; their quality and performance dictate the caliber and robustness of ligand binding assays. After immunization, polyclonal B cells generate a diverse antibody repertoire against constant and variable regions of the therapeutic antibody immunogen. Herein we describe a comprehensive and multifactorial screening strategy to eliminate undesirable constant region-specific antibodies and select for anti-idiotypic antibodies with specificity for the unique variable region. Application of this strategy is described for the therapeutic antibody Mab-A case study. Five different factors were evaluated to select a final antibody pair for the quantification of therapeutics in biological matrices: (i) matrix effect in preclinical and clinical matrices, (ii) assay sensitivity with lower limit of quantification goal of single-digit ng/ml (low pM) at a signal-to-background ratio greater than 5, (iii) epitope distinction or nonbridging antibody pair, (iv) competition with target and inhibitory capacity enabling measurement of free drug, and (v) neutralizing bioactivity using bioassay. The selected antibody pair demonstrated superior assay sensitivity with no or minimal matrix effect in common biological samples, recognized two distinct binding epitopes on the therapeutic antibody variable region, and featured inhibitory and neutralizing effects with respect to quantification of free drug levels.

Data quality in drug discovery: the role of analytical performance in ligand binding assays.

Despite its importance and all the considerable efforts made, the progress in drug discovery is limited. One main reason for this is the partly questionable data quality. Models relating biological activity and structures and in silico predictions rely on precisely and accurately measured binding data. However, these data vary so strongly, such that only variations by orders of magnitude are considered as unreliable. This can certainly be improved considering the high analytical performance in pharmaceutical quality control. Thus the principles, properties and performances of biochemical and cell-based assays are revisited and evaluated. In the part of biochemical assays immunoassays, fluorescence assays, surface plasmon resonance, isothermal calorimetry, nuclear magnetic resonance and affinity capillary electrophoresis are discussed in details, in addition radiation-based ligand binding assays, mass spectrometry, atomic force microscopy and microscale thermophoresis are briefly evaluated. In addition, general sources of error, such as solvent, dilution, sample pretreatment and the quality of reagents and reference materials are discussed. Biochemical assays can be optimized to provide good accuracy and precision (e.g. percental relative standard deviation <10 %). Cell-based assays are often considered superior related to the biological significance, however, typically they cannot still be considered as really quantitative, in particular when results are compared over longer periods of time or between laboratories. A very careful choice of assays is therefore recommended. Strategies to further optimize assays are outlined, considering the evaluation and the decrease of the relevant error sources. Analytical performance and data quality are still advancing and will further advance the progress in drug development.

New fluorescent probes for ligand-binding assays of odorant-binding proteins.

Fluorescence-linked binding assays allow determination of dissociation constants at equilibrium and have recently become increasingly popular, thanks to their ease of operation. Currently used probes, such as 1-aminoanthracene and N-phenyl-1-naphthylamine, are excited and emit in the ultraviolet region, but alternative ligands operating in the visible spectrum would be highly desirable for applications in biosensing devices. Based on the two above structures, we have designed and synthesised six new fluorescent probes to be used in ligand-binding assays. The compounds are derivatives of naphatalene, anthracene and fluoranthene and present two aromatic moieties linked by an amine nitrogen. We have measured the emission spectra of the new probes and their binding to three odorant-binding proteins. The probes bind the tested proteins with different affinities, generally with dissociation constants about one order of magnitude lower than the parent compounds. The extended aromatic systems present in the new compounds produced a shift of both excitation and emission peaks at higher wavelength, close or within the visible spectrum, thus facilitating measurements in biosensors for odorants and small organic molecules using optical devices.

Precise, fast, and flexible determination of protein interactions by affinity capillary electrophoresis: part 3: anions.

The binding of physiologically anionic species or negatively charged drug molecules to proteins is of great importance in biochemistry and medicine. Since affinity capillary electrophoresis (ACE) has already proven to be a suitable analytical tool to study the influence of ions on proteins, this technique was applied here for comprehensively studying the influence of various anions on proteins of BSA, ß-lactoglobulin, ovalbumin, myoglobin, and lysozyme. The analysis was performed using different selected anions of succinate, glutamate, phosphate, acetate, nitrate, iodide, thiocyanate, and pharmaceuticals (salicylic acid, aspirin, and ibuprofen) that exist in the anionic form at physiological pH 7.4. Due to the excellent repeatability and precision of the ACE measurements, not necessarily strong but significant influences of the anions on the proteins were found in many cases. Different influences in the observed bindings indicated change of charge, mass, or conformational changes of the proteins due to the binding with the studied anions. Combining the mobility-shift and pre-equilibrium ACE modes, rapidity and reversibility of the protein-anion bindings were discussed. Further, circular dichroism has been used as an orthogonal approach to characterize the interactions between the studied proteins and anions to confirm the ACE results. Since phosphate and various anions from amino acids and small organic acids such as succinate or acetate are present in very high concentrations in the cellular environment, even weak influences are certainly relevant as well.

Structure-dependent binding and activation of perfluorinated compounds on human peroxisome proliferator-activated receptor γ.

Perfluorinated compounds (PFCs) have been shown to disrupt lipid metabolism and even induce cancer in rodents through activation of peroxisome proliferator-activated receptors (PPARs). Lines of evidence showed that PPARα was activated by PFCs. However, the information on the binding interactions between PPARγ and PFCs and subsequent alteration of PPARγ activity is still limited and sometimes inconsistent. In the present study, in vitro binding of 16 PFCs to human PPARγ ligand binding domain (hPPARγ-LBD) and their activity on the receptor in cells were investigated. The results showed that the binding affinity was strongly dependent on their carbon number and functional group. For the eleven perfluorinated carboxylic acids (PFCAs), the binding affinity increased with their carbon number from 4 to 11, and then decreased slightly. The binding affinity of the three perfluorinated sulfonic acids (PFSAs) was stronger than their PFCA counterparts. No binding was detected for the two fluorotelomer alcohols (FTOHs). Circular dichroim spectroscopy showed that PFC binding induced distinctive structural change of the receptor. In dual luciferase reporter assays using transiently transfected Hep G2 cells, PFCs acted as hPPARγ agonists, and their potency correlated with their binding affinity with hPPARγ-LBD. Molecular docking showed that PFCs with different chain length bind with the receptor in different geometry, which may contribute to their differences in binding affinity and transcriptional activity.

Binding characteristics and structural dynamics of two general odorant-binding proteins with plant volatiles in the olfactory recognition of Glyphodes pyloalis.

Glyphodes pyloalis Walker (Lepidoptera: Pyralidae) is the most destructive pest, causing severe damage to mulberry production in China's sericulture industry. The insecticide application in mulberry orchards poses a significant risk of poisoning to Bombyx mori. Shifting from insecticides to odor attractants is a beneficial alternative, but not much data is available on the olfactory system of G. pyloalis. We identified 114 chemosensory genes from the antennal transcriptome database of G. pyloalis, with 18 odorant-binding protein (OBP) and 17 chemosensory protein (CSP) genes significantly expressed in the antennae. Ligand-binding assays for two antennae-biased expressed general odorant-binding proteins (GOBPs) showed high binding affinities of GOBP1 to hexadecanal, ß-ionone, and 2-ethylhexyl acrylate, while GOBP2 exhibited binding to 4-tert-octylphenol, benzyl benzoate, ß-ionone, and farnesol. Computational simulations indicated that van der Waal forces predominantly contributed to the binding free energy in the binding processes of complexes. Among them, Phe12 of GOBP1 and Phe19 of GOBP2 were demonstrated to play crucial roles in their bindings to plant volatiles using site-directed mutagenesis experiments. Moreover, hexadecanal and ß-ionone attracted G. pyloalis male moths in the behavioral assays, while none of the candidate plant volatiles significantly affected female moths. Our findings provide a comprehensive understanding of the molecular mechanisms underlying olfactory recognition in G. pyloalis, setting the groundwork for novel mulberry pests control strategies based on insect olfaction.