Multidimensional chromatographic separation of estrogen receptors

Analyses of estrogen and progesterone receptors in biopsies of breast carcinoma play a vital role in the selection of patients likely to respond to hormone manipulation. Sucrose density gradient centrifugation has been the reference method in the determination of estrogen receptors in human breast carcinoma cytosols. To reduce assay time and circumvent prolonged manipulation of labile receptor preparations, high performance liquid chromatography techniques in the size-exclusion and ion-exchange modes were compared as potential alternate methods for the rapid separation of receptor isoforms. Multidimensional analyses were performed by reapplying estrogen receptor isoforms obtained from high performance size-exclusion and ion-exchange chromatography to sucrose density gradients and vice versa. This confirmed that the estrogen-binding components identified by high performance liquid chromatography appear to correspond to estrogen receptor species from sucrose density gradients.     

Multiplexed immunoassays for the analysis of breast cancer biopsies

Within the last decade, protein microarray technology has been successfully used for the simultaneous quantification of target proteins from minimal amounts of samples in basic and applied proteome research. The robustness and appropriate sensitivity of these miniaturized assays have been demonstrated and thus the transfer to routine and high-throughput applications is now possible. In this study, multiplexed bead-based sandwich immunoassays were used to determine the concentrations of 54 protein analytes, including HER 2 and the estrogen receptor, from ultrasound-guided large-core needle biopsies (LCNBs) from breast cancer patients. Expression levels for HER 2, estrogen receptors and progesterone receptors were also assessed by immunohistochemical routine staining, performed in the clinic on corresponding biopsy samples. The high concordance of the data sets generated with the bead-based protein arrays and by conventional immunohistochemical assessment of HER 2 and the estrogen receptor expressed by breast cancer cells present in the biopsies was demonstrated.     

A technique for relative quantitation of cancer biomarkers in formalin‐fixed,paraffin‐embedded (FFPE) tissue using stable‐isotope‐label based mass spectrometry imaging (SILMSI)

We developed a novel technique for the relative quantitation of pairs of cancer biomarkers in formalin‐fixed paraffin‐embedded (FFPE) tissue. The method utilizes stable isotope labeled (SIL) chromogens deposited during the standard immunohistochemistry (IHC) tissue staining process. The labeled chromogens are precipitated on tissue enzymatically using the standard IHC protocols. The tissue is then imaged with matrix‐free laser desorption ionization time‐of‐flight mass spectrometry, and peak intensities of reporter ions are used to estimate the relative quantitation of protein biomarkers across the tissue. The relative abundance of two breast cancer biomarkers, estrogen receptor (ER) and progesterone receptor (PgR), were quantitated using their ratio of expression in xenograft models, and the ratios were found to be reproducible both within and across serial sections. The relative quantification of multiple biomarkers in situ across a single tissue section adds an additional dimension in cancer histological evaluation by allowing a visual and statistical assessment of tumor heterogeneity. Copyright © 2015 John Wiley & Sons, Ltd.     

Enzyme-Amplified Receptor Assay (ERA): A Novel Approach to Drug Detection

Abstract

Isolated acetylcholine receptors from Torpedo nobiliana are used as recognition elements for phencyclidine (PCP), a potent hallucinogen with high affinity for the ion channel site associated with the receptor, in a novel enzyme-amplified receptor assay. A fixed amount of PCP labelled enzyme competes with free PCP for a limited number of receptor sites resulting in changes of observed enzyme activity proportional to free drug concentration. The proposed concept combines the use of receptors with an enzyme amplification step to yield an exceptionally simple and convenient assay.     

Selective Targeting of Breast Cancer by Tafuramycin A Using SMA-Nanoassemblies

Triple-negative breast cancer (TNBC) is a heterogeneous subtype of tumors that tests negative for estrogen receptors, progesterone receptors, and excess HER2 protein. The mainstay of treatment remains chemotherapy, but the therapeutic outcome remains inadequate. This paper investigates the potential of a duocarmycin derivative, tafuramycin A (TFA), as a new and more effective chemotherapy agent in TNBC treatment. To this extent, we optimized the chemical synthesis of TFA, and we encapsulated TFA in a micellar system to reduce side effects and increase tumor accumulation. In vitro and in vivo studies suggest that both TFA and SMA–TFA possess high anticancer effects in TNBC models. Finally, the encapsulation of TFA offered a preferential avenue to tumor accumulation by increasing its concentration at the tumor tissues by around four times in comparison with the free drug. Overall, the results provide a new potential strategy useful for TNBC treatment.     

Design and Synthesis of Novel Nonsteroidal Phytoestrogen‐based Probes as Potential Biomarker: Evaluation of Anticancer Activity and Docking Studies

Two series of novel flavones and flavone‐tanaproget conjugates were designed, synthesized, ¹²⁵I­radiolabeled, and evaluated for their antiproliferative activity against human cancer cell lines (MCF‐7, MDA‐MB‐231, and HeLa). They exhibited good‐to‐excellent in vitro antiproliferative activity. For improving the receptor‐binding affinity of synthesized compounds, flavone‐tanaproget moieties were linked together through alkyl‐alkenyl chain spacer, and the butyl chain was substituted at C‐3 position of oxa‐ and aza‐flavones to enhance the lypophilicity of the molecule. These compounds are hypothesized to bind with estrogen and progesterone receptors by docking studies, and binding affinities were shown to increase significantly compared with the reference ligands.     

Structure-Functions Relationships of the Benzodiazepine and Serotonine Receptors Ligands

Abstract

States of anxiety and fear are controlled in organism by GABA-ergic and serotonine- ergic systems. Concepts about the structure and functions of GABA_A receptor channel, benzodiazepine and serotonine receptors have been considered. Structural and conformational peculiarities of ligands of these receptors determine their role (agonists, antagonists, inverse agonists, partial agonists, partial inverse agonists) at the formation of supramolecular complexes ?ligand-receptor’ and, as a result, pharmacological effects of ligands.     

Environmental Analysis Using Antibody and Receptor Based Techniques

Abstract

Immunochemical analysis relies on the selective binding of antibodies to defined targets such as environmental compounds. Commercial applications in the environmental field are still restricted to a limited number of immunoassays and a few immunochromatographic applications. The main barrier to a broader exploitation is seen in the generation of a sufficient number of antibodies within an acceptable time period. Recombinant technologies are expected to eventually replace the circumstantial approach to obtain new Abs by new immunizations. Examples for recombinant singlechain fragments (scFv) and antigen binding fragments (Fab) directed against herbicidal s-triazines are given. If antibodies are replaced by receptors and other functional ligands, biological effects monitoring becomes available. As an example an enzyme-linked receptor assay (ELRA) for endocrine disruptors providing estrogen equivalents is presented. Finally, the concept of bioresponse-linked instrumental analysis is introduced for the tight coupling of effect monitoring to chemical analysis. In the first step, analyte binding by functional biomolecules is recorded to provide binding equivalents. The second step is targeted at chemical analysis of bioeffective analytes bound to the functional biomolecules.     

Multiple-injection affinity capillary electrophoresis to estimate binding constants of receptors to ligands

Multiple-injection affinity capillary electrophoresis (MIACE) is used to determine binding constants (K _b) between receptors and ligands using as model systems vancomycin and teicoplanin from Streptomyces orientalis and Actinoplanes teichomyceticus, respectively, and their binding to D-Ala-D-Ala peptides and carbonic anhydrase B (CAB. EC 4.2.1.1) and the binding of the latter to arylsulfonamides. A sample plug containing a non-interacting standard is first injected followed by multiple plugs of sample containing the receptor and then a final injection of sample containing a second standard. Between each injection of sample, a small plug of buffer is injected which contains an increasing concentration of ligand to effect separation between the multiple injections of sample. Electrophoresis is then carried out in an increasing concentration of ligand in the running buffer. Continued electrophoresis results in a shift in the migration time of the receptor in the sample plugs upon binding to their respective ligand. Analysis of the change in the relative migration time ratio (RMTR) or electrophoretic mobility (μ) of the resultant receptor–ligand complex relative to the non-interacting standards, as a function of the concentration of ligand yields a value for K _b. The MIACE technique is a modification in the ACE method that allows for the estimation of binding affinities between biological interactions on a timescale faster than that found for standard ACE. In addition sample volume requirements for the technique are reduced compared to traditional ACE assays. These findings demonstrate the advantage of using MIACE to estimate binding parameters between receptors and ligands.     

A comparison of progestin and androgen receptor binding using the CoMFA technique

Summary A series of 48 steroids has been studied with the SYBYL QSAR module using Relative Binding Affinities (RBAs) to progesterone and androgen receptors obtained from the literature. Models for the progesterone and androgen data were developed. Both models show regions where sterics and electrostatics correlate to binding affinity but are different for androgen and progesterone which suggests differences possibly important for receptor selectivity. The progesterone model is more predictive than the androgen (predictive r² of 0.725 vs. 0.545 for progesterone and androgen, respectively).     

HPLC‐MS/MS method for quantitative determination of the novel dual inhibitor of FGF and VEGF receptors E‐3810 in tumor tissues from xenograft mice and human biopsies

We developed and validated a high‐performance liquid chromatography–tandem mass spectrometry analytical method to measure E‐3810, a novel dual inhibitor of fibroblast growth factor receptor 1 and vascular endothelial growth factor receptor 1–3 in tissues and determined the drug concentration in a biopsy of human breast cancer for the first time. The method is a modification of our previous one in plasma to study the clinical pharmacokinetics of the drug during the phase I/II trial. In view of the changes in matrix, we applied a partial validation protocol to determine recovery, sensitivity, range of linearity, precision, accuracy and stability of the method over three runs in a mouse tumor tissue and liver. The recovery of E‐3810 from liver or tumor homogenate was >69%, and the lower limit of quantification was 5 ng/ml. The method was linear in the concentration range 5.0–500.0 ng/ml, as demonstrated by a determination coefficient R² ≥ 0.9955. The range of the calibration curve was appropriate for the analysis, as demonstrated by the accuracy, which was between 91.4% and 106.7%. Interday precision and accuracy on quality control samples at 9, 30 and 300 ng/ml were 3.1‐11.2% and 98.3–111.4%, respectively. The assay was applied successfully to determine the intratumor concentration of E‐3810 in different mouse xenograft tumor models and in a biopsy of a patient with breast cancer included in the phase I/II trial of the drug. In mouse tumors, the concentrations of E‐3810 were higher than necessary to exert antitumor activity in vitro (1 µM). Even more of interest was the result obtained in a human biopsy of few milligrams, where E‐3810 reached 4.9 µg/g (11 µM). Copyright © 2014 John Wiley & Sons, Ltd.     

A Conductimetric System Based on Polyaniline for Determination of Ammonia in Fertilizers

Abstract

A simple conductimetric system to determine ammonia concentration using a sensor based on a conductor polymer was developed. The sensitive element to ammonia is a thin polyaniline film deposited by chemical synthesis in an acrylic substrate prepared before hand with two graphite electrodes. The conductance of the polyaniline film decreases when exposed to the ammonia gas and this variation can be related to the ammonia concentration. To determine ammonia in fertilizer samples a system consisting of a measurement cell, a conductivity meter and a strip chart recorder was used. The results were compared with those obtained by three different laboratories employing a Kjeldahl method and are in good agreement. The detection range of the system was 0.6 to 3.7 μg.mL^?1 with a response time of 4 minutes. The relative standard deviation of the proposed method was about 5%.     

Application of structure‐based focusing to the estrogen receptor

We present the Cerius² Structure‐Based Focusing (SBF) application. This application was applied to the estrogen receptor. A series of three‐dimensional queries were generated for the binding site of the receptor. The queries consist of combinations of hydrogen bond donors and acceptors, and lipophilic features for the binding site along with excluded volume regions occupied by the receptor atoms. A database of 31 ligands with known relative binding affinities for the estrogen receptor was used to examine the selectivity of the queries. The objective of the study was to determine if queries generated by the Cerius² SBF method could differentiate between the more and less active ligands of the training set. Results are promising, with the generated queries showing greater selectivity toward the more active ligands. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 993–1003, 2001     

Inhibition by protease inhibitors of binding of adrenal and sex steroid hormones.

Binding of steroid hormones is inhibited by protease inhibitors and substrates. The protease inhibitors phenylmethyl sulphonylfluoride, tosyl-lysine chloromethyl ketone, and tosylamide-phenylethyl-chloromethyl ketone and the protease substrates tosyl arginine methyl ester and tryptophan methyl ester eliminate specific binding of aldosterone, dexamethasone, dihydrotestosterone, estrogen, and progesterone to their respective receptors. These protease inhibitors and substrates also inhibit binding of progesterone to the 20,000 molecular weight mero-receptor formed from the progesterone receptor in chick oviduct. The binding of estradiol to rat alpha-fetoprotein is inhibited by the protease inhibitors and substrates but not by tryptophan or tryptophan amide, indicating the importance of an ester structure in the inhibition of steroid binding. Our results suggest that all steroid hormone receptors have a site with both common structural features and a role in the regulation of steroid hormone binding.     

Virtual screening using docking and molecular dynamics of cannabinoid analogs against CB1 and CB2 receptors

BackgroundCannabis sativa has been attributed to different pharmacological properties. A number of secondary metabolites such as tetrahydrocannabinol (THC), cannabinol (CBD), and different analogs, with highly promising biological activity on CB₁ and CB₂ receptors, have been identified.MethodsThus, this study aimed was to evaluate the activity of THC, CBD, and their analogs using molecular docking and molecular dynamics simulations (MD) methods. Initially, the molecules (ligands) were selected by bioinformatics searches in databases. Subsequently, CB₁ and CB₂ receptors were retrieved from the protein data bank database. Afterward, each receptor and its ligands were optimized to perform molecular docking. Then, MD Simulation was performed with the most stable ligand-receptor complexes. Finally, the Molecular Mechanics-Generalized Born Surface Area (MM-PBSA) method was applied to analyze the binding free energy between ligands and cannabinoid receptors.ResultsThe results obtained showed that ligand LS-61176 presented the best affinity in the molecular docking analysis. Also, this analog could be a CB₁ negative allosteric modulator like CBD and probably an agonist in CB₂ like THC and CBD according to their dynamic behavior in silico. The possibility of having a THC and a CBD analog (LS-61176) as a promising molecule for experimental evaluation since it could have no central side-effects on CB₁ and have effects of CB₂ useful in pain, inflammation, and some immunological disorders. Docking results were validate using ROC curve for both cannabinoids receptor where AUC for CB1 receptor was 0.894±0.024, and for CB2 receptor AUC was 0.832±0032, indicating good affinity prediction.     

Linkage effects on binding affinity and activation of GPR30 and estrogen receptors ERalpha/beta with tridentate pyridin-2-yl hydrazine tricarbonyl-Re/(99m)Tc(I) chelates

We describe a new structural class of neutral tridentate pyridin-2-yl hydrazine chelates for labeling with tricarbonyl Re/99mTc(I) under aqueous conditions and investigate the receptor binding of synthetic estradiol derivatives with the novel G-protein-coupled receptor GPR30 and estrogen receptors ERalpha/beta. The steroid linkage affected the affinity and selectivity of estrogen binding with these receptors. Fluorescence assays based on calcium signaling demonstrate that membrane-permeable chelates 2 and 3 interact with the receptors in whole cells. These results suggest that in vitro assays will facilitate the development of targeted imaging agents for intracellular receptors and the feasibility of targeting GPR30 and ERalpha/beta for diagnostic tumor imaging.     

Homology model of the rainbow trout estrogen receptor (rtERα) and docking of endocrine disrupting chemicals (EDCs)‖

A model for rainbow trout (Oncorhynchus mykiss) estrogen receptor (rtERα) was built by homology with the human estrogen receptor (hERα). A high level of sequence conservation between the two receptors was found with 64% and 80% of identity and similarity, respectively. Selected endocrine disrupting chemicals were docked into the ligand binding domain (LBD) of rtERα and the corresponding free binding energies Δ(ΔG_bind) values were calculated. A Quantitative Structure-Activity Relationship (QSAR) model between the relative binding affinity data and the Δ(ΔG_bind) values was derived in order to predict which further organic pollutants are likely to bind to rtERα.