Academic literature on the topic 'Miniaturized receptors'

The measurement of intracellular calcium response transients in living mammalian cells is a popular functional assay for identification of agonists and antagonists to receptors or channels of pharmacological interest. In recent years, advances in fluorescence-based detection techniques and automation technologies have facilitated the adaptation of this assay to 384-well microplate format high-throughput screening (HTS) assays. However, the cost and time required performing the intracellular calcium HTS assays in the 384-well format can be prohibitive for HTS campaigns of greater than 1 × 106 wells. For these reasons, it is attractive to miniaturize intracellular calcium functional assays to the 1536-well microplate format, where assay volumes and plate throughput can be decreased by several fold. The focus of the research described in this article is the miniaturization of an intracellular calcium assay to 1536-well plate format. This was accomplished by modifying the hardware and software of a fluorometric imaging plate reader (FLIPR) to enable transfer of nanoliters of test compound directly to a 1536-well assay plate, and measure the resulting calcium response from all 1536 wells simultaneously. An intracellular calcium functional assay against the rat muscarinic acetylcholine receptor subtype 1 (rmAchR1) G-protein coupled receptor (GPCR) was miniaturized and executed on this modified instrument. In experiments measuring the activity of known muscarinic receptor agonists and antagonists, the miniaturized FLIPR assay gave EC50 and IC50 values and rank order potency comparable to the 384-well format assays. Calculated Z′ factors for the miniaturized agonist and antagonist assays were, respectively, 0.56 ± 0.21 and 0.53 ± 0.22, which were slightly higher (Z′agonist = 0.55 ± 0.33) and lower (Z′antagonist = 0.70 ± 0.18) than the corresponding values in the 384-well assays. A mock agonist HTS campaign against the muscarinic receptor in miniaturized format was able to identify all wells spiked with the rmAchR1 agonist carbachol.

We have developed homogeneous miniaturized assays to measure ligand binding to either intact cells or receptor-containing membrane fragments by analysis of particle brightness. As an example, the affinities and inhibition constants of fluorescently labeled interleukin-8 (IL-8) and a low-molecular-weight antagonist toward the receptors CXCR1 and CXCR2, which belong to the superfamily of G protein-coupled receptors (GPCRs), were determined. Although the results were generally comparable between the two approaches, the cell-based measurements revealed a more complex pattern of both ligand and inhibitor titration curves, pointing to the influence of intracellular regulatory events. Both the vesicle- and cell-based membrane receptor assays were successfully miniaturized to a total volume of 1,l without compromising their sensitivity, indicating that screening of transmembrane receptors in these formats is feasible. This is the first report of a cellular ligand-binding assay performed in such low volumes. The resulting savings in reagent could potentially enable the use of primary cells for future HTS/ultra-HTS efforts.

Cell membrane receptors play a central role in controlling cellular functions, making them the target of drugs for a wide variety of diseases. This report describes how a recently developed method, fluorescence intensity distribution analysis (FIDA), can be used to develop homogeneous, nonradioactive high throughput screening assays for membrane receptors. With FIDA, free ligand and ligand accumulated on receptor-bearing membrane vesicles can be distinguished on the basis of their particle brightness. This allows the concentration of both bound and free ligand to be determined reliably from a single measurement, without any separation. We demonstrate that ligand affinity, receptor expression level, and potency of inhibitors can be determined using the epidermal growth factor and β2-adrenergic receptors as model systems. Highly focused confocal optics enable single-molecule sensitivity, and sample volumes can thus be reduced to 1,IL without affecting the quality of the fluorescence signal. Our results demonstrate that FIDA is an ideal method for membrane receptor assays offering substantial benefits for assay development and high throughput pharmaceutical screening.

A flexible electrochemical sensor based on the carbon felt (CF) functionalized with Bisphenol A (BPA) synthetic receptors was developed. The artificial Bisphenol A receptors were grafted on the CF by a simple thermal polymerization molecular imprinting process. Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM) and electrochemical characterizations were used to analyze the receptors. Characterization results demonstrated that the Bisphenol A synthetic receptors successfully formed on the CFs surface. Because the synthetic receptor and the porous CFs were successfully combined, the sensor displayed a better current response once Bisphenol A was identified. The sensor’s linear range was determined to be from 0.5 to 8.0 nM and 10.0 to 300.0 nM, with a detection limit of 0.36 nM. Even after being bent and stretched repeatedly, the electrode’s performance was unaffected, demonstrating the robustness, adaptability and viability of installing the sensor on flat or curved surfaces for on-site detection. The designed electrochemical sensor has been used successfully to identify Bisphenol A in milk samples with satisfactory results. This work provided a promising platform for the design of implantable, portable and miniaturized sensors.

Cell-based β-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 μL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC50/IC50 precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.

Mass sensitive sensors were applied for fast and label-free detection of bio-analytes. Robust and miniaturized sensor devices were fabricated by combining bio-mimetic imprinted surfaces with quartz crystal microbalances for the analysis of yeast and bacteria cells. These sensors allow us to differentiate between different growing stages of yeast cells. Moreover, the viability of cells was detected by structuring quartz crystal microbalance electrodes like a grid. Artificial yeast cells were produced to pattern the recognition layer, giving reversible enrichment of the respective bio-analytes. This approach was followed to ensure the reproducibility of the identical sensitive material in each case, because the properties of each cell depend on its growth stage, which varies over time. The strategy was further applied to develop a sensitive system for Escherichia coli. Structuring of these materials by soft lithography allows differentiation between cell strains, e.g. E. coli (strain W & B) with a five-fold selectivity.

G protein–coupled receptors (GPCRs) exist as collections of conformations in equilibrium, and the efficacy of drugs has been proposed to be associated with their absolute and relative affinities for these different conformations. The serotonin 2A (5-HT2A) receptor regulates multiple physiological functions, is involved in the pathophysiology of schizophrenia, and serves as an important target of atypical antipsychotic drugs. This receptor was one of the first GPCRs for which the functional selectivity phenomenon was observed, with its various ligands exerting differential effects on the phospholipase A2 (PLA2) and phospholipase C (PLC) signaling pathways. We aimed to develop a multiplex functional assay in 96-well plates for the simultaneous measurement of the PLA2 and PLC pathways coupled to 5-HT2A receptors; this approach enables the detection of either functional selectivity or cooperativity phenomena in early drug screening stages. The suitability of the method for running screening campaigns was tested using the Prestwick Chemical Library, and 22 confirmed hits with activities of more than 90% were identified; 11 of these hits produced statistically significant differences between the two effector pathways. Thus, we have developed a miniaturized multiplex assay in 96-well plates to measure functional selectivity for 5-HT2A receptors in the early stages of the drug discovery process.

Environmentally relevant contaminants endowed with chirality may include pharmaceutical compounds, flame retardants, perfluoroalkyl chemicals, pesticides, and polychlorinated biphenyls. Despite having similar physicochemical properties, enantiomers may differ in their biochemical interactions with enzymes, receptors, and other chiral molecules leading to different biological responses. In this work, we have designed a wireless miniaturized stereoselective light-emitting system able to qualitatively detect a chiral contaminant (3,4-dihydroxyphenylalanine, DOPA) dissolved in reduced volumes (in the microliters range), through bipolar electrochemistry. The diastereomeric environment was created by mixing the enantiomers of an inherently chiral inductor endowed with helical shape (7,8-dipropyltetrathia[7]helicene) and the chiral probe (DOPA) in micro-solutions of a commercial ionic liquid. The synergy between the inductor, the applied electric field, and the chiral pollutant was transduced by the light emission produced from a miniaturized light-emitting diode (LED) exploited in such an approach as a bipolar electrode.

G Protein-coupled receptors (GPCRs) represent one of the most important target classes for drug discovery. Various assay formats are currently applied to screen large compound libraries for agonists or antagonists. However, the development of nonradioactive, miniaturizable assays that are compatible with the requirements of ultra-high throughput screening (uHTS) has so far been slow. In this report we describe homogeneous fluorescence-based binding assays that are highly amenable to miniaturization. Fluorescence intensity distribution analysis (FIDA) is a single-molecule detection method that is sensitive to brightness changes of individual particles, such as those induced by binding of fluorescent ligands to membrane particles with multiple receptor sites. As a confocal detection technology, FIDA inherently allows reduction of the assay volume to the microliter range and below without any loss of signal. Binding and displacement experiments are demonstrated for various types of GPCRs, such as chemokine, peptide hormone, or small-molecule ligand receptors, demonstrating the broad applicability of this method. The results correlate quantitatively with radioligand binding data. We compare FIDA with fluorescence anisotropy (FA), which is based on changes of molecular rotation rates upon binding of fluorescent ligands to membranes. While FA requires a higher degree of binding, FIDA is sensitive down to lower levels of receptor expression. Both methods are, within these boundary conditions, applicable to uHTS.

2012/2013
Coffee is a worldwide diffused beverage and it has a very high impact on the global economy. The success of coffee is due to the perfect combination between the overall involvement of our senses and the well-known stimulating effect. During the years, many studies focused on its volatile fraction, but our knowledge about the taste responsible compounds is still nowadays scarce. Among such compounds present in the coffee beans, chlorogenic acids (CGAs), that belong to the polyphenols family, acquire more and more importance, due to their key-role in determining the coffee flavor, particularly its bitterness, their beneficial health properties, and their possible use as marker to control the industrial process. The roasting of green coffee beans causes a partial loss of CGAs due to the formation of degradation products, such as the corresponding lactones, whose amount depends on the roasting degree; the identification and the quantification of acids and lactones in coffee could be therefore a useful parameter to evaluate the final quality of the beverage. This PhD project arises from the interest in searching new methodologies for the selective analysis of the lactone fraction, exploiting the recognition properties of biomolecules, proteins or peptides, or of designed sensing elements with high affinity for such lactonic compounds. The chosen molecules, not commercially available, were first synthesized and fully characterized, namely: 3,4-O-dicaffeoyl-1,5-gamma-quinide, 3-O-[3,4-(dimethoxy)cinnamoyl]-1,5-gamma-quinide, 3,4-O-bis[3,4-(dimethoxy)cinnamoyl]-1,5-gamma-quinide, and 1,3,4-O-tris[3,4-(dimethoxy)cinnamoyl]-1,5-gamma-quinide. A direct synthesis of the tri-substituted quinide was also tuned up, starting from D-(-)-quinic acid and 3,4-dimethoxycinnamoyl chloride. Considering the potential biological activities of polyphenols, the antiviral properties of these compounds against many viruses have been also evaluated. A possible approach to develop a selective biosensor is to use natural peptide scaffolds, with stable and highly organized conformations, and reduce its dimensions down to the limit of receptor stability, exploiting even the randomization of the aminoacid within the binding site to improve its ligand properties. In this perspective, the specific binding constants to Human Serum Albumin (HSA), more exactly to its Sudlow site I, of caffeic acid, ferulic acid, 3,4-dimethoxycinnamic acid, 5-O-caffeoyl quinic acid and of the four synthesized quinides were measured in physiological conditions by fluorescence spectroscopy, reaching promising KD values, in the micromolar range; moreover 3,4-O-dicaffeoyl-1,5-gamma-quinide gave a peculiar result, showing a very interesting double binding in the same active site of the protein. The two diester quinides were also used to test, always by means of fluorescence spectroscopy, the binding ability of a functional 100 aminoacids fragment that replicates the binding site I of the whole protein: this peptide, called HSA100, has been obtained by our research group; even some mutants of HSA100 have been prepared and four of them, randomly chosen, were used in the fluorescence assays. The obtained binding constants with HSA100 and its four mutants remain in the micromolar range, close to those measured with the whole protein, suggesting that proper mutations could lead to a selective biosensor with high affinity for the quinides. The Surface Plasmon Resonance (SPR) technique could offer a way for the fast screening of the binding properties. In the perspective of setting up a SPR method to quickly select the hits of the mutants libraries, a linker-equipped quinide was synthesized and immobilized on a gold chip, then the binding with HSA was tested in this way. Considering the well-known interaction between polyphenols compounds, such as chlorogenic acid, and caffeine in aqueous solution, NMR titrations were performed to study the behavior of caffeine with the synthesized quinides. This kind of binding may be exploited to develop a selective biosensor for quinides by introducing several molecules of caffeine or other xanthines on a suitable scaffold.
XXVI Ciclo
1986

2011/2012
The project deals with the design and development of artificial receptors for monitoring of drugs used for the HIV “Anti-Retroviral Therapy” (ART). Efavirenz, a RT-inhibitor, and its analogues were synthesised to screen with several techniques (surface plasmon resonance, NMR, fluorescence and UV spectroscopy) the new receptors obtained from different approaches. One exploits computational tools to model peptides with high affinity for small molecules. These designed peptides will then be incorporated in polymeric matrix for biosensor purposes. Molecularly imprinted polymers with efavirenz-binding properties were performed during my experience at Queen Mary University of London. Another approach is based on miniaturized receptors obtained from the reduction of the known sequence of a natural receptor as human serum albumin.
XXV Ciclo
1984