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Fluorescent Approaches for Understanding Interactions of Ligands with G Protein Coupled Receptors
Abstract
G Protein Coupled Receptors (GPCRs) are responsible for a wide variety of signaling responses in diverse cell types. Despite major advances in the determination of structures of this class of receptors, the underlying mechanisms by which binding of different types of ligands specifically elicits particular signaling responses remains unclear. The use of fluorescence spectroscopy can provide important information about the process of ligand binding and ligand dependent conformational changes in receptors, especially kinetic aspects of these processes, that can be difficult to extract from x-ray structures. We present an overview of the extensive array of fluorescent ligands that have been used in studies of GPCRs and describe spectroscopic approaches for assaying binding and probing the environment of receptor-bound ligands with particular attention to examples involving yeast pheromone receptors. In addition, we discuss the use of fluorescence spectroscopy for detecting and characterizing conformational changes in receptors induced by the binding of ligands. Such studies have provided strong evidence for diversity of receptor conformations elicited by different ligands, consistent with the idea that GPCRs are not simple on and off switches. This diversity of states constitutes an underlying mechanistic basis for biased agonism, the observation that different stimuli can produce different responses from a single receptor. It is likely that continued technical advances will allow fluorescence spectroscopy to play an important role in continued probing of structural transitions in GPCRs. This article is part of a Special Issue entitled: Structural and biophysical characterisation of membrane protein-ligand binding.
... Ligand binding studies with fluorescent probes can be used on native, endogenously expressed GPCRs, but have found significant utility in pharmacological studies when tested in cells expressing genetically-modified GPCRs, typically fused at the N-terminus to fluorescent or luminescent proteins (including the aforementioned SNAP domains and the extremely bright luciferase NanoLuc R ), to assess ligand-receptor interactions in real time via proximity-based techniques that exploit Förster resonance energy transfer (FRET) or bioluminescent resonance energy transfer (BRET) [4]. Data generated with these methods are often consistent with those measured using radioligands and are sensitive enough to reveal parameters such as drug residence time, important due to its potential to predict how ligands confer physiological effects and clinical outcomes [5]. ...
... To complement binding studies, fluorescence imaging techniques such as high-content imaging, whole cell confocal microscopy, single molecule microscopy [2] and fluorescence correlation spectroscopy [6] are aided by the use of fluorescent probes to report invaluable information about the behavior of GPCRs in a complex cellular environment, and how receptors or ligands diffuse, using either live or fixed cells. Together, fluorescent probes underpin many technologies that have become essential for drug discovery; consequently, this exciting and expanding field has been the focus of many excellent reviews [4,[7][8][9][10]. ...
... Fluorescent probes have been generated for more than 40 distinct receptors, with peptide-based GPCR ligands featuring most heavily in early endeavors to generate fluorescent tools [4]. This is due to the relative ease of modifying the N-or C-terminus or side chains of amino acids that are known to be noncritical for ligand activity and because the dyes used are typically relatively small relative to the labeled peptide. ...
G protein-coupled receptors (GPCRs) are essential signaling proteins and tractable therapeutic targets. To develop new drug candidates, GPCR drug discovery programs require versatile, sensitive pharmacological tools for ligand binding and compound screening. With the availability of new imaging modalities and proximity-based ligand binding technologies, fluorescent ligands offer many advantages and are increasingly being used, yet labeling small molecules remains considerably more challenging relative to peptides. Focusing on recent fluorescent small molecule studies for family A GPCRs, this review addresses some of the key challenges, synthesis approaches and structure–activity relationship considerations, and discusses advantages of using high-resolution GPCR structures to inform conjugation strategies. While no single approach guarantees successful labeling without loss of affinity or selectivity, the choice of fluorophore, linker type and site of attachment have proved to be critical factors that can significantly affect their utility in drug discovery programs, and as discussed, can sometimes lead to very unexpected results.
... The indolinium-type cyanine dye-labeled NT(8−13) derivatives 6,9,11,12,16, and 17 were prepared by treatment of the amino-functionalized precursor peptides 4, 7, or 15, 19 containing an N ω -carbamoylated arginine either in position 8 (4,7) or in position 9 (15) with the succinimidyl esters of the respective dyes (5, 8, or 10) (Scheme 1). The pyridinium dye-labeled peptide 14 was obtained by treatment of 7 with the pyrylium derivative 13 32 in the presence of triethylamine (Scheme 1). ...
... As reported for 3, 19 the fluorescent NT(8−13) derivatives 6,9,11,12,14,16, and 17 exhibited high NTS 1 R affinities with pK i values of 8.15−9.12 (Table 1; competition binding curves shown in Figure S9, Supporting Information). ...
... NTS 1 R Binding Data (pK i ) of1,3,6,9,11,12,14,16, and 17, NTS 2 R Binding Data (pK i ) of 1, 3, 6, 9, 12, and 17, pK d Values of 3, 6, 9, and 12 Determined by Saturation Binding (hNTS 1 R), and hNTS 1 R Potencies (pEC 50 ) of 1, 3, 6, and 12 from Ca 2+ Assays ...
Fluorescence-labeled receptor ligands have emerged as valuable molecular tools, being indispensable for studying receptor-ligand interactions by fluorescence-based techniques, such as high-content imaging, fluorescence microscopy or fluorescence polarization. Applying a new labeling strategy for peptides, a series of fluorescent neurotensin(8-13) derivatives was synthesized by attaching red-emitting fluorophores (indolinium- and pyridinium-type cyanine dyes) to carbamoylated arginine residues in neurotensin(8-13) analogs, yielding fluorescent probes with high NTS1R affinity (pKi values: 8.15-9.12) and potency (pEC50 values (Ca²⁺-mobilization): 8.23-9.43). Selected fluorescent ligands were investigated by flow cytometry and high-content imaging (saturation binding, kinetic studies, competition binding) as well as by confocal microscopy using intact CHO-hNTS1R cells. The study demonstrates the applicability of the fluorescent probes as molecular tools to obtain, for example, information about the localization of receptors in cells and to determine binding affinities of non-labeled ligands.
... Binding of a ligand to a GPCR is also routinely assessed by labelling the ligand of interest, either with a radioactive isotope or fluorescent marker [6][7][8]. The use of radio-labeled ligands in either kinetic, saturation, or competition binding assays provides a reliable way to determine binding affinities between said ligand and a particular receptor. ...
... However, due to issues involved with the safety, expense, and general handling of radioactive isotopes, efforts have been made in recent years to move away from the use of radioactive isotopes and replace them with fluorescent labels. The usefulness of fluorescently labelled ligands [9][10][11] can actually extend beyond a simple replacement of radioactive isotopes in binding assays and be used for other more in depth studies [7,8], such as localization of bound receptors using traditional fluorescence microscopes to more sophisticated approaches that quantify the geometry of ligand-receptor and receptor-receptor complexes using Forster resonance energy transfer [12]. However, attachment of a fluorescent probe to the ligand can potentially alter the nature of the ligand's interaction with receptors. ...
Using radiofrequency dielectric spectroscopy, we have investigated the impact of the interaction between a G protein-coupled receptor (GPCR), the sterile2 α-factor receptor protein (Ste2), and its cognate agonist ligand, the α-factor pheromone, on the dielectric properties of the plasma membrane in living yeast cells (Saccharomyces cerevisiae). The dielectric properties of a cell suspension containing a saturating concentration of α-factor were measured over the frequency range 40Hz–110 MHz and compared to the behavior of a similarly prepared suspension of cells in the absence of α-factor. A spherical three-shell model was used to determine the electrical phase parameters for the yeast cells in both types of suspensions. The relative permittivity of the plasma membrane showed a significant increase after exposure to α-factor (by 0.06 ± 0.05). The equivalent experiment performed on yeast cells lacking the ability to express Ste2 showed no change in plasma membrane permittivity. Interestingly, a large change also occurred to the electrical properties of the cellular interior after the addition of α-factor to the cell suspending medium, whether or not the cells were expressing Ste2. We present a number of different complementary experiments performed on the yeast to support these dielectric data and interpret the results in terms of specific cellular reactions to the presence of α-factor.
... A very important step in the early stages of developing novel ligands for GPCRs is to characterize their binding properties. Representing an attractive alternative to radioligand binding assays [1], fluorescencebased techniques have gained popularity in this field during the last decade [2,3]. Classical radioligand binding experiments have to be performed in a heterogeneous manner, i.e. the receptor-bound ligand has to be separated from the unbound ligand [4], precluding equilibrium conditions during measurement. ...
... Furthermore, the use of radiolabeled compounds has some practical and financial drawbacks, especially with respect to handling and waste management. Several different fluorescence-based readouts are now available to study ligand binding to GPCRs [2,3,6], including Abbreviations: BBV, budded baculovirus; BRET, bioluminescence resonance energy transfer; BSA, bovine serum albumin; c final , final compound concentration used in an assay; CHO, Chinese hamster ovary cells; DIBA, dibenzodiazepinone; DMEM, Dulbecco's Modified Eagle's Medium; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetate; FA, fluorescence anisotropy; FBS, fetal bovine serum; G418, geneticin; GPCR, G protein-coupled receptor; HEK293T, human embryonic kidney cells; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; k off , dissociation rate constant; k obs , observed association rate constant; k on , association rate constant; λ max , wavelength, at which peak bioluminescence occurs; L-15, Leibovitz' L-15 medium; MOI, multiplicity of infection; MR, muscarinic acetylcholine receptor; NLuc, NanoLuc®; IC 50 , concentration causing half-maximal inhibition; K d equilibrium , equilibrium dissociation constant determined in saturation binding fluorescence anisotropy (FA) [7][8][9][10], fluorescence correlation spectroscopy [11,12] or (time-resolved) Förster or bioluminescence resonance energy transfer ((TR)-FRET [13][14][15][16] or BRET [17][18][19][20][21]). For BRET binding assays (see Fig. 1A), the NanoLuc® (NLuc) [22] is generally fused to the N-terminus of the investigated GPCR [17,23]. ...
BRET and fluorescence anisotropy (FA) are two fluorescence-based techniques used for the characterization of ligand binding to G protein-coupled receptors (GPCRs) and both allow monitoring of ligand binding in real-time. In this study, we present the first direct comparison of BRET-based and FA-based binding assays using the human M2 muscarinic acetylcholine receptor (M2R) and two TAMRA (5-carboxytetramethylrhodamine)-labeled fluorescent ligands as a model system. The determined fluorescent ligand affinities from both assays were in good agreement with results obtained from radioligand competition binding experiments. The assays yielded real-time kinetic binding data revealing differences in the mechanism of binding for the investigated fluorescent probes. Furthermore, the investigation of various unlabeled M2R ligands yielded pharmacological profiles in accordance with earlier reported data. Taken together, this study showed that BRET- and FA-based binding assays represent valuable alternatives to radioactivity-based methods for screening purposes and for a precise characterization of binding kinetics supporting the exploration of binding mechanisms.
... The development of fluorescent target occupancy probes has allowed target engagement to be visualized in real time in living cells and has provided new insights into the molecular pharmacology of two major families of membrane-bound receptors: GPCRs (47,48) and receptor tyrosine kinases (RTKs) (49). Fluorescent probes are composed of a pharmacophore (be that an agonist or Fluorescence imaging of compound engagement with live cells. ...
... Fluorescence anisotropy (FA) or polarization (FP) has also been used to detect target engagement, but applications for live cells have been limited. In FA/FP, linearly polarized light is used to excite fluorescent molecules that, if stationary, will emit fluorescence with a linear polarization with respect to the light source (47). However, if the fluorescent molecule is free to rotate (e.g., unbound fluorescent probe), its emission will be depolarized. ...
The binding affinity and kinetics of target engagement are fundamental to establishing structure–activity relationships (SARs) for prospective therapeutic agents. Enhancing these binding parameters for operative targets, while minimizing binding to off-target sites, can translate to improved drug efficacy and a widened therapeutic window. Compound activity is typically assessed through modulation of an observed phenotype in cultured cells. Quantifying the corresponding binding properties under common cellular conditions can provide more meaningful interpretation of the cellular SAR analysis. Consequently, methods for assessing drug binding in living cells have advanced and are now integral to medicinal chemistry workflows. In this review, we survey key technological advancements that support quantitative assessments of target occupancy in cultured cells, emphasizing generalizable methodologies able to deliver analytical precision that heretofore required reductionist biochemical approaches.
Expected final online publication date for the Annual Review of Biochemistry, Volume 89 is June 22, 2020. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Intrinsic fluorescence can also be used to study the binding interactions between proteins and potential aggregation inhibitors or chaperone molecules. Changes in fluorescence can indicate alterations in the binding affinity or mode of interaction [29]. Anisotropy measurements in intrinsic fluorescence spectroscopy investigate the rotational mobility of tryptophan residues within aggregated proteins [30]. ...
Aberrant accumulation of protein misfolding can cause aggregation and fibrillation and is one of the primary characteristic features of neurodegenerative diseases. Because they are disordered, misfolded, and aggregated proteins pose a significant setback in drug designing. The structural study of intermediate steps in these kinds of aggregated proteins will allow us to determine the conformational changes as well as the probable pathways encompassing various neurodegenerative disorders. The analysis of protein aggregates involved in neurodegenerative diseases relies on a diverse toolkit of biophysical techniques, encompassing both morphological and non-morphological methods. Additionally, Thioflavin T (ThT) assays and Circular Dichroism (CD) spectroscopy facilitate investigations into aggregation kinetics and secondary structure alterations. The collective application of these biophysical techniques empowers researchers to comprehensively unravel the intricate nature of protein aggregates associated with neurodegeneration. Furthermore, the topics covered in this review have summed up a handful of well-established techniques used for the structural analysis of protein aggregation. This multifaceted approach advances our fundamental understanding of the underlying mechanisms driving neurodegenerative diseases and informs potential therapeutic strategies.
Keywords
Neurodegenerative diseasesProtein aggregationStructural analysis
... Several articles [14][15][16][17][18][19] reported the binding of L-tryptophan with plasma proteins, a crucial step in its general digestion in vivo. Due to their importance as building blocks in living systems, the proximity of free amino acids concentrations to various physiological processes and the metabolic pathways of proteins and peptides, there has been an an increase in interest in their detection across several disciplines, such as clinical chemistry, biochemistry and pharmaceutical chemistry [20][21][22][23]. This is because the 20 natural amino acids serve a vital role in daily living [24][25][26]. ...
The binding of L-tryptophan (L-Trp) and hesperidin (HES) was investigated by UV, fluorescence, synchronous fluorescence, time-resolved fluorescence, Fourier transform infrared (FTIR) spectroscopies, FRET, antibacterial activity, anticancer activity and molecular docking study. The flavonoid glycoside known as hesperidin has been shown to have therapeutic properties for a variety of disorders, including cancer diseases. Its low solubility and bioavailability cause it to be little absorbed, which means that a delivery mechanism is necessary in order for it to reach its therapeutic goal. Fluorescence data revealed that the fluorescence quenching mechanisms of L Trp by hesperidin are all static quenching procedures. Synchronous fluorescence spectroscopy shows the interaction between hesperidin and L-Trp changes the hydrophobicity of the microenvironment of tryptophan (Trp) residues. The anticancer activity effect of hesperidin and L-Trp on human cervical cancer cell lines was assessed using MTT and crystal violet assays.
... The SSF quenching of aromatic amino acids (Matyus et al., 2006;Sudlow et al., 1975) is a proficient approach for investigating the binding affinity and mechanism underlying ligand-protein interactions. The utilization of fluorescence quenching presents numerous advantages in comparison to other biophysical and biochemical methodologies for the examination of protein/ligand interaction (Dos Santos Rodrigues et al., 2023;Sridharan et al., 2014). For instance, quantitative measurements can be obtained as the fluorescence intensity is directly related to the quantity of fluorophores in a sample (Rabbani et al., 2015(Rabbani et al., , 2014. ...
The utilization of BRAF and MEK inhibitors in combination therapy has demonstrated superior outcomes in the treatment of melanoma as compared to monotherapy. In the present scenario, the combination therapy of Encorafenib (ENC), a BRAF inhibitor, and Binimetinib (BINI), a MEK inhibitor, has been identified as one of the most efficacious treatment modalities for this malignancy. Investigations of protein binding, particularly with human serum albumin (HSA), are essential to understand drug performance and enhance therapeutic outcomes. The investigation of the interplay between small molecule drugs and HSA is of paramount importance, given that such interactions can exert a substantial influence on the pharmacokinetics of these therapeutic agents. The present study aims to bridge these lacunae by implementing a comprehensive approach that integrates fluorescence spectroscopy (FS), isothermal titration calorimetry (ITC), far-ultraviolet circular dichroism (far-UV CD), and molecular simulations. Through analysis of the fluorescence quenching of HSA at three distinct temperatures, it was ascertained that the association constants for the complexes formed between drugs and HSA were of the magnitude of 104 M-1. This suggests that the interactions between the compounds and albumin were moderate and comparable. Simultaneously, the investigation of fluorescence indicated a contrasting binding mechanism for the two inhibitors: ENC predominantly binds to HSA through enthalpic interaction, while BINI/HSA is stabilized by entropic contributions. The data obtained was confirmed through experimental procedures conducted using the ITC method. The results of ligand-competitive displacement experiments indicate that ENC and BINI can bind to HSA within subdomain IIA, specifically Sudlow site I. However, far-UV CD studies show that there are no notable alterations in the structure of HSA upon binding with either of the two inhibitors. Ultimately, the results were supported by computational molecular analysis, which identified the key interactions that contribute to the stabilization of the two ligand/HSA complexes.
... 17−20 Each of them relies on monitoring the time-dependent evolution of a signal in response to the binding event. 21 The first strategy revolves around the radio- 22 and spectroscopic 20,23 labeling of ligands and includes techniques such as fluorescent resonance energy transfer 24 and bioluminescence resonance energy transfer. 25 An alternative approach revolves around the exploitation of label-free approaches such as surface plasmon resonance, 26,27 nuclear magnetic resonance, 28 surface acoustic wave methods, 29 and various declinations of isothermal titration calorimetry. ...
The prediction of ligand efficacy has long been linked to thermodynamic properties such as the equilibrium dissociation constant, which considers both the association and the dissociation rates of a defined protein-ligand complex. In the last 15 years, there has been a paradigm shift, with an increased interest in the determination of kinetic properties such as the drug-target residence time since they better correlate with ligand efficacy compared to other parameters. In this article, we present thermal titration molecular dynamics (TTMD), an alternative computational method that combines a series of molecular dynamics simulations performed at progressively increasing temperatures with a scoring function based on protein-ligand interaction fingerprints for the qualitative estimation of protein-ligand-binding stability. The protocol has been applied to four different pharmaceutically relevant test cases, including protein kinase CK1δ, protein kinase CK2, pyruvate dehydrogenase kinase 2, and SARS-CoV-2 main protease, on a variety of ligands with different sizes, structures, and experimentally determined affinity values. In all four cases, TTMD was successfully able to distinguish between high-affinity compounds (low nanomolar range) and low-affinity ones (micromolar), proving to be a useful screening tool for the prioritization of compounds in a drug discovery campaign.
... β-lactoglobulin was a compact globular structure, due to this fact, it has great resistance to breakdown in the gastric environment [145]. Curcumin molecules can not only be easily connected on the surface of proteins through a facile complexation method [112], but also be incorporated into the interior side of protein, because the structure of a protein is highly dependent on the pH medium used [146]. Fluorescence measurements, Raman spectroscopy, and Fourier transform infra-red (FTIR) spectroscopy have shown that the complexation took place through the hydrogen bonding and hydrophobic interactions between CCM and proteins [147]. ...
Curcumin (CCM) is one of the most frequently explored plant compounds with various biological actions such as antibacterial, antiviral, antifungal, antineoplastic, and antioxidant/anti-inflammatory properties. The laboratory data and clinical trials have demonstrated that the bioavailability and bioactivity of curcumin are influenced by the feature of the curcumin molecular complex types. Curcumin has a high capacity to form molecular complexes with proteins (such as whey proteins, bovine serum albumin, β-lactoglobulin), carbohydrates, lipids, and natural compounds (e.g., resveratrol, piperine, quercetin). These complexes increase the bioactivity and bioavailability of curcumin. The current review provides these derivatization strategies for curcumin in terms of biological and physico-chemical aspects with a strong focus on different type of proteins, characterization methods, and thermodynamic features of protein–curcumin complexes, and with the aim of evaluating the best performances. The current literature review offers, taking into consideration various biological effects of the CCM, a whole approach for CCM-biomolecules interactions such as CCM-proteins, CCM-nanomaterials, and CCM-natural compounds regarding molecular strategies to improve the bioactivity as well as the bioavailability of curcumin in biological systems.
... The liquid jet approach also opens up an opportunity to synergistically combine orthogonal biophysical methods such as optical pump-probe and spectroscopy with XFMS. Fluorescence can provide information on changes in size, shape, flexibility, and conformation, as well as knowledge about the proximity of biding partners [40][41][42][43][44] , whereas Raman can fingerprint the changes associated with secondary structure and protein-pigment interactions [45][46][47][48][49][50] . These photoluminescence spectroscopy methods are non-invasive, highly sensitive, and adaptable to the microfluidic configuration of XFMS, and therefore most appropriate to complement solvent accessibility data. ...
X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method’s overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.
... With the rapid development of confocal microscopy, the study of receptor biology using fluorescently labeled ligands has become popular [70,71]. Fluorescently labeled ligands have proven to be very useful in monitoring receptor binding sites, kinetics, regulation, clustering, dynamics, and trafficking [72][73][74][75][76]. Fluorescent ligands are often more attractive than radioligands, since affinity measurements can be performed by flow cytometry and receptor dynamics can be studied both at the cellular and the molecular level. ...
Serotonin is a neurotransmitter that plays a crucial role in the regulation of several behavioral and cognitive functions by binding to a number of different serotonin receptors present on the cell surface. We report here the synthesis and characterization of several novel fluorescent analogs of serotonin in which the fluorescent NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl) group is covalently attached to serotonin. The fluorescent ligands compete with the serotonin1A receptor specific radiolabeled agonist for binding to the receptor. Interestingly, these fluorescent ligands display a high environmental sensitivity of their fluorescence. Importantly, the human serotonin1A receptor stably expressed in CHO-K1 cells could be specifically labeled with one of the fluorescent ligands with minimal nonspecific labeling. Interestingly, we show by spectral imaging that the NBD-labeled ligand exhibits a red edge excitation shift (REES) of 29 nm when bound to the receptor, implying that it is localized in a restricted microenvironment. Taken together, our results show that NBD-labeled serotonin analogs offer an attractive fluorescent approach for elucidating the molecular environment of the serotonin binding site in serotonin receptors. In view of the multiple roles played by the serotonergic systems in the central and peripheral nervous systems, these fluorescent ligands would be useful in future studies involving serotonin receptors.
... The availability of amine-reactive and thiol-reactive derivatives of fluorescent probes enabled site-specific bioconjugation of labels to the proteins and oligonucleotides. This enabled FRET based studies involving single molecule, protein folding, protein-protein interaction, and ligand interaction studies in vitro (Jäger et al. 2005;Jäger et al. 2006;Sridharan et al. 2014). Furthermore, sitespecific labeling followed by FRET measurements enabled the study on intramolecular distances inside the proteins (Karolin et al. 1998). ...
In the last two decades, fluorescent proteins have become one of the most widely studied and exploited protein in biochemistry and cell biology. Fluorescent protein is a protein that upon excitation at low wavelength light emits fluorescence at higher wavelength. Its ability to generate high intracellular visibility together with the stable internal fluorophore and non-invasive measurement technologies made it the finest tool to monitor cellular processes and molecular events in living cells at its normal physiological conditions. Protein engineering and identification of novel fluorescent proteins have resulted in the development of color variants ranging from the blue to near-infrared region of the spectrum. Protein engineering has also lead to the development of highly stable fluorescent proteins with improved photochemical properties and sensing abilities.
The fluorescent proteins have made a strong impact in cell biology research due to its ability to participate in energy transfer interactions, such as Fluorescence resonance energy transfer (FRET) and thus allowing to measure and study molecular-scale distances and dynamics through changes in fluorescence. Development of novel FRET based techniques, FRET sensors and FRET pairs will provide opportunity to understand the cellular processes and dynamics with high precision at nano-scale level. This thesis focusses on FRET studies by developing novel FRET based sensor, novel FRET pairs and analyzing intramolecular FRET. The study also focuses on analyzing the potential of fluorescent proteins in sensing applications outside the cell environment, an area which has not yet been exploited. This was accomplished by protein engineering of fluorescent proteins with specific objectives followed by steady-state and time-resolved fluorescence spectroscopy measurements.
In one of the specific objective, intramolecular FRET in fluorescent proteins was studied by demonstrating FRET between fluorescent protein and conjugated chemical fluorophores whereby FRET occurs from inside to outside of the protein and vice versa. For this study, novel FRET pairs MDCC−Citrine and Citrine− Alexafluor 568 was generated. FRET analyzed using steady-state and ultra-fast time-resolved spectroscopy measurements revealed strong intramolecular FRET with high efficiencies. To my knowledge, this is the first and only study on bidirectional FRET between fluorescent protein and conjugated chemical labels. This study was made possible by genetically engineering Citrine to incorporate cysteine residues on the surface of the protein and this enabled site-specific bioconjugation of the labels to the fluorescent protein.
The surface exposed cysteine on the fluorescent protein was also exploited in this study to generate self-assembled monolayer (SAM) of Citrine on the surface of etched optical fibers (EOF). The conjugation of Citrine to the surface of EOF demonstrated a proof-of-concept for the use of this bio-conjugated protein in in vitro bio-sensing applications. To the best of our knowledge, this is the first and only study on the formation of fluorescent protein SAM on EOF. Steady-state and fluorescence lifetime measurements confirm the formation of SAM on EOF and revealed that the bioconjugation is site-specific and covalent in nature. The study also demonstrates that the proteins retains its photochemical properties on bioconjugation and are stable at physiological conditions.
The engineered surface exposed cysteine was further used in this study for the development of a FRET based redox sensor. This was developed aiming to overcome the disadvantages of the current FRET based redox sensors which includes low FRET efficiency and dynamic range, and to monitor the redox status in bacteria. For the sensor development, fluorescent proteins Citrine and Cerulean were genetically engineered to expose reactive cysteine residues on the protein surface. The proteins were fused using a biotinylation domain as a linker to generate the FRET sensor. The redox titrations and the fluorescence measurements confirmed the redox response and reversibility of the sensor. The FRET sensor exhibited high FRET efficiency and dynamic range in intensity based measurements. Intracellular studies with Escherichia coli revealed the capability of the FRET sensor in detecting real-time redox variations at single cell level.
In the final study, novel FRET pairs were developed aiming at improved fluorescence lifetime dynamic range and high FRET efficiency for the use in fluorescence lifetime imaging microscopy (FLIM) studies. The fluorescent protein with the longest reported fluorescence lifetime NowGFP was used as a FRET donor and various red-fluorescent protein variants were screened for the optimal FRET acceptor. Among the FRET pairs screened, NowGFP-tdTomato and NowGFP-mRuby2 were found to be superior FRET pairs with high lifetime dynamic range and FRET efficiency. NowGFP-tdTomato pair was found to have the highest reported Förster radius and fluorescence lifetime dynamic range for any fluorescent protein based FRET pairs yet used in biological studies.
In summary, we have developed novel FRET based tools and in vitro techniques using fluorescent proteins which can assist in deepening the knowledge on intracellular environment and dynamics, and also in developing novel fluorescent protein based sensors which can be used outside the cellular environment.
... The presence of fluorescent amino acids (e.g. tyrosine [Tyr], tryptophan [Trp], and phenylalanine [Phe]) in protein structure facilitate the fluorimetric titrations (Abdollahpour et al., 2011), which has been applied frequently for ligand binding studies to the proteins with intrinsic fluorescence (Sridharan et al., 2014). The interaction between drugs and plasma proteins possesses a significant impact on the distribution, excretion, metabolism, and pharmacodynamic of drugs. ...
The molecular mechanism and thermodynamic properties of the interaction between diltiazem (DTZ) and human serum albumin (HSA), has been studied in vitro using spectroscopic techniques (UV-Vis, fluorescence, FTIR), and molecular docking methods. The effect of acidic and basic pH, glucose, urea, and metal ions on the DTZ-HSA binding has been investigated as well. According to the results, there is a 1:1 interaction between DTZ and HSA, while the quenching mechanism is static up to 313 K. The apparent binding constant was 2.09 × 10⁶ M⁻¹ that indicates a strong binding between DTZ and HSA. DTZ binding was increased in acidic pH while its binding was slowly decreased in the presence of glucose, urea, and metal ions. Thermodynamic studies showed that DTZ binds to HSA via an exothermic and spontaneous reaction via hydrogen bonding and electrostatic interactions. The conformational alteration of HSA is obvious according to the FTIR study. The site marker competitive study confirmed the binding of DTZ to the warfarin binding site. Molecular docking studies showed that DTZ binds to subdomain IB (−9.22 kcal mol⁻¹) and subdomain IIIA (−9.03 kcal mol⁻¹) with a higher tendency. Also, the results showed that the oxygen and nitrogen atoms of hydroxyl and amino functional groups of DTZ facilitate hydrogen bond formation.
• Highlights
• Strong binding of diltiazem to HSA was studied and confirmed by fluorescence quenching titrations.
• Diltiazem binding to HSA reduces in the presence of metal ions, glucose, urea and alkaline pH.
• Diltiazem binding to HSA is exothermic and spontaneous.
... In the presence of increased catalase concentration, the intensity of the emission maximum of PCB declined, as shown in Fig. 4A. One possible explanation for this finding can be the Förster resonance energy transfer (FRET) effect [39]. This effect is present when the acceptor's absorption spectrum (in this case, the heme in catalase has a small absorption peak at 625 nm) overlaps with the donor's emission spectrum (PCB at 640 nm), suggesting possible dipoledipole interactions between heme and PCB. ...
Phycocyanobilin is a dark blue linear tetrapyrrole chromophore covalently attached to protein subunits of phycobiliproteins present in the light-harvesting complexes of the cyanobacteria Arthrospira platensis (Spirulina “superfood”). It shows exceptional health-promoting properties and emerging use in various fields of bioscience and industry. This study aims to examine the mutual impact of phycocyanobilin interactions with catalase, a life-essential antioxidant enzyme. Fluorescence quenching experiments demonstrated moderate binding (Ka of 3.9 x 10⁴ M⁻¹ at 25°C; n = 0.89) (static type), while van't Hoff plot points to an enthalpically driven ligand binding (ΔG = –28.2 kJ mol⁻¹; ΔH = –41.9 kJ mol⁻¹). No significant changes in protein secondary structures (α-helix content ∼ 22%) and thermal protein stability in terms of enzyme tetramer subunits (Tm ∼ 64°C) were detected upon ligand binding. Alterations in the tertiary catalase structure were found without adverse effects on enzyme activity (∼ 2 x 10⁶ IU/mL). The docking study results indicated that the ligand most likely binds to amino acid residues (Asn141, Arg 362, Tyr369 and Asn384) near the cavity between the enzyme homotetramer subunits not related to the active site. Finally, complex formation protects the pigment from free-radical induced oxidation (bleaching), suggesting possible prolongation of its half-life and bioactivity in vivo if bound to catalase.
... The binding of ligands with albumin is a major determinant of the ligands' distribution in the body. Binding to albumin has a significant effect on ligand dynamics since only unbound ligands are free to interact with other molecules or receptors [6]. ...
... The same authors also used label-free intrinsic tryptophan fluorescence spectroscopy to demonstrate ligand-specific effects on conformational change. There are several reports using this approach that have broadened our understanding of molecular pharmacology (e.g., efficacy and ligand bias) via ligand-specific conformational dynamics with detergent-purified GPCRs (as reviewed in ref [52]). ...
G-protein-coupled receptor (GPCR) drug research is presently hindered by the technical challenges associated with generating purified receptors. Consequently, the application of critical modern discovery technologies has been limited, and the vast untapped opportunity for new GPCR-directed medicines is not being realised. A simple but transformative solution is to purify receptors without removing them from their native phospholipid environment by using polymer lipid particle (PoLiPa) technology, with reagents such as styrene-maleic acid co-polymer (SMA). Compared with contemporary detergent-based and stabilising mutagenesis methods, the PoLiPa approach is simple and generic and, therefore, offers huge advantages, with the potential to revolutionise GPCR research by facilitating the availability of the purified receptors that are required for structural biology, biophysical, and panning technologies.
... 8 However, either FP or FRET is relatively sensitive to autofluorescence of test compounds. 9 An alternative assay should be available to validate the bioactivity of hits that are filtered by FP or FRET screening. ...
Development of Keap1–Nrf2 interaction inhibitors is a promising strategy for the discovery of therapeutic agents against oxidative stress-mediated diseases. Two motifs of Nrf2, ETGE and DLG motif, are responsible for Keap1-Nrf2 binding. Previously, ETGE peptide or ETGE-derived peptide-based approaches were used to detect Keap1-Nrf2 interaction; however, these approaches are not able to monitor Keap1-DLG motif binding. We first report here a novel Enzyme-linked Immunosorbent Assay (ELISA) approach to detect the protein-protein interaction of full length Keap1 and Nrf2. In our assay, the test compounds can target either ETGE or DLG binding site, therefore facilitating the exploration of diverse Keap1-Nrf2 inhibitors. Three FDA-approved drugs, zafirlukast, dutasteride and ketoconazole, were found to inhibit the Keap1-Nrf2 interaction with IC50 of 5.87, 2.81 and 1.67 μM, respectively. Additionally, these three drugs also activated Nrf2 pathway in neuroblasts and lipopolysaccharide (LPS)-challenged mice. The results presented here indicate that the ELISA approach has the capacity to identify Keap1-Nrf2 inhibitors.
... [45][46][47][48] Within this field, the development of novel fluorescent probes to label wild-type receptors is of high importance. [47][48][49][50][51][52][53][54] However, studies 4 on developing KOR selective fluorescent probes have been particularly scarce. Most available probes are non-selective derivatives of wide-spectrum OR antagonists like naloxone or βnaltrexamine. ...
Opioid receptors (ORs) are among the best studied G protein-coupled receptors due to their involvement in neurological disorders and important role in pain treatment. Contrary to the classical monomeric model, indirect evidence suggests that ORs might form dimers, which could be endowed with a distinct pharmacological profile, and, thus, be targeted to develop innovative pharmacological therapies. However, direct evidence for the spontaneous formation of OR dimers in living cells under physiological conditions is missing. Despite a growing interest in the kappa opioid receptor (KOR), KOR-selective fluorescent probes are particularly scarce in literature. Herein, we present the first set of fluorescent KOR-selective probes with antagonistic properties. Two of these were employed in single molecule microscopy (SMM) experiments to investigate KOR homodimerization, localization and trafficking. Our findings indicate that most KORs labelled with the new fluorescent probes are present as apparently freely diffusing monomers on the surface of a simple cell model.
... GPCRs can be activated by different ligands to transduce similar signaling pathways. Previous studies employing various techniques including fluorescence microscopy, crystallography or theoretical modeling have revealed aspects of GPCR signaling transduction at the molecular level [6][7][8][9][10][11]. However, since the receptortransducer complex is highly dynamic and can adopt many conformational transitions, it is virtually impossible to use crystallographic techniques alone to capture all conformational states of either bound or unbound GPCRs. ...
Numbers of activated receptor dictate efficacy of neurotransmitter stimulation. Many PLC coupled receptors activated by ligands elicit canonical downstream Gq/11 pathway to induce endogenous Ca2+ gated chloride channels. The coupling from receptors to effectors was analyzed in Xenopus oocytes expressing genetically modified angiotensin receptor type 1 receptor (AT1R). The latency between ATII binding and Ca2+-induced Cl- current surge was inversely correlated. AT1R activation triggered a chain of chemical reactions, of which the products were playing messengers for subsequent events. Messenger accumulation must rate-limit the agonism. For accurate quantification the speed of ATII triggered the i Cl-. The T-form AT1R-IRK1 fusion exhibits faster induction compared to the M-form. The latency of the recorded none vanished i Cl-, marking the lowest genuine calcium activation, took place at earlier time point by the timer time. The evoked i Cl- however reached similar maximal amplitudes. This kinetic effect raises the possibility to use temporal coding to complement amplitude coding (analogous to FM versus AM radio transmission) for receptor-agonist pairs.
... This increase in availability, coupled with improvements in affinity and selectivity, has widened their suitability for a range of techniques from single-molecule microscopy to high-throughput binding assays, leading to a significant rise in their use. Although there have been several reviews on fluorescent ligands for GPCRs (Sridharan, Zuber, Connelly, Mathew, & Dumont, 2014;Stoddart, Kilpatrick, Briddon, & Hill, 2015), the rapid progress of research in both ligand design and GPCR pharmacology warranted an updated discussion, and therefore, this review will focus on publications in this area from the last eight years. We discuss the diverse ways that fluorescent ligands have been used experimentally to probe emerging paradigms in GPCR pharmacology with an emphasis on ligand-binding kinetics, allosteric modulation, and ligand bias. ...
In recent years several novel aspects of G protein‐coupled receptor (GPCR) pharmacology have been described, which are thought to play a role in determining the in vivo efficacy of a compound. Fluorescent ligands have been used to study many of these, which have also required the development of new experimental approaches. Fluorescent ligands offer the potential to use the same fluorescent probe to perform a broad range of experiments, from single molecule microscopy to in vivo bioluminescence resonance energy transfer. This review provides an overview of the in vitro use of fluorescent ligands in further understanding emerging pharmacological paradigms within the GPCR field, including ligand binding kinetics, allosterism and intracellular signalling, along with the use of fluorescent ligands to study physiologically relevant therapeutics.
... This would allow for study of desensitization, recycling and homo-or oligo-merization of the receptors [19]. A description of the techniques used to study GPCRs was recently reviewed and comprised fluorescence polarization, confocal microscopy, fluorescence correlation spectroscopy (FCS), flow cytometry, FRET and bioluminescence resonance energy transfer (BRET) [18,19,[61][62][63]. Nowadays, thanks to the progress in both synthetic and instrumental techniques, a plethora of fluorophores is available, with a diversity of applicable conjugation strategies that simplify the development of new specific fluorescent ligands [64][65][66]. ...
Research on the adenosine receptors has been supported by the continuous discovery of new chemical probes characterized by more and more affinity and selectivity for the single adenosine receptor subtypes (A1, A2A, A2B and A3 adenosine receptors). Furthermore, the development of new techniques for the detection of G protein-coupled receptors (GPCR) requires new specific probes. In fact, if in the past radioligands were the most important GPCR probes for detection, compound screening and diagnostic purposes, nowadays, increasing importance is given to fluorescent and covalent ligands. In fact, advances in techniques such as fluorescence resonance energy transfer (FRET) and fluorescent polarization, as well as new applications in flow cytometry and different fluorescence-based microscopic techniques, are at the origin of the extensive research of new fluorescent ligands for these receptors. The resurgence of covalent ligands is due in part to a change in the common thinking in the medicinal chemistry community that a covalent drug is necessarily more toxic than a reversible one, and in part to the useful application of covalent ligands in GPCR structural biology. In this review, an updated collection of available chemical probes targeting adenosine receptors is reported.
Intrinsic tryptophan fluorescence spectroscopy is an important tool for examining the effects of molecular crowding and confinement on the structure, dynamics, and function of proteins. Synthetic crowders such as dextran, ficoll, polyethylene glycols, polyvinylpyrrolidone, and their respective monomers are used to mimic crowded intracellular environments. Interactions of these synthetic crowders with tryptophan and the subsequent impact on its fluorescence properties are therefore critically important for understanding the possible interference created by these crowders. In the present study, the effects of polymer and monomer crowders on tryptophan fluorescence were assessed by using experimental and computational approaches. The results of this study demonstrated that both polymer and monomer crowders have an impact on the tryptophan fluorescence intensity; however, the molecular mechanisms of quenching were different. Using Stern–Volmer plots and a temperature variation study, a physical basis for the quenching mechanism of commonly used synthetic crowders was established. The quenching of free tryptophan was found to involve static, dynamic, and sphere-of-action mechanisms. In parallel, computational studies employing Kohn–Sham density functional theory provided a deeper insight into the effects of intermolecular interactions and solvation, resulting in differing quenching modes for these crowders. Taken together, the study offers new physical insights into the quenching mechanisms of some commonly used monomer and polymer synthetic crowders.
The succinate receptor (SUCNR1) has emerged as a potential target for the treatment of various metabolic and inflammatory diseases, including hypertension, inflammatory bowel disease, and rheumatoid arthritis. While several ligands for this receptor have been reported, species differences in pharmacology between human and rodent orthologs have limited the validation of SUCNR1's therapeutic potential. Here, we describe the development of the first potent fluorescent tool compounds for SUCNR1 and use these to define key differences in ligand binding to human and mouse SUCNR1. Starting from known agonist scaffolds, we developed a potent agonist tracer, TUG-2384 (22), with affinity for both human and mouse SUCNR1. In addition, we developed a novel antagonist tracer, TUG-2465 (46), which displayed high affinity for human SUCNR1. Using 46 we demonstrate that three humanizing mutations on mouse SUCNR1, N181.31E, K2697.32N, and G84EL1W, are sufficient to restore high-affinity binding of SUCNR1 antagonists to the mouse receptor ortholog.
One way that an excited molecule can return to the ground state is to transfer the excitation energy to another molecule. This process, resonance energy transfer, plays a particularly important role in photosynthetic organisms. Extended arrays of pigment-protein complexes in the membranes of plants and photosynthetic bacteria absorb sunlight and transfer energy to the reaction centers, where the energy is trapped in electron-transfer reactions [1–3]. In other organisms, photolyases, which use the energy of blue light to repair ultraviolet damage in DNA, contain a pterin or deazaflavin that transfers energy efficiently to a flavin radical in the active site [4]. A similar antenna is found in cryptochromes, which appear to play a role in circadian rhythms [5]. Because the rate of resonance energy transfer depends on the distance between the energy donor and acceptor, the process also is used experimentally to probe intermolecular distances in biophysical systems [6]. Typical applications are to measure the distance between two proteins in a multienzyme complex or between ligands bound at two sites on a protein or to examine the rate at which components from two membrane vesicles mingle in a fused vesicle. An inquiry into the mechanism of resonance energy transfer also provides insight into the electronic coupling that underlies other time-dependent processes such as electron transfer.
This chapter explores the binding interactions that occur between macromolecules, such as proteins and other molecules, often of lower molecular weight. These binding events are the initiators of most of the biochemical reactions observed both in vitro and in vivo. The chapter defines the smaller molecular weight partner in the binding interaction as the ligand and the macromolecular binding partner as the receptor . It provides a brief survey of experimental methods for studying protein–ligand interactions. The chapter then defines the equilibrium between free and ligand‐bound receptor molecules. Over the years several graphical methods have been employed in data analysis for receptor–ligand binding experiments. The chapter reviews some of these methods. Any ligand that binds in the same pocket as the substrate molecule is referred to as an orthosteric ligand.
Luminescence-based techniques play an increasingly important role in all areas of biochemical research, including investigations on G protein-coupled receptors (GPCRs). One quite recent and popular addition has been made by introducing bioluminescence resonance energy transfer (BRET)-based binding assays for GPCRs, which are based on the fusion of nanoluciferase (Nluc) to the N-terminus of the receptor and the occurring energy transfer via BRET to a bound fluorescent ligand. However, being based on BRET, the technique is strongly dependent on the distance/orientation between the luciferase and the fluorescent ligand. Here we describe an alternative strategy to establish BRET-based binding assays for GPCRs, where the N-terminal fusion of Nluc did not result in functioning test systems with our fluorescent ligands (e.g., for the neuropeptide Y Y1 receptor (Y1R) and the neurotensin receptor type 1 (NTS1R)). Instead, we introduced Nluc into their second extracellular loop and we obtained binding data for the fluorescent ligands and reported standard ligands (in saturation and competition binding experiments, respectively) comparable to data from the literature. The strategy was transferred to the angiotensin II receptor type 1 (AT1R) and the M1 muscarinic acetylcholine receptor (M1R), which led to affinity estimates comparable to data from radioligand binding experiments. Additionally, an analysis of the binding kinetics of all fluorescent ligands at their respective target was performed using the newly described receptor/Nluc-constructs.
A conserved intracellular allosteric binding site (IABS) has recently been identified at several G protein‐coupled receptors (GPCRs). Ligands targeting the IABS, so‐called intracellular allosteric antagonists, are highly promising compounds for pharmaceutical intervention and currently evaluated in several clinical trials. Beside co‐crystal structures that laid the foundation for the structure‐based development of intracellular allosteric GPCR antagonists, small molecule tools that enable an unambiguous identification and characterization of intracellular allosteric GPCR ligands are of utmost importance for drug discovery campaigns in this field. Herein, we discuss recent approaches that leverage cellular target engagement studies for the IABS and thus play a critical role in the evaluation of IABS‐targeted ligands as potential therapeutic agents.
Protein aggregation is a process wherein misfolded proteins accumulate to assemble various forms of oligomeric or aggregated structures. This process has been closely related to a variety of human diseases, primarily represented by neurodegenerative diseases. This review provides an overview of fluorescent methods, in particular fluorogenic approaches, that can detect and follow the multiple steps of protein aggregation both in vitro and in vivo . To emphasize the advantage of these approaches, they are compared with other non-fluorescent methods, including western blotting, spectroscopy, and dynamic light scattering. Furthermore, chemical mechanisms that enable the development of these fluorogenic probes are discussed, with a focus on design principles and physical mechanisms underlying fluorescent signal changes that are induced by the process of protein aggregation. In the end, a perspective of challenges and opportunities are discussed to potentiate future method development to study protein aggregation, as well as related biological and biomedical questions.
In the last decades, new evidence on brain structure and function has been acquired by morphological investigations based on synergic interactions between biochemical anatomy approaches, new techniques in microscopy and brain imaging, and quantitative analysis of the obtained images. This effort produced an expanded view on brain architecture, illustrating the central nervous system as a huge network of cells and regions in which intercellular communication processes, involving not only neurons but also other cell populations, virtually determine all aspects of the integrative function performed by the system. The main features of these processes are described. They include the two basic modes of intercellular communication identified (i.e., wiring and volume transmission) and mechanisms modulating the intercellular signaling, such as cotransmission and allosteric receptor–receptor interactions. These features may also open new possibilities for the development of novel pharmacological approaches to address central nervous system diseases. This aspect, with a potential major impact on molecular medicine, will be also briefly discussed.
Chemokines regulate directed cell migration, proliferation and survival and are key components in various physiological and pathological processes. They exert their functions by interacting with seven-transmembrane domain receptors that signal through G proteins (GPCRs). Atypical chemokine receptors (ACKRs) play important roles in the chemokine–receptor network by regulating chemokine bioavailability for the classical receptors through chemokine sequestration, scavenging or transport. Currently, this subfamily of receptors comprises four members: ACKR1, ACKR2, ACKR3 and ACKR4. They differ notably from the classical chemokine receptors by their inability to elicit G protein-mediated signaling, which precludes the use of classical assays relying on the activation of G proteins and related downstream secondary messengers to investigate ACKRs. There is therefore a need for alternative approaches to monitor ACKR activation, modulation and trafficking. This chapter details sensitive and versatile methods based on Nanoluciferase Binary Technology (NanoBiT) and Nanoluciferase Bioluminescence Resonance Energy Transfer (NanoBRET) to monitor ACKR2 and ACKR3 activity through the measurement of β-arrestin and GRK recruitment, and receptor trafficking, including internalization and delivery to early endosomes.
Mas related G-protein-coupled receptor member X2 (MrgX2) has been identified as the crucial receptor in drug induced pseudo-allergic reactions and allergic diseases. In this research, the first type of fluorescent agonists (ZX1, ZX2 and ZX3) for MrgX2 were developed by conjugating environment-sensitive fluorophore coumarin to MrgX2 selective agonists (R)-ZINC-3573. Their environment-sensitive property was confirmed by the dramatically increase of fluorescent intensity after binding to the hydrophobic ligand binding domain MrgX2, which help to overcome the high background signal. Based on these characteristics, they can be used for selective visualization of MrgX2 in living cells even with their own background interference. Among these fluorescent agonists, compound ZX2 possessed splendid spectroscopic properties, outstanding pharmacological activities (EC50 = 0.93 μM, KD = 1.97 μM). And a competitive binding assay was established with ZX2 to analysis the binding affinity of MrgX2 agonists, which shown high coherence with the results of cell membrane chromatography. To our knowledge, these probes are the first fluorescent ligands of MrgX2 with agonistic activity and environment-sensitive property, which is expected to use for the development of MrgX2 molecular pharmacology and serve as a convenient high-throughput screening tool for the drug candidates targeting MrgX2.
Cellular quiescence is a reversible state of cell cycle arrest whereby cells are temporarily maintained in the non‐dividing phase. Inducing quiescence in cancer cells by targeting growth receptors is a treatment strategy to slow cell growth in certain aggressive tumors, which in turn increases the efficacy of treatments such as surgery or systemic chemotherapy. However, ligand interactions with cell receptors induce receptor mediated endocytosis followed by proteolytic degradation, which limits the duration of cellular quiescence. Here, we report the effects of targeted covalent affibody photoconjugation to EGFR on EGFR‐positive MDA‐MB‐468 breast cancer cells. First, covalently conjugating affibodies to cells increased doubling time two‐fold and reduced ERK activity by 30% as compared to cells treated with an FDA‐approved anti‐EGFR antibody Cetuximab, which binds to EGFR noncovalently. The distribution of cells in each phase of the cell cycle was determined, and cells conjugated with the affibody demonstrated an accumulation in the G1 phase, indicative of G1 cell cycle arrest. Finally, the proliferative capacity of the cells was determined by the incorporation of 5‐ethynyl‐2‐deoxyuridine (EdU) and Ki67 Elisa assay, which showed that the percentage of proliferative cells with photoconjugated affibody was half of that found for the untreated control. This article is protected by copyright. All rights reserved.
G protein coupled receptors (GPCRs) comprise a large superfamily of transmembrane receptors responsible for transducing responses to the binding of a wide variety of hormones, neurotransmitters, ions, and other small molecules. There is extensive evidence that GPCRs exist as homo-and hetero-oligomeric complexes, however, in many cases, the role of oligomerization and the extent to which it occurs at low, physiological, levels of receptor expression in cells remain unclear. We report here the use of flow cytometry to detect receptor-receptor interactions based on fluorescence resonance energy transfer between fluorescently-labeled cell-impermeant ligands bound to yeast α-mating pheromone receptors that are members of the GPCR superfamily. A novel procedure was used to analyze energy transfer as a function of receptor occupancy by donor and acceptor ligands. Measurements of loss of donor fluorescence due to energy transfer in cells expressing high levels of receptors were used to calibrate measurements of enhanced acceptor emission due to energy transfer in cells expressing low levels of receptors. The procedure allows determination of energy transfer efficiencies over a 50-fold range of expression of full-length receptors at the surface of living cells without the need to create fluorescent or bioluminescent fusion proteins. Energy transfer efficiencies for fluorescently-labeled derivatives of the receptor agonist α-factor do not depend on receptor expression level and are unaffected by C-terminal truncation of receptors. Fluorescently-labeled derivatives of α-factor that act as receptor antagonists exhibit higher transfer efficiencies than those for labeled agonists. While the approach cannot determine the number of receptors per oligomer, these results demonstrate that ligand-bound, native α-factor receptors exist as stable oligomers in the cell membranes of intact yeast cells at normal physiological expression levels and that the extent of oligomer formation is not dependent on the concentration of receptors in the membrane.
So far, only little is known about the internalization process of the histamine H2 receptor (H2R). One promising approach to study such dynamic processes is the use of agonistic fluorescent ligands. Therefore, a series of carbamoylguanidine-type H2R agonists containing various fluorophores, heterocycles, and linkers (28-40) was synthesized. The ligands were pharmacologically characterized in several binding and functional assays. These studies revealed a significantly biased efficacy (Emax) for some of the compounds, e.g. 32: whereas 32 acted as strong partial (Emax: 0.77, mini-Gs recruitment) or full agonist (Emax: 1.04, [³⁴S]GTPγS binding) with respect to G protein activation, it was only a weak partial agonist regarding β-arrestin1/2 recruitment (Emax: 0.09-0.12) and failed to promote H2R internalization (confocal microscopy). On the other hand, H2R internalization was observed for compounds that exhibited moderate agonistic activity in the β-arrestin1/2 pathways (Emax ≥ 0.22). The presented differently-biased fluorescent ligands are versatile molecular tools for future H2R studies on receptor trafficking and internalization e.g. using fluorescence microscopy.
Despite the fact that transmembrane proteins represent the main therapeutic targets for decades, complete and in‐depth knowledge about their biochemical and pharmacological profiling is not fully available. In this regard, target‐tailored small‐molecule fluorescent ligands are a viable approach to fill in the missing pieces of the puzzle. Such tools, coupled with the ability of high‐precision optical techniques to image with an unprecedented resolution at a single‐molecule level, helped unraveling many of the conundrums related to plasma proteins’ life‐cycle and druggability. Herein, we review the recent progress made during the last two decades in fluorescent ligand design and potential applications in fluorescence microscopy of voltage‐gated ion channels, ligand‐gated ion channels and G‐coupled protein receptors.
G protein‐coupled receptors (GPCRs) are the largest family of membrane receptors and major targets for FDA‐approved drugs. The ability to quantify GPCR expression and ligand binding characteristics in different cell types and tissues is therefore important for drug discovery. The advent of genome editing along with developments in fluorescent ligand design offers exciting new possibilities to probe GPCRs in their native environment. This review provides an overview of the recent technical advances employed to study the localisation and ligand binding characteristics of genome‐edited and endogenously expressed GPCRs.
Fluorescent ligands are versatile tools for the study of G protein-coupled receptors. Depending on the fluorophore, they can be used for a range of different applications, including fluorescence microscopy and bioluminescence or fluorescence resonance energy transfer (BRET or FRET) assays. Starting from phenylpiperazines and indanylamines, privileged scaffolds for dopamine D2-like receptors, we developed dansyl-labeled fluorescent ligands that are well accommodated in the binding pockets of D2 and D3 receptors. These receptors are the target proteins for the therapy for several neurologic and psychiatric disorders, including Parkinson’s disease and schizophrenia. The dansyl-labeled ligands exhibit binding affinities up to 0.44 nM and 0.29 nM at D2R and D3R, respectively. When the dansyl label was exchanged for sterically more demanding xanthene or cyanine dyes, fluorescent ligands 10a-c retained excellent binding properties and, as expected from their indanylamine pharmacophore, acted as agonists at D2R. While the Cy3B-labeled ligand 10b was used to visualize D2R and D3R on the surface of living cells by total internal reflection microscopy, ligand 10a comprising a rhodamine label showed excellent properties in a NanoBRET binding assay at D3R.
The cell membrane possesses an extensive library of proteins, carbohydrates, and lipids that control a significant portion of inter- and intracellular functions, including signaling, proliferation, migration, and adhesion, among others. Augmenting the cell surface composition would open possibilities for advances in therapy, tissue engineering, and probing fundamental cell processes. While genetic engineering has proven effective for many in vitro applications, these techniques result in irreversible changes to cells and are difficult to apply in vivo. Another approach is to instead attach exogenous functional groups to the cell membrane without changing the genetic nature of the cell. This review focuses on more recent approaches of nongenetic methods of cell surface modification through metabolic pathways, anchorage by hydrophobic interactions, and chemical conjugation. Benefits and drawbacks of each approach are considered, followed by a discussion of potential applications for nongenetic cell surface modification and an outlook on the future of the field.
The present study aims at synthesizing three new copper(II) complexes of maltol in the presence of 1,10-phenanthroline-, 2,2′-bipyridine- and 4,4-dibromo-2,2′-bipyridine ligands. The structure of the Cu(II) complexes was characterized by FTIR, CHN analysis and X-ray crystallography. The interaction of the Cu(II) complexes with human serum albumin (HSA) was studied using various methods including fluorescence spectroscopy, UV-visible spectroscopy and molecular docking. The cytotoxic activity of the complexes was investigated using human breast cancer cells (MCF-7), and the results were compared with the activity of cis-platin as a positive control. The data showed that the complex 1, [Cu(mal)(4,4-dibromo-2,2′-bpy)(H2O)]·NO3, has the highest cytotoxicity among the compounds. The experiment indicated that the quenching process of HSA fluorescence by complexes 1–4 and the maltol ligand is a static mechanism. In addition, the results provided information about thermodynamic parameters and the number of binding sites. The high values of Kb show that the complexes can bind strongly with HSA. The results from the UV-Visible spectroscopy studies demonstrated that conformational alterations occurred in the secondary structure of HSA due to binding with the complexes.
Dopamine receptors are G protein‐coupled receptors that have several essential functions in the central nervous system. A better understanding of the regulatory mechanisms of ligand binding to the receptor may open new possibilities to affect the downstream signal transduction pathways. The majority of the available ligand binding assays use either membrane preparations, cell suspensions, or genetically modified receptors, which may give at least partially incorrect understanding of ligand binding. In this study, we implemented an assay combining fluorescence and bright‐field microscopy to measure ligand binding to dopamine D3 receptors in live mammalian cells. For fluorescence intensity quantification from microscopy images, we developed a machine learning‐based user‐friendly software Membrane Tools and incorporated into a data management software Aparecium that has been previously developed in our workgroup. For the experiments, a fluorescent ligand NAPS‐Cy3B was synthesized by conjugating a dopaminergic antagonist N‐(p‐aminophenethyl)spiperone with a fluorophore Cy3B. The subnanomolar affinity of NAPS‐Cy3B makes it a suitable ligand for the characterization of D3 receptors in live HEK293 cells. Using a microplate compatible automated widefield fluorescence microscope, together with the Membrane Tools software, enables the detection and quantification of ligand binding with a high‐throughput. The live cell assay is suitable for the characterization of fluorescent ligand binding and also in the competition experiments for the characterization of novel unlabeled dopaminergic ligands. We propose that this simple yet more native‐like approach is feasible in GPCR research, as it enables the detection of ligand binding in an environment containing more components involved in the signal transduction cascade.
Fluorescence labeled ligands have been gaining importance as molecular tools, enabling receptor-ligand-binding studies by various fluorescence-based techniques. Aiming at red-emitting fluorescent ligands for the hH2R, a series of squaramides labeled with pyridinium or cyanine fluorophores (19-27), was synthesized and characterized. The highest hH2R affinities in radioligand competition binding assays were obtained in the case of pyridinium labeled antagonists 19-21 (pKi: 7.71-7.76) and cyanine labeled antagonists 23 and 25 (pKi: 7.67, 7.11). These fluorescent ligands proved to be useful tools for binding studies (saturation and competition binding as well as kinetic experiments), using confocal microscopy, flow cytometry and high content imaging. Saturation binding experiments revealed pKd values comparable to the pKi values. The fluorescent probes 21, 23 and 25 could be used to localize H2 receptors in HEK cells and to determine the binding affinities of unlabeled compounds.
Mass spectrometry (MS) binding assays are a label-free alternative to radioligand or fluorescence binding assays, so the readout is based on direct mass spectrometric detection of the test ligand. The study presented here describes the development and validation of a highly sensitive, rapid, and robust MS binding assay for the quantification of the binding of the metabotropic glutamate 5 (mGlu5) negative allosteric modulator (NAM), MPEP (2-methyl-6-phenylethynylpyridine) at the mGlu5 allosteric binding site. The LC-ESI-MS/MS (liquid chromatography-electrospray ionization-tandem mass spectrometric) analytical method was established and validated with a deuterated analogue of MPEP as an internal standard. The developed MS binding assay described here allowed for the determination of MS binding affinity estimates that were in agreement with affinity estimates obtained from a tritiated MPEP radioligand saturation binding assay, indicating the suitability of this methodology for determining affinity estimates for compounds that target mGlu5 allosteric binding sites.
Graphical abstract
We have developed a rapid and sensitive single-well dual-parametric method introduced in linked RAS nucleotide exchange and RAS/RAF-RBD interaction assay. RAS mutations are frequent drivers of multiple different human cancers, but the development of therapeutic strategies has been challenging. Traditionally, efforts to disrupt RAS function have focused on nucleotide exchange inhibitors, GTP-RAS interaction inhibitors, and activators increasing GTPase activity of mutant RAS proteins. As the amount of biological knowledge grows, targeted biochemical assays enabling high-throughput screening have become increasingly interesting. We have previously introduced a homogeneous quenching resonance energy transfer (QRET) assay for nucleotide binding studies with RAS and heterotrimeric G proteins. Here, we introduce a novel homogeneous signaling technique called QTR-FRET, which combine QRET technology and time-resolved Förster resonance energy transfer (TR-FRET). The dual-parametric QTR-FRET technique enables the linking of guanine nucleotide exchange factor induced Eu3+-GTP association to RAS, monitored at 615 nm, and subsequent Eu3+-GTP loaded RAS interaction with RAF-RBD-Alexa680 monitored at 730 nm. Both reactions were monitored in a single-well assay applicable for inhibitor screening and real-time reaction monitoring. This homogeneous assay enables separable detection of both nucleotide exchange and RAS/RAF interaction inhibitors using low nanomolar protein concentrations. To demonstrate a wider applicability as a screening and real-time reaction monitoring method, the QTR-FRET technique was also applied for G(i)α GTP-loading and pertussis toxin-catalyzed ADP-ribosylation of G(i)α, for which we synthetized a novel γ-GTP-Eu3+ molecule. The study indicates that the QTR-FRET detection technique presented here can be readily applied to dual-parametric assays for various targets.
A novel approach on fluorescence quenching of tyrosine and l‐tryptophan is presented for spectrofluorimetric determination of aniracetam in drug substances and products. The quenching mechanism was investigated using Stern–Volmer plots and ultraviolet spectra figures of quencher–fluorophore mixtures. Binding constant and stoichiometry were calculated using double‐log plots. The spectrofluorimetric method was optimized for the experimental conditions affecting fluorescence quenching including fluorophore concentration, diluent, and reaction time. Moreover, the pH‐rate profile of aniracetam was studied using simple kinetics and found to be stable within the pH range 5–8. Fluorescence quenching of tyrosine and l‐tryptophan were observed on addition of aniracetam in aqueous medium at pH 5.5–6.5. Aniracetam quenched the fluorescence of tyrosine and l‐tryptophan in the concentration range 1–20 μg/ml and 0.3–20 μg/ml, respectively, with binomial relationships between quenching values (ΔF) and aniracetam concentration. Limits of detection were found to be 0.10 μg/ml for tyrosine–aniracetam and 0.14 μg/ml for l‐tryptophan–aniracetam. Method validation was performed as per ICH guidelines and demonstrated that the developed spectrofluorimetric method was accurate, precise, specific, and suitable for analysis of aniracetam in routine quality control laboratories. All experimental materials and solvents used are eco‐friendly, indicating that the cited spectrofluorimetric procedure is an excellent green method.
P>Background: Albumin was reported to engage nearly 95% of plasma Amyloid β (Aβ) and to reverse Aβ fibril formation in brain.
Objective: Since O-glycosylated LRP family of receptors capture Aβ in brain we compared Aβ binding to electrophoretically purified albumin and to O-glycoproteins AOP1 and AOP2 that adhere noncovalently to plasma albumin.
Methods: Strength of Aβ-protein interaction was measured as fluorescence increase in Fluorescentlabeled Aβ (F-Aβ) resulting from conformational changes. Alternatively, differential segregation of free and protein-bound Aβ in Density Gradient Ultracentrifugation (DGUC) was also examined.
Results: Fluorescence enhancement in F-Aβ was significantly greater by AOP1 and AOP2 than by known Aβ reactants α -synuclein and β -cyclodextrin, but nil by albumin. In DGUC Aβ migrated with the O-glycoproteins but not with albumin. Free O-glycoproteins unlike their albumin-bound forms were blocked by LDL from capturing F-Aβ. Associated albumin did not affect Aβ binding of O-glycoproteins. De-O-glycosylation of AOP1/AOP2 enhanced their Aβ binding showing that peptide sequences at O-glycosylated regions were recognized by Aβ. Unlike albumin, AOP1 and AOP2 were immunologically cross-reactive with LRP. Albumin sample used earlier to report albumin-Aβ interaction contained two O-glycoproteins cross-reactive with human LRP and equal in size to human AOP1 or AOP2.
Conclusion: Unlike albumin, albumin-bound O-glycoproteins, immunologically cross-reactive with LRP, bind plasma Aβ. These O-glycoproteins are potential anti-amyloidogenic therapeutics if they inhibit Aβ aggregation as other Aβ reactants do. Circulating immune complexes of albuminbound O-glycoproteins with O-glycoprotein-specific natural antibodies can bind further to LRP-like membrane proteins and are possible O-glycoprotein transporters to tissues.</P
The calcitonin gene‐related peptide (CGRP) system provides a significant opportunity for the treatment of migraine. However, further understanding of the function of CGRP receptors is required. Fluorescent ligands are valuable probes that enable deeper understanding of peptide‐receptor interactions and receptor function. We herein report the synthesis and biological activity of a potent fluorescent CGRP that allowed direct observation of CGRP receptor internalization in a transfected cell system. The potent activity of this fluorescent CGRP thus enables access to a valuable pharmacological tool for further understanding the CGRP system.
Aim: To propose newer combinations of antibiotics effective against NDM-1-producing bacterial strains. Materials & methods: Antibiotics combinations were tested by checkerboard assay. NDM-1 protein/enzyme was expressed and purified to perform enzyme kinetics, circular dichroism and fluorescence spectroscopic studies. Results: Doripenem-cefoxitin combination and doripenem-tetracycline combination showed synergistic effect toward NDM-1-producing strains. The catalytic efficiency of NDM-1 enzyme was decreased drastically by 96.6% upon doripenem-cefoxitin treatment and by 35.54% after doripenem-tetracycline treatment. Conformational changes were observed in NDM-1 upon combination treatment. Conclusion: NDM-1-producing bacterial strains show resistance to multiple antibiotics but the combination of doripenem-cefoxitin and doripenem-tetracycline are effective against them. The combination of a carbapenem and cephamycin antibiotic is proposed for future treatment options against bacteria-producing NDM-1.
Fluorescence polarization assays in 384-well microtiter plates have been demonstrated. The performance is suitable for high throughput drug screening applications with respect to speed of analysis, displaceable signal, precision, and sensitivity to various reagents. Rank order of potency was maintained relative to ['251]-ligand filtration assays, and the effects of the highly colored compounds, tartrazine and Chicago Sky Blue, were insignificant on the polarization signal up to a concentration of 1 tiM. These attributes suggest that accurate assessment of drug binding can be obtained.
G protein-coupled receptors (GPCRs) comprise the largest family of transmembrane proteins. For GPCR drug discovery, it is important that ligand affinity is determined in the correct cellular environment and preferably using an unmodified receptor. We developed a live cell high-content screening assay that uses a fluorescent antagonist, CA200645, to determine binding affinity constants of competing ligands at human adenosine-A(1) and -A(3) receptors. This method was validated as a tool to screen a library of low molecular weight fragments, and identified a hit with submicromolar binding affinity (K(D)). This fragment was structurally unrelated to substructures of known adenosine receptor antagonists and was optimized to show selectivity for the adenosine-A(3) receptor. This technology represents a significant advance that will allow the determination of ligand and fragment affinities at receptors in their native membrane environment.
Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor® 647), for example, kept high affinities for their cognate receptors. These ligands turn out to be interesting tools to develop FRET-based binding assays. We also used these fluorescent ligands to analyse GPCR oligomerization by measuring FRET between ligands bound to receptor dimers. In contrast with FRET strategies, on the basis of receptor labelling, the ligand-based approach we developed is fully compatible with the study of wild-type receptors and therefore with receptors expressed in native tissues. Therefore, by using fluorescent analogues of oxytocin, we demonstrated the existence of oxytocin receptor dimers in the mammary gland of lactating rats.
G-protein-coupled receptors (GPCRs) constitute the largest family of receptors and major pharmacological targets. Whereas many GPCRs have been shown to form di-/oligomers, the size and stability of such complexes under physiological conditions are largely unknown. Here, we used direct receptor labeling with SNAP-tags and total internal reflection fluorescence microscopy to dynamically monitor single receptors on intact cells and thus compare the spatial arrangement, mobility, and supramolecular organization of three prototypical GPCRs: the β(1)-adrenergic receptor (β(1)AR), the β(2)-adrenergic receptor (β(2)AR), and the γ-aminobutyric acid (GABA(B)) receptor. These GPCRs showed very different degrees of di-/oligomerization, lowest for β(1)ARs (monomers/dimers) and highest for GABA(B) receptors (prevalently dimers/tetramers of heterodimers). The size of receptor complexes increased with receptor density as a result of transient receptor-receptor interactions. Whereas β(1)-/β(2)ARs were apparently freely diffusing on the cell surface, GABA(B) receptors were prevalently organized into ordered arrays, via interaction with the actin cytoskeleton. Agonist stimulation did not alter receptor di-/oligomerization, but increased the mobility of GABA(B) receptor complexes. These data provide a spatiotemporal characterization of β(1)-/β(2)ARs and GABA(B) receptors at single-molecule resolution. The results suggest that GPCRs are present on the cell surface in a dynamic equilibrium, with constant formation and dissociation of new receptor complexes that can be targeted, in a ligand-regulated manner, to different cell-surface microdomains.
Protease-activated receptor 1 (PAR1) is the prototypical member of a family of G-protein-coupled receptors that mediate cellular responses to thrombin and related proteases. Thrombin irreversibly activates PAR1 by cleaving the amino-terminal exodomain of the receptor, which exposes a tethered peptide ligand that binds the heptahelical bundle of the receptor to affect G-protein activation. Here we report the 2.2-Å-resolution crystal structure of human PAR1 bound to vorapaxar, a PAR1 antagonist. The structure reveals an unusual mode of drug binding that explains how a small molecule binds virtually irreversibly to inhibit receptor activation by the tethered ligand of PAR1. In contrast to deep, solvent-exposed binding pockets observed in other peptide-activated G-protein-coupled receptors, the vorapaxar-binding pocket is superficial but has little surface exposed to the aqueous solvent. Protease-activated receptors are important targets for drug development. The structure reported here will aid the development of improved PAR1 antagonists and the discovery of antagonists to other members of this receptor family.
Agonist binding to G protein-coupled receptors is believed to promote a conformational change that leads to the formation of the active receptor state. However, the character of this conformational change which provides the important link between agonist binding and G protein coupling is not known. Here we report evidence that agonist binding to the 2 adrenoceptor induces a conformational change around 125Cys in transmembrane domain (TM) III and around 285Cys in TM VI. A series of mutant 2 adrenoceptors with a limited number of cysteines available for chemical derivatization were purified, site-selectively labeled with the conformationally sensitive, cysteine-reactive fluorophore IANBD and analyzed by fluorescence spectroscopy. Like the wild-type receptor, mutant receptors containing 125Cys and/or 285Cys showed an agonist-induced decrease in fluorescence, while no agonist-induced response was observed in a receptor where these two cysteines were mutated. These data suggest that IANBD bound to 125Cys and 285Cys are exposed to a more polar environment upon agonist binding, and indicate that movements of transmembrane segments III and VI are involved in activation of G protein-coupled receptors.
Ligand recognition of the NK1 receptor (substance P receptor) by peptide agonist and non-peptide antagonist has been investigated
and compared by the use of fluorescent ligands and spectrofluorometric methods. Analogues of substance P (SP) labeled with
the environment-sensitive fluorescent group 5-dimethylaminonaphthalene-1-sulfonyl (dansyl) at either position 3, 8, or 11
or with fluorescein at theN
α position were synthesized and characterized. Peptides modified at the α-amino group or at positions 3 or 11 conserved a relatively
good affinity for NK1 and agonistic properties. Modification at position 8 resulted in an 18,000-fold decrease in affinity.
A fluorescent dansyl analogue of the non-peptide antagonist CP96,345 was prepared and characterized. The quantum yield of
fluorescence for dansyl-CP96,345 was much higher than for any of the dansyl-labeled peptides indicating that the micro-environment
of the binding site is more hydrophobic for the non-peptide antagonist than for the peptide agonists. Comparison of collisional
quenching of fluorescence by the water-soluble hydroxy-Tempo compound showed that dansyl-CP96,345 is buried and virtually
inaccessible to aqueous quenchers, whereas dansyl- or fluoresceinyl-labeled peptides were exposed to the solvent. Anisotropy
of all fluorescent ligands increased upon binding to NK1 indicating a restricted motional freedom. However, this increase
in anisotropy was more pronounced for the dansyl attached to the non-peptide antagonist CP96,345 than for the fluorescent
probes attached to different positions of SP. In conclusion, our data indicate that the environment surrounding non-peptide
antagonist and peptide agonists are vastly different when bound to the NK1 receptor. These results support recent observations
by mutagenesis and cross-linking work suggesting that peptide agonists have their major interaction points in the N-terminal
extension and the loops forming the extracellular face of the NK1 receptor. Our data also suggest that neither the C terminus
nor the N terminus of SP appears to penetrate deeply below the extracellular surface in the transmembrane domain of the receptor.
The β2 adrenergic receptor (β2AR) is a prototypical family A G protein-coupled receptor (GPCR) and an excellent model system for studying the mechanism
of GPCR activation. The β2AR agonist binding site is well characterized, and there is a wealth of structurally related ligands with functionally diverse
properties. In the present study, we use catechol (1,2-benzenediol, a structural component of catecholamine agonists) as a
molecular probe to identify mechanistic differences between β2AR activation by catecholamine agonists, such as isoproterenol, and by the structurally related non-catechol partial agonist
salbutamol. Using biophysical and pharmacologic approaches, we show that the aromatic ring of salbutamol binds to a different
site on the β2AR than the aromatic ring of catecholamines. This difference is important in receptor activation as it has been hypothesized
that the aromatic ring of catecholamines plays a role in triggering receptor activation through interactions with a conserved
cluster of aromatic residues in the sixth transmembrane segment by a rotamer toggle switch mechanism. Our experiments indicate
that the aromatic ring of salbutamol does not activate this mechanism either directly or indirectly. Moreover, the non-catechol
ring of partial agonists does not interact optimally with serine residues in the fifth transmembrane helix that have been
shown to play an important role in activation by catecholamines. These results demonstrate unexpected differences in binding
and activation by structurally similar agonists and partial agonists. Moreover, they provide evidence that activation of a
GPCR is a multistep process that can be dissected into its component parts using agonist fragments.
The insertion of a stable soluble protein into loops of transmembrane proteins has proved to be a successful approach for enhancing their stabilities and crystallization, and may also be useful in contexts where the inserted proteins can modulate or report on the activities of membrane proteins. While the use of T4 lysozyme to replace portions of the third intracellular loops of G protein-coupled receptors (GPCRs) has allowed determination of the structures of members of this important class of receptors, the creation of such fusion proteins generally leads to loss of signaling function of the resulting fusion protein, since the third intracellular loops of GPCRs play critical roles in their interactions with G proteins. We describe here a random screening approach allowing insertion of T4 lysozyme into diverse positions in the third loop of the yeast α-pheromone receptor, a GPCR encoded by the yeast STE2 gene. Insertions were accompanied by varying extents of deletion or duplication of the loop. A set of phenotypic screens allow detection of potentially rare variant receptors that are expressed, bind to agonist and are capable of signal transduction via activation of the cognate G protein. A large fraction of screened full-length receptor variants containing at least partial duplications of the loop on either side of the inserted T4 lysozyme retain the ability to activate the downstream signaling pathway in response to binding of ligand. However, we were unable to identify any receptors with truncated C-termini that retain significant signaling function in the presence of inserted T4 lysozyme. Our results establish the feasibility of creating functional receptors containing insertions of T4 lysozyme in their third intracellular loops.
Neurotensin (NTS) is a 13-amino-acid peptide that functions as both a neurotransmitter and a hormone through the activation of the neurotensin receptor NTSR1, a G-protein-coupled receptor (GPCR). In the brain, NTS modulates the activity of dopaminergic systems, opioid-independent analgesia, and the inhibition of food intake; in the gut, NTS regulates a range of digestive processes. Here we present the structure at 2.8 Å resolution of Rattus norvegicus NTSR1 in an active-like state, bound to NTS(8-13), the carboxy-terminal portion of NTS responsible for agonist-induced activation of the receptor. The peptide agonist binds to NTSR1 in an extended conformation nearly perpendicular to the membrane plane, with the C terminus oriented towards the receptor core. Our findings provide, to our knowledge, the first insight into the binding mode of a peptide agonist to a GPCR and may support the development of non-peptide ligands that could be useful in the treatment of neurological disorders, cancer and obesity.
We describe the design, construction and validation of a fluorescence sensor to measure activation by agonist of the m1 muscarinic cholinergic receptor, a prototypical class I G(q)-coupled receptor. The sensor uses an established general design in which Förster resonance energy transfer (FRET) from a circularly permuted CFP mutant to FlAsH, a selectively reactive fluorescein, is decreased 15-20% upon binding of a full agonist. Notably, the sensor displays essentially wild-type capacity to catalyze activation of Gα(q), and the purified and reconstituted sensor displays appropriate regulation of affinity for agonists by G(q). We describe the strategies used to increase the agonist-driven change in FRET while simultaneously maintaining regulatory interactions with Gα(q), in the context of the known structures of Class I G protein-coupled receptors. The approach should be generally applicable to other Class I receptors which include numerous important drug targets.
The G protein-coupled β2-adrenoreceptor (β2AR) signals through the heterotrimeric G proteins Gs and Gi and β-arrestin. As such, the energy landscape of β2AR-excited state conformers is expected to be complex. Upon tagging Cys-265 of β2AR with a trifluoromethyl probe, 19F NMR was used to assess conformations and possible equilibria between states. Here, we report key differences in β2AR conformational dynamics associated with the detergents used to stabilize the receptor. In dodecyl maltoside (DDM) micelles,
the spectra are well represented by a single Lorentzian line that shifts progressively downfield with activation by appropriate
ligand. The results are consistent with interconversion between two or more states on a time scale faster than the greatest
difference in ligand-dependent chemical shift (i.e. >100 Hz). Given that high detergent off-rates of DDM monomers may facilitate conformational exchange between functional states
of β2AR, we utilized the recently developed maltose-neopentyl glycol (MNG-3) diacyl detergent. In MNG-3 micelles, spectra indicated
at least three distinct states, the relative populations of which depended on ligand, whereas no ligand-dependent shifts were
observed, consistent with the slow exchange limit. Thus, detergent has a profound effect on the equilibrium kinetics between
functional states. MNG-3, which has a critical micelle concentration in the nanomolar regime, exhibits an off-rate that is
4 orders of magnitude lower than that of DDM. High detergent off-rates are more likely to facilitate conformational exchange
between distinct functional states associated with the G protein-coupled receptor.
Allosteric ligands that modulate how G protein-coupled receptors respond to traditional orthosteric drugs are an exciting
and rapidly expanding field of pharmacology. An allosteric ligand for the cannabinoid receptor CB1, Org 27569, exhibits an
intriguing effect; it increases agonist binding, yet blocks agonist-induced CB1 signaling. Here we explored the mechanism behind this behavior, using a site-directed fluorescence labeling
approach. Our results show that Org 27569 blocks conformational changes in CB1 that accompany G protein binding and/or activation,
and thus inhibit formation of a fully active CB1 structure. The underlying mechanism behind this behavior is that simultaneous
binding of Org 27569 produces a unique agonist-bound conformation, one that may resemble an intermediate structure formed
on the pathway to full receptor activation.
Yeast assays for G-protein-coupled receptors have many attractions due to their simplicity, low cost, and lack of endogenous receptors. Since the first report of functional coupling of the human g 2 adrenergic receptor to the yeast pheromone-response pathway in 1990, the technology has developed to a point at which more than 30 heterologous GPCRs are now published to couple. Major breakthroughs have come from an understanding of receptor-G protein interactions, alongside advances in knowledge of the structure of heterotrimeric G proteins. Yeast screens have been used to identify ligands both from compound collections and through the autocrine expression of peptide libraries. Yeast genetics has also been applied to a functional analysis of GPCRs and peptide ligands. In this review we describe the historical development of yeast GPCR assay systems and their current applications.
Recombinant proteins containing four cysteines at the i,i + 1, i + 4, and i + 5 positions of an α helix were fluorescently labeled in living cells by extracellular administration of 4′,5′-bis(1,3,2-dithioarsolan-2-yl)fluorescein.
This designed small ligand is membrane-permeant and nonfluorescent until it binds with high affinity and specificity to the
tetracysteine domain. Such in situ labeling adds much less mass than does green fluorescent protein and offers greater versatility
in attachment sites as well as potential spectroscopic and chemical properties. This system provides a recipe for slightly
modifying a target protein so that it can be singled out from the many other proteins inside live cells and fluorescently
stained by small nonfluorescent dye molecules added from outside the cells.
Single-molecule studies of the conformations of the intact beta(2) adrenergic receptor were performed in solution. Photon bursts from the fluorescently tagged adrenergic receptor in a micelle were recorded. A photon-burst algorithm and a Poisson time filter were implemented to characterize single molecules diffusing across the probe volume of a confocal microscope. The effects of molecular diffusion and photon number fluctuations were deconvoluted by assuming that Poisson distributions characterize the molecular occupation and photon numbers. Photon-burst size histograms were constructed, from which the source intensity distributions were extracted. Different conformations of the beta(2) adrenergic receptor cause quenching of the bound fluorophore to different extents and hence produce different photon-burst sizes. An analysis of the photon-burst histograms shows that there are at least two distinct substates for the native adrenergic membrane receptor. This behavior is in contrast to one peak observed for the dye molecule, rhodamine 6G. We test the reliability and robustness of the substate number determination by investigating the application of different binning criteria. Conformational changes associated with agonist binding result in a marked change in the distribution of photon-burst sizes. These studies provide insight into the conformational heterogeneity of G protein-coupled receptors in the presence and absence of a bound agonist.
Structural analysis of class B G-protein-coupled receptors (GPCRs), cell-surface proteins that respond to peptide hormones, has been restricted to the amino-terminal extracellular domain, thus providing little understanding of the membrane-spanning signal transduction domain. The corticotropin-releasing factor receptor type 1 is a class B receptor which mediates the response to stress and has been considered a drug target for depression and anxiety. Here we report the crystal structure of the transmembrane domain of the human corticotropin-releasing factor receptor type 1 in complex with the small-molecule antagonist CP-376395. The structure provides detailed insight into the architecture of class B receptors. Atomic details of the interactions of the receptor with the non-peptide ligand that binds deep within the receptor are described. This structure provides a model for all class B GPCRs and may aid in the design of new small-molecule drugs for diseases of brain and metabolism.
In response to peptide pheromones, yeast cells prepare themselves for mating; changes include arrest of the cell cycle and induction of transcription. Proteins involved in this signal transduction pathway include the pheromone receptors, subunits of a G protein, protein kinases and DNA-binding proteins. Understanding of this pathway has been facilitated by yeast genetics, which has allowed the genes encoding all of these proteins to be identified and characterized.
Development of CXCR4-specific ligands is an important issue in chemotherapy of HIV infection, cancer metastasis, and rheumatoid arthritis, and numerous potential ligands have been developed to date. However, it is difficult to assess their binding mode and specificity because of uncertainties in the structure of the CXCR4-ligand complexes. To address this problem, we have synthesized fluorophore labeled Ac-TZ14011, which is derived from T140, a powerful CXCR4 antagonist. Binding of Ac-TZ14011 to CXCR4 on the cell membrane was observed by fluorescence microscope, and analysis of the binding data produced IC 50 values of several ligands comparable to those obtained in RI-based assays. This fluorescence-based assay is applicable to explore new pharmacophores of CXCR4-specific ligands with high-throughput screening and also to screening of the other GPCR binding ligands.
The majority of extracellular physiologic signaling molecules act by stimulating GTP-binding protein (G-protein)-coupled receptors (GPCRs). To monitor directly the formation of the active state of a prototypical GPCR, we devised a method to site specifically attach fluorescein to an endogenous cysteine (Cys-265) at the cytoplasmic end of transmembrane 6 (TM6) of the adrenergic receptor (2AR), adjacent to the G-protein-coupling domain. We demonstrate that this tag reports agonist-induced conformational changes in the receptor, with agonists causing a decline in the fluorescence intensity of fluorescein-2AR that is proportional to the biological efficacy of the agonist. We also find that agonists alter the interaction between the fluorescein at Cys-265 and fluorescence-quenching reagents localized to different molecular environments of the receptor. These observations are consistent with a rotation and/or tilting of TM6 on agonist activation. Our studies, when compared with studies of activation in rhodopsin, indicate a general mechanism for GPCR activation; however, a notable difference is the relatively slow kinetics of the conformational changes in the 2AR, which may reflect the different energetics of activation by diffusible ligands.
1 This study examines the cellular localization of α 1 ‐adrenoceptors and demonstrates that binding to intracellular receptive binding sites in native smooth muscle cells may influence the pharmacological profile of agonists or antagonists. The example tissue studied was rat basilar artery.
2 An α 1 ‐adrenoceptor antagonist and fluorescent analogue of prazosin, BODIPY‐FL prazosin (QAPB) allowed visualization, with high resolution, of both plasma membrane and cytosolic binding sites on live native cells, as previously shown in cells harbouring recombinant receptors. QAPB‐associated fluorescence binding was both time‐ and concentration‐ dependent in rat basilar smooth muscle cells and affinity for α 1 ‐adrenoceptors was calculated from specific binding curves as 1.1 nM.
3 Concentration‐dependent binding of QAPB detected in smooth muscle cells dissociated from rat basilar arteries was composed of diffuse and clustered fluorescence. Visually the diffuse component of fluorescence was the more evident up to a concentration of 5 nM QAPB. Confocal visualization of an optical section through the cell showed that the clustered component was located mainly intracellularly. In rat basilar artery smooth muscle cells the intracellular binding sites were located in close proximity to the nuclear membrane.
4 3D models of QAPB‐associated fluorescence demonstrate that a high proportion of effective binding sites are intracellular, showing not only that a high proportion of receptors are located inside the cell but also that in this location they can bind ligands. This has implications for. pharmacological analysis in relation to the consequences of intracellular binding per se and for differential effects upon the pharmacology of particular ligands according to whether they can enter the cell.
The smoothened (SMO) receptor, a key signal transducer in the hedgehog signalling pathway, is responsible for the maintenance of normal embryonic development and is implicated in carcinogenesis. It is classified as a class frizzled (class F) G-protein-coupled receptor (GPCR), although the canonical hedgehog signalling pathway involves the GLI transcription factors and the sequence similarity with class A GPCRs is less than 10%. Here we report the crystal structure of the transmembrane domain of the human SMO receptor bound to the small-molecule antagonist LY2940680 at 2.5 Å resolution. Although the SMO receptor shares the seven-transmembrane helical fold, most of the conserved motifs for class A GPCRs are absent, and the structure reveals an unusually complex arrangement of long extracellular loops stabilized by four disulphide bonds. The ligand binds at the extracellular end of the seven-transmembrane-helix bundle and forms extensive contacts with the loops.
Dissecting Serotonin Receptors
Serotonin receptors are the targets for many widely used drugs prescribed to treat ailments from depression to obesity and migraine headaches (see the Perspective by Palczewski and Kiser ). C. Wang et al. (p. 610 , published online 21 March) and Wacker et al. (p. 615 , published online 21 March) describe crystal structures of two members of the serotonin family of receptors bound to antimigraine medications or to a precursor of the hallucinogenic drug LSD. Subtle differences in the way particular ligands bind to the receptors cause substantial differences in the signals generated by the receptor and the consequent biological responses. The structures reveal how the same ligand can activate one or both of the two main serotonin receptor signaling mechanisms, depending on which particular receptor it binds.
Agonists of seven-transmembrane receptors, also known as G protein-coupled receptors (GPCRs), do not uniformly activate all cellular signalling pathways linked to a given seven-transmembrane receptor (a phenomenon termed ligand or agonist bias); this discovery has changed how high-throughput screens are designed and how lead compounds are optimized for therapeutic activity. The ability to experimentally detect ligand bias has necessitated the development of methods for quantifying agonist bias in a way that can be used to guide structure-activity studies and the selection of drug candidates. Here, we provide a viewpoint on which methods are appropriate for quantifying bias, based on knowledge of how cellular and intracellular signalling proteins control the conformation of seven-transmembrane receptors. We also discuss possible predictions of how biased molecules may perform in vivo, and what potential therapeutic advantages they may provide.
Various fluorescent nucleoside agonists of the A(3) adenosine receptor (AR) were compared as high affinity probes using radioligands and flow cytometry (FCM). They contained a fluorophore linked through the C2 or N(6) position and rigid A(3)AR-enhancing (N)-methanocarba modification. A hydrophobic C2-(1-pyrenyl) derivative MRS5704 bound nonselectively. C2-Tethered cyanine5-dye labeled MRS5218 bound selectively to hA(3)AR expressed in whole CHO cells and membranes. By FCM, binding was A(3)AR-mediated (blocked by A(3)AR antagonist, at least half through internalization), with t(1/2) for association 38min in mA(3)AR-HEK293 cells; 26.4min in sucrose-treated hA(3)AR-CHO cells (K(d) 31nM). Membrane binding indicated moderate mA(3)AR affinity, but not selectivity. Specific accumulation of fluorescence (50nM MRS5218) occurred in cells expressing mA(3)AR, but not other mouse ARs. Evidence was provided suggesting that MRS5218 detects endogenous expression of the A(3)AR in the human promyelocytic leukemic HL-60 cell line. Therefore, MRS5218 promises to be a useful tool for characterizing the A(3)AR.
Interest is increasing in developing fluorescent ligands for characterization of adenosine receptors (ARs), which hold a promise of usefulness in the drug discovery process. The size of a strategically labeled AR ligand can be greatly increased after the attachment of a fluorophore. The choice of dye moiety (e.g. Alexa Fluor 488), attachment point and linker length can alter the selectivity and potency of the parent molecule. Fluorescent derivatives of adenosine agonists and antagonists (e.g. XAC and other heterocyclic antagonist scaffolds) have been synthesized and characterized pharmacologically. Some are useful AR probes for flow cytometry, fluorescence correlation spectroscopy, fluorescence microscopy, fluorescence polarization, fluorescence resonance energy transfer, and scanning confocal microscopy. Thus, the approach of fluorescent labeled GPCR ligands, including those for ARs, is a growing dynamic research field.
During the past few years, crystallography of G protein-coupled receptors (GPCRs) has experienced exponential growth, resulting in the determination of the structures of more than 16 distinct receptors-9 of them in the first half of 2012 alone. Including closely related subtype homology models, this coverage amounts to approximately 12% of the human GPCR superfamily. The adrenergic, rhodopsin, and adenosine receptor systems are also described by agonist-bound active-state structures, including a structure of the receptor-G protein complex for the β2-adrenergic receptor. Biochemical and biophysical techniques, such as nuclear magnetic resonance and hydrogen-deuterium exchange coupled with mass spectrometry, are providing complementary insights into ligand-dependent dynamic equilibrium between different functional states. Additional details revealed by high-resolution structures illustrate the receptors as allosteric machines that are controlled not only by ligands but also by sodium, lipids, cholesterol, and water. This wealth of data is helping redefine our knowledge of how GPCRs recognize such a diverse array of ligands and how they transmit signals 30 angstroms across the cell membrane; it also is shedding light on a structural basis of GPCR allosteric modulation and biased signaling. Expected final online publication date for the Annual Review of Pharmacology and Toxicology Volume 53 is January 06, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
Through binding to parathyroid hormone (PTH), PTH1R interacts with kidney-specific scaffold proteins, including the sodium hydrogen exchanger regulatory factors 1 and 2 (NHERFs), and ezrin. To facilitate in vivo localization, tetramethylrhodamine-labeled PTH (PTH-TMR) was used as a fluorescent probe. In mice, PTH-TMR localizes to luminal surfaces of tubular S1 segments that overlap PTH1R immunostaining, but does not directly overlap with megalin-specific antibodies. PTH-TMR staining directly overlaps with Npt2a in nascent, endocytic vesicles, marking the location of transporter regulation. PKA substrate antibodies display marked staining increases in segments labeled with PTH-TMR, demonstrating a functional effect. In the presence of secondary hyperparathyroidism, PTH-TMR staining is markedly reduced and shifts to co-localizing with megalin. At 15min post-injection, PTH-TMR-labeled vesicles do not co-localize with either NHERF or ezrin, suggesting PTH1R dissociation from the scaffold complex. At the 5min time point, PTH-TMR stains the base of microvilli where it localizes with both NHERF2 and ezrin, and only partially with NHERF1. Strikingly, the bulk of ezrin protein becomes undetectable with the polyclonal, CS3145 antibody, revealing a PTH-induced conformational change in the scaffold. A second ezrin antibody (3C12) is capable of detecting the altered ezrin protein. The CS3145 antibody only binds to the active form of ezrin and fails to recognize the inactive form, while the 3C12 reagent can detect either active or inactive ezrin. Here we show that the PTH1R is part of the ezrin scaffold complex and that acute actions of PTH suggest a rapid inactivation of ezrin in a spatially defined manner.
G-protein-coupled receptors serve as key signal transduction conduits, linking extracellular inputs with diverse cellular responses. These receptors eluded structural characterization for decades following their identification. A landmark structure of rhodopsin provided a basis for structure-function studies and homology modeling, but advances in receptor biology suffered from a lack of receptor-specific structural insights. The recent explosion in GPCR structures confirms some features predicted by rhodopsin-based models, and more importantly, it reveals unexpected ligand-binding modes and critical aspects of the receptor activation process. The new structures also promise to foster studies testing emerging models for GPCR function such as receptor dimerization and ligand-biased signaling.
Fluorescent antagonists for human 5-HT4 receptors were synthesized based on ML10302 1, a potent 5-HT4 receptor agonist and on piperazine analogue 2. These molecules were derived with three fluorescent moieties, dansyl, naphthalimide, and NBD (7-nitrobenz-2-oxa-1,3-diazol-4-yl), through alkyl chains. The synthesized molecules were evaluated in binding assays on the recently cloned human 5-HT4(e) receptor isoform stably expressed in C6 glial cells with [3H]GR113808 as the radioligand. The affinity values depended upon the basal structure together with the alkyl chain length. The derivatives based on ML10302 were more potent ligands than the derivatives based on piperazine analogue. For ML10302-based ligands, dansyl and NBD derivatives attached through a chain length of one carbon atom 17a and 32, respectively, led to affinities close to the affinity of ML10302. The most potent compounds 17a, 28, and 32 produced an inhibition of the 5-HT stimulated cyclic AMP synthesis in the same cellular system with nanomolar Kb values. Fluorescent properties of 17a, 28, and 32 were more particularly studied. Interactions of the fluorescent ligand 28 with the h5-HT4(e) receptor were indicated using h5-HT4(e) receptor transfected C6 glial cell membranes and entire cells. Ligand 28 was also used in fluorescence microscopy experiments in order to label h5-HT4(e) receptor transfected C6 glial cells, and subcellular localization of these receptors was more precisely determined using confocal microscopy.
Three analogues of the α-mating factor pheromone of Saccharomyces cerevisiae containing the 7-nitrobenz-2-oxa-1,3-diazol-4-yl (NBD) group were synthesized that had high binding affinity to the receptor and retained biological activity. The fluorescence emission maximum of the NBD group in [K7(NBD),Nle12]-α-factor was blue shifted by 35 nm compared to buffer when the pheromone bound to its receptor. Fluorescence quenching experiments revealed that the NBD group in [K7(NBD),Nle12]-α-factor bound to the receptor was shielded from collision with iodide anion when in aqueous buffer. In contrast, the emission maximum of NBD in [K7(ahNBD),Nle12]-α-factor or [Orn7(NBD),Nle12]-α-factor was not significantly shifted and iodide anion efficiently quenched the fluorescence of these derivatives when they were bound to receptor. The fluorescence investigation suggests that when the α-factor is bound to its receptor, K7 resides in an environment that has both hydrophobic and hydrophilic groups within a few angstroms of each other.
Biologically inactive, truncated analogues of the Saccharomyces cerevisiae alpha-mating factor (WHWLQLKPGQPMY) either antagonized or synergized the activity of the native pheromone. An amino-terminal truncated pheromone [WLQLKPGQP(Nle)Y] had no activity by itself, but the analogue acted as an antagonist by competing with binding and activity of the mating factor. In contrast, a carboxyl-terminal truncated pheromone [WHWLQLKPGQP] was not active by itself nor did the peptide compete with alpha-factor for binding to the alpha-factor receptor, but it acted as a synergist by causing a marked increase in the activity of alpha-factor. The observation that residues near the amino terminus may be involved in signal transduction whereas those near the carboxyl terminus influence binding allows us to separate binding and signal transduction in the yeast pheromone response pathway. If found for other hormone-receptor systems, synergists may have potential as therapeutic compounds.
We present the synthesis and characterization of the somatostatin receptor-specific peptide H2N-(d-Phe)-cyclo[Cys-Phe-(d-Trp)-Lys-Thr-Cys]-Thr-OH, which is labeled with a carboxylated indodicarbo- and an indotricarbocyanine dye at the N-terminal amino group. The preparation was performed by automated solid-phase synthesis, with subsequent attachment of the cyanine dye and cleavage of the entire conjugate from the resin. The compounds display high molar absorbance and fluorescence quantum yields typical for cyanine dyes and are thus suitable receptor-targeted contrast agents for molecular optical imaging. The ability of these agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, in which the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. In contrast, the corresponding linearized derivative of the sequence H2N-(d-Phe)-Met-Phe-(d-Trp)-Lys-Thr-Met-Thr-OH produced only minimal cell fluorescence, hence confirming the specificity of the cyclic somatostatin analogue. Intracellular localization could be visualized by near-infrared (NIR) fluorescence microscopy. In conclusion, receptor-specific peptides are promising tools for designing site-directed optical contrast agents for use in molecular optical imaging.
A series of fluorescent benzazepine ligands for the arginine-vasopressin V(2) receptor (AVP V(2)R) was synthesized using "Click" chemistry. Their in vitro pharmacological profile at AVP V(2)R, V(1a)R, V(1b)R, and oxytocin receptor was measured by binding assay and functional studies. Compound 9p, labeled with Lissamine Rhodamine B using novel solid-phase organic tagging (SPOrT) resin, exhibited a high affinity for V(2)R (4.0 nM), an excellent selectivity toward V(2)R and antagonist properties. By changing the nature of the dye, DY647 and Lumi4-Tb probes 44 and 47 still display a high affinity for V(2)R (5.6 and 5.8 nM, respectively). These antagonists constitute the first high-affinity selective nonpeptidic fluorescent ligands for V(2)R. They enabled the development of V(2)R time-resolved FRET-based assay readily amenable to high-throughput screening. Taking advantage of their selectivity, these compounds were also successfully involved in the study of V(1a)R-V(2)R dimerization on cell surface.
G protein-coupled receptors (GPCRs) comprise a large protein family of transmembrane receptors involved in many physiological processes. They are engaged in various transduction processes of extracellular signals into intracellular responses. Due to their involvement in numerous diseases they represent an important pharmacological target. Fluorescence correlation spectroscopy (FCS) poses a very sensitive analytical technique well-suited for the investigation of GPCRs. It is minimally invasive and operates on a single molecular level. It further provides detailed pharmacological information on receptor kinetics and quantities of activated receptors on the cell membrane. In addition, FCS allows distinguishing between different receptor states based on different diffusion time constants. In order to be applicable for FCS, the molecule of interest has to be fluorescently labeled. This review focuses on the physical requirements for dyes intended for FCS, their influence on the binding characteristics of coupled ligands and strategies to generate dye labeled ligands, exemplified on GPCR ligands.
By occupying specific surface receptors, adenosine and adenosine analogues modulate neutrophil functions; in particular, functional and biochemical studies have shown that A1 adenosine receptors modulate chemotaxis in response to chemotactic peptides. Until now, the characteristics of the specific agonist binding and the visualization of A1 receptors in human neutrophils have not been investigated. In the present study, we used the agonist [3H] CHA for radioligand binding studies and a CHA-biotin XX probe in order to visualize the A1 binding sites in human neutrophils, ultrastructurally, by conjugation with colloidal gold-streptavidin. [3H] CHA bound A1 adenosine receptors with selectivity and specificity, although with a low binding capacity. Scatchard analysis showed a Kd value of 1.4 ± 0.08 nM and a maximum density of binding sites of 7.1 ± 0.37 fmol/mg of proteins. The good affinity and selectivity of the CHA-biotin XX probe for A1 adenosine receptors allowed us to visualize them, after conjugation with colloidal gold-streptavidin, as electron-dense gold particles on the neutrophil surface and inside the cell. The internalization of the ligand-receptor complex was followed in a controlled temperature system, and occurred through a receptor-mediated pathway. The kinetics of the intracellular trafficking was fast, taking less than 5 min. These data suggest that the CHA-biotin XX-streptavidin-gold complex is a useful marker for the specific labelling of A1 binding sites and to follow the intracellular trafficking of these receptors. J. Cell. Biochem.75:235–244, 1999. © 1999 Wiley-Liss, Inc.
Human M1 muscarinic receptor chimeras were designed (i) to allow detection of their interaction with the fluorescent antagonist pirenzepine labelled with Bodipy [558/568], through fluorescence resonance energy transfer, (ii) to investigate the structure of the N-terminal extracellular moiety of the receptor and (iii) to set up a fluorescence-based assay to identify new muscarinic ligands. Enhanced green (or yellow) fluorescent protein (EGFP or EYFP) was fused, through a linker, to a receptor N-terminus of variable length so that the GFP barrel was separated from the receptor first transmembrane domain by six to 33 amino-acids. Five fluorescent constructs exhibit high expression levels as well as pharmacological and functional properties superimposable on those of the native receptor. Bodipy-pirenzepine binds to the chimeras with similar kinetics and affinities, indicating a similar mode of interaction of the ligand with all of them. From the variation in energy transfer efficiencies determined for four different receptor-ligand complexes, relative donor (EGFP)-acceptor (Bodipy) distances were estimated. They suggest a compact architecture for the muscarinic M1 receptor amino-terminal domain which may fold in a manner similar to that of rhodopsin. Finally, this fluorescence-based assay, prone to miniaturization, allows reliable detection of unlabelled competitors.
We report on an in vivo single-molecule study of the signaling kinetics of G protein-coupled receptors (GPCR) performed using the neurokinin 1 receptor (NK1R) as a representative member. The NK1R signaling cascade is triggered by the specific binding of a fluorescently labeled agonist, substance P (SP). The diffusion of single receptor–ligand complexes in plasma membrane of living HEK 293 cells is imaged using fast single-molecule wide-field fluorescence microscopy at 100 ms time resolution. Diffusion trajectories are obtained which show intra- and intertrace heterogeneity in the diffusion mode. To investigate universal patterns in the diffusion trajectories we take the ligand-binding event as the common starting point. This synchronization allows us to observe changes in the character of the ligand–receptor-complex diffusion. Specifically, we find that the diffusion of ligand–receptor complexes is slowed down significantly and becomes more constrained as a function of time during the first 1000 ms. The decelerated and more constrained diffusion is attributed to an increasing interaction of the GPCR with cellular structures after the ligand–receptor complex is formed.
A review. The practical implementation of many techniques, including DNA and cell prepn. and culture, library selection and clone characterization is addressed. Detailed protocols are given for the most common techniques. Engineering of the fibronectin type III domain mol. recognition scaffold is used as an example, although the translation to other systems should be readily apparent. [on SciFinder(R)]
A new fluorescent -blocker, 9-amino-acridin propranolol (9-AAP), was administered i.v. to rats. Multiple fluorescent 9-AAP binding sites were observed on cardiac muscle cells in frozen sections. Intensity and density of cardiac 9-AAP fluorescence were markedly reduced following pretreatment with ()- and (–)-propranolol but not with (+)-propranolol. Our findings suggest that 9-AAP may label -adrenergic receptor sites in rat myocardium.
Structural studies of human G protein-coupled receptors (GPCRs) have recently been accelerated through the use of a fusion partner that was inserted into the third intracellular loop. Using chimeras of the human β(2)-adrenergic and human A(2A) adenosine receptors, we present the methodology and data for the initial selection of an expanded set of fusion partners for crystallizing GPCRs. In particular, use of the thermostabilized apocytochrome b(562)RIL as a fusion partner displays certain advantages over previously utilized fusion proteins, resulting in a significant improvement in stability and structure of GPCR-fusion constructs.
