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STUDY QUESTION Can a combination of the focussed protein kinase assays and a wide-scale proteomic screen pinpoint novel, clinically relevant players in decidualization in vitro and in vivo? SUMMARY ANSWER Rho-dependent protein kinase (ROCK) activity is elevated in response to the combined treatment with progesterone and 8-Br-cAMP during in vitro decidualization, mirrored by increase of ROCK2 mRNA and protein levels and the phosphorylation levels of its downstream target Cofilin-1 (CFL1) in secretory versus proliferative endometrium. WHAT IS KNOWN ALREADY Decidualization is associated with extensive changes in gene expression profile, proliferation, metabolism and morphology of endometrium, yet only a few underlying molecular pathways have been systematically explored. In vitro decidualization of endometrial stromal cells (ESCs) can be reportedly induced using multiple protocols with variable physiological relevance. In our previous studies, cyclic AMP (cAMP)/cAMP-dependent protein kinase (PKA)/prolactin axis that is classically upregulated during decidualization showed dampened activation in ESCs isolated from polycystic ovary syndrome (PCOS) patients as compared to controls. STUDY DESIGN, SIZE, DURATION In vitro decidualization studies were carried out in passage 2 ESCs isolated from controls (N = 15) and PCOS patients (N = 9). In parallel, lysates of non-cultured ESCs isolated from proliferative (N = 4) or secretory (N = 4) endometrial tissue were explored. The observed trends were confirmed using cryo-cut samples of proliferative (N = 3) or secretory endometrium (N = 3), and in proliferative or secretory full tissue samples from controls (N = 8 and N = 9, respectively) or PCOS patients (N = 10 for both phases). PARTICIPANTS/MATERIALS, SETTING, METHODS The activities of four target kinases were explored using kinase-responsive probes and selective inhibitors in lysates of in vitro decidualized ESCs and non-cultured ESCs isolated from tissue at different phases of the menstrual cycle. In the latter lysates, wide-scale proteomic and phosphoproteomic studies were further carried out. ROCK2 mRNA expression was explored in full tissue samples from controls or PCOS patients. The immunofluorescent staining of phosphorylated CFL1 was performed in full endometrial tissue samples, and in the in vitro decidualized fixed ESCs from controls or PCOS patients. Finally, the cellular migration properties were explored in live in vitro decidualized ESCs. MAIN RESULTS AND THE ROLE OF CHANCE During in vitro decidualization, the activities of PKA, protein kinase B (Akt/PKB), and ROCK are increased while the activity of casein kinase 2 (CK2) is decreased; these initial trends are observable after 4-day treatment (P LARGE SCALE DATA Proteomic and phosphoproteomic data are available via ProteomeXchange with identifier PXD026243. LIMITATIONS, REASONS FOR CAUTION The number of biological samples was limited. The duration of protocol for isolation of non-cultured ESCs from tissue can potentially affect phosphorylation pathways in cells, yet the possible artefacts were minimized by the identical treatment of proliferative and secretory samples. WIDER IMPLICATIONS OF THE FINDINGS The study demonstrated the benefits of combining the focussed kinase activity assay with wide-scale phosphoproteomics and showed the need for detailed elaboration of the in vitro decidualization protocols. ROCK was identified as the novel target of interest in decidualization, which requires closer attention in further studies—including the context of decidualization-related subfertility and infertility. STUDY FUNDING/COMPETING INTEREST(S) This study was funded by the Estonian Ministry of Education and Research, and the Estonian Research Council (PRG1076, PRG454, PSG230 and PSG608), Enterprise Estonia (EU48695), Horizon 2020 innovation grant (ERIN, Grant no. EU952516) of the European Commission, the COMBIVET ERA Chair, H2020-WIDESPREAD-2018-04 (Grant agreement no. 857418), the Academy of Finland (Project grants 315921 and 321763), the Finnish Medical Foundation and The Sigrid Juselius Foundation. The authors confirm that they have no conflict of interest with respect to the content of this article.

Studying mechanisms of receptor-ligand interactions has remained challenging due to several limitations of different measurement methods. Here we present a total internal reflection fluorescence microscopy-based method that maintains the right balance between retaining the receptors in the natural lipid environment, sufficient throughput for ligand screening, high sensitivity, and offering more detailed view into the ligand-binding process. The novel method combines G protein-coupled receptor display in budded baculovirus particles and the immobilization of the particles to a functionalized coverslip. We adapted and validated the functionalized coverslip preparation process to achieve selective immobilization of budded baculovirus particles. The selectivity of budded baculovirus immobilization was validated with budded baculovirus particles displaying either Frizzled 6 receptors labeled with mCherry or neuropeptide Y Y1 receptors. To scale the system for ligand binding assays, we developed both open-source multiwell systems and image analysis software SPOTNIC for flexible assay design. The neuropeptide Y Y1 receptor was used for further receptor-ligand binding studies with high-affinity TAMRA labeled fluorescent ligand UR-MC026. The affinities of the fluorescent ligand and four unlabeled ligands (BIBO3304, UR-MK299, PYY, pNPY) were obtained with the developed method and followed a similar trend with both the parallel measurements with fluorescence anisotropy method and the data published earlier. The novel method could be extended for various advanced assays utilizing multidimensional detection modes, integrating super-resolution methods for single molecule detection and microfluidic devices for kinetic measurements.

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 membrane fluorescence intensity quantification from microscopy images, we developed a machine learning-based user-friendly software membrane tools and incorporated it 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 screening 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.

Melanocortin-4 receptors (MC4 R) are unique among G-protein-coupled receptors (GPCRs) as they have endogenous ligands that can exhibit inverse agonistic properties in the case of elevated basal activity. It is known that the constitutive activity of GPCRs strongly affects the ligand-dependent physiological responses, but little is known about these regulatory mechanisms. Since several metal ions have been shown to be important modulators of the signal transduction of GPCRs, we hypothesized that metal ions regulate the basal activity of MC4 Rs. Implementation of a fluorescence anisotropy assay and novel redshifted fluorescent peptides enabled kinetic characterization of ligand binding to MC4 R expressed on budded baculoviruses. We show that Ca2+ is required for high-affinity ligand binding, but Zn2+ and Cu2+ in the presence of Ca2+ behave as negative allosteric modulators of ligand binding to MC4 R. FRET-based cAMP biosensor was used to measure the activation of MC4 R stably expressed in CHO-K1 cells. At low micromolar concentrations, Zn2+ caused MC4 R-dependent activation of the cAMP pathway, whereas Cu2+ reduced the activity of MC4 R even below the basal level. These findings indicate that at physiologically relevant concentrations can Zn2+ and Cu2+ function as MC4 R agonists or inverse agonists, respectively. This means that depending on the level of constitutive activity induced by Zn2+ ions, the pharmacological effect of orthosteric ligands of MC4 R can be switched from a partial to an inverse agonist. OPEN SCIENCE BADGES: This article has received a badge for *Open Materials* because it provided all relevant information to reproduce the study in the manuscript. More information about the Open Science badges can be found at https://cos.io/our-services/open-science-badges/.

Sigma-1 receptor (Sig1R) is an endoplasmic reticulum (ER)-related membrane protein, that forms heteromers with other cellular proteins. As the mechanism of action of this chaperone protein remains unclear, the aim of the present study was to detect and analyze the intracellular dynamics of Sig1R in live cells using super-resolution imaging microscopy. For that, the Sig1R-yellow fluorescent protein conjugate (Sig1R-YFP) together with fluorescent markers of cell organelles were transfected into human ovarian adenocarcinoma (SK-OV-3) cells with BacMam technology. Sig1R-YFP was found to be located mainly in the nuclear envelope and in both tubular and vesicular structures of the ER but was not detected in the plasma membrane, even after activation of Sig1R with agonists. The super-resolution radial fluctuations approach (SRRF) performed with a highly inclined and laminated optical sheet (HILO) fluorescence microscope indicated substantial overlap of Sig1R-YFP spots with KDEL-mRFP, slight overlap with pmKate2-mito and no overlap with the markers of endosomes, peroxisomes, lysosomes, or caveolae. Activation of Sig1R with (+)-pentazocine caused a time-dependent decrease in the overlap between Sig1R-YFP and KDEL-mRFP, indicating that the activation of Sig1R decreases its colocalization with the marker of vesicular ER and does not cause comprehensive translocations of Sig1R in cells.

Tumor extracellular matrix (ECM) is a high-capacity and genetically stable target for the precision delivery of affinity ligand-guided drugs and imaging agents. Recently, we developed a PL1 peptide (sequence: PPRRGLIKLKTS) for systemic targeting of malignant ECM. Here we map the dynamics of PL1 binding to its receptors Fibronectin Extra Domain B (FN-EDB) and Tenascin C C-isoform (TNC-C) by computational modeling and cell-free binding studies on mutated receptor proteins, and study cellular binding and internalization of PL1 nanoparticles in cultured cells. Molecular dynamics simulation and docking analysis suggested that the engagement of PL1 peptide with both receptors is primarily driven by electrostatic interactions. Substituting acidic amino acid residues with neutral amino acids at predicted PL1 binding sites in FN-EDB (D52N-D49N-D12N) and TNC-C (D39N-D45N) resulted in the loss of binding of PL1 nanoparticles. Remarkably, PL1-functionalized nanoparticles (NPs) were not only deposited on the target ECM but bound the cells and initiated a robust cellular uptake via a pathway resembling macropinocytosis. Our studies establish the mode of engagement of the PL1 peptide with its receptors and suggest applications for intracellular delivery of nanoscale payloads. The outcomes of this work can be used for the development of PL1-derived peptides with improved stability, affinity and specificity for precision targeting of the tumor ECM and malignant cells.One Sentence SummaryPL1 peptide is recruited to the acidic surfaces on oncofetal fibronectin EDB and tenascin C-C isoform, triggering cellular uptake of PL1-functionalized nanoparticles.

Cyclic adenosine monophosphate (cAMP) serves as a second messenger for numerous G-protein-coupled receptors. Changes in cellular cAMP levels reflect the biological activity of various GPCR-specific agents, including protein hormones. cAMP biosensors based on detection of Förster-type resonance energy transfer (FRET) offer unique advantages including the ratiometric nature of measurement, adjustable affinity toward detected molecule, capability of monitoring kinetics of cAMP release, and compatibility with the multi-well format and fluorescence plate reader platforms. In this chapter, we introduce the optimized version of the previously reported method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system. As a practical challenge, we address the applicability of the designed assay for screening of biological activity of human hormones, including human chorionic gonadotropin (hCG) bearing different posttranslational modifications.

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC

Cyclic adenosine monophosphate (cAMP) serves as a second messenger for numerous G-protein-coupled receptors. Changes in cellular cAMP levels reflect the biological activity of various GPCR-specific agents, including protein hormones. cAMP biosensors based on detection of Forster-type resonance energy transfer (FRET) offer unique advantages including the ratiometric nature of measurement, adjustable affinity toward detected molecule, capability of monitoring kinetics of cAMP release, and compatibility with the multi-well format and fluorescence plate reader platforms. In this chapter, we introduce the optimized version of the previously reported method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system. As a practical challenge, we address the applicability of the designed assay for screening of biological activity of human hormones, including human chorionic gonadotropin (hCG) bearing different posttranslational modifications.

During the past decade, fluorescence methods have become valuable tools for characterizing ligand binding to G protein-coupled receptors (GPCRs). However, only a few of the assays enable studying wild-type receptors and monitor the ligand binding in real time. One of the approaches that is inherently suitable for this purpose is the fluorescence anisotropy (FA) assay. In the FA assay, the change of ligand's rotational freedom connected with its binding to the receptor can be monitored with a conventional fluorescence plate reader equipped with suitable optical filters. To achieve the high receptor concentration required for the assay and the low autofluorescence levels essential for reliable results, budded baculoviruses that display GPCRs on their surfaces can be used. The monitoring process generates a substantial amount of kinetic data, which is usually stored as a proprietary file format limiting the flexibility of data analysis. To solve this problem, we propose the use of the data curation software Aparecium ( http://gpcr.ut.ee/aparecium.html ), which integrates experimental data with metadata in a Minimum Information for Data Analysis in Systems Biology (MIDAS) format. Aparecium enables data export to different software packages for fitting to suitable kinetic or equilibrium models. A combination of the FA assay with the novel data analysis strategy is suitable for screening new active compounds, but also for modeling complex systems of ligand binding to GPCRs. We present the proposed approach using different fluorescent probes and assay types to characterize ligand binding to melanocortin 4 (MC4) receptor.

Extracellular vesicles (EVs), including exosomes and microvesicles (

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 D

Extracellular matrix in solid tumors has emerged as a specific, stable, and abundant target for affinity-guided delivery of anticancer drugs. Here we describe the homing peptide that interacts with the C-isoform of Tenascin-C (TNC-C) upregulated in malignant tissues. TNC-C binding PL3 peptide (amino acid sequence: AGRGRLVR) was identified by in vitro biopanning on recombinant TNC-C. Besides TNC-C, PL3 interacts via its C-end Rule (CendR) motif with cell-and tissue penetration receptor neuropilin-1 (NRP-1). Functionalization of iron oxide nanoworms (NWs) and metallic silver nanoparticles (AgNPs) with PL3 peptide increased tropism of systemic nanoparticles towards glioblastoma (GBM) and prostate carcinoma xenograft lesions in nude mice (eight and five-fold respectively). Treatment of glioma-bearing mice with proapoptotic PL3-guided NWs improved the survival of the mice, whereas treatment with untargeted particles had no effect. PL3-coated nanoparticles were found to accumulate in TNC-C and NRP-1-positive areas in clinical tumor samples, suggesting a translational relevance. The systemic tumor-targeting properties and binding of PL3-NPs to the clinical tumor sections, suggest that the PL3 peptide may have applications as a targeting moiety for the selective delivery of imaging and therapeutic agents to solid tumors.

Dopamine receptors, which belong to the family of G protein-coupled receptors, are very substantial regulators in the brain and therefore important targets in drug discovery. Radioligand binding assay has been the method of choice for screening novel dopaminergic drugs for decades. We demonstrate that fluorescent ligand BodipyFL-SKF83566 binding to dopamine D1 receptors expressed in budded baculovirus particles can be characterized with fluorescent anisotropy (FA) based assay and that this is a valuable alternative to the radioligand binding assay. High binding affinity (KD = 5.2 nM) and fast association and dissociation kinetics (half-lives 40 s and 70 s, respectively) make BodipyFL-SKF83566 a suitable fluorescent ligand for this type of experiments. Good correlation between pKi values of 11 different dopaminergic ligands determined in competition binding experiments with radioligand [3H]SCH23390 or BodipyFL-SKF83566 (R2 = 0.96, slope not significantly different from unity) further validates the FA assay. In addition to competitor's affinity, the method also enables to quantify the apparent pIC50 values in time and hence kinetic properties of an unlabeled ligand can be estimated from the same competition binding experiment. Due to the homogenous nature of the FA assay reactions can be monitored in real time without any risk of precipitation of receptors in budded baculovirus particles. Despite the lack of coupled G proteins, the proposed novel assay system allows on-line monitoring of ligand binding to dopamine D1 receptors that could be easily applicable in early drug screening.

Melanocortin 4 (MC4) receptors are important drug targets as they regulate energy homeostasis, eating behaviour and sexual functions. The ligand binding process to these G protein-coupled receptors is subject to considerable complexity. Different steps in the complex dynamic regulation can be characterized by ligand binding kinetics. Optimization of these kinetic parameters in terms of on-rate and residence time can increase the rapid onset of drug action and reduce off-target effects. Fluorescence anisotropy (FA) is one of the homogeneous fluorescence-based assays that enable continuous online monitoring of ligand binding kinetics. FA has been implemented for the kinetic study of melanocortin MC4 receptors expressed on budded baculoviruses. However, the slow dissociation of the fluorescently labelled peptide NDP-α-MSH does not enable reaching equilibrium nor enable more in-depth study of the binding mechanisms. To overcome this problem, two novel red-shifted fluorescent ligands were designed. These cyclized heptapeptide derivatives (UTBC101 and UTBC102) exhibited nanomolar affinity toward melanocortin MC4 receptors but had relatively different kinetic properties. The dissociation half-lives of UTBC101 (τ1/2=160 min) and UTBC102 (τ1/2=7 min) were shorter compared to that what was previously reported for Cy3B-NDP-α-MSH (τ1/2=224 min). The significantly shorter dissociation half-life of UTBC102 enables equilibrium in screening assays, whereas the higher affinity of UTBC101 helps to resolve a wider range of competitor potencies. These two ligands are suitable for further kinetic screening of novel melanocortin MC4 receptor specific ligands and could complement each other in these studies.

Glyphosate is the most widespread herbicide and its global use is steadily increasing. Although glyphosate is considered to have low toxicity, its wide application has raised concerns about its effects on human health. The extensive use of glyphosate has risen a need of its continuous monitoring in drinking and surface waters to assure in accordance with the set standards. Within the present study, we have developed a novel assay for the on-site detection of glyphosate by combining flow-through technology with the high specificity of immunorecognition. The proposed biosensing system was based on the detection of fluorescence signal generated by the quantitative replacement of glyphosate in antigen-antibody complex with IgY-type anti-glyphosate antibodies on microbeads by synthetic 5-carboxytetramethylrhodamine (5-TAMRA) conjugated glyphosate. The working range of this assay was in low millimolar range and the time required for glyphosate detection around 0.5 h. The applicability of the immunoassay for glyphosate detection in surface water was tested and the biosensor results were validated with high-performance liquid chromatography.

We recently identified a tumor-homing peptide (mUNO, sequence: “CSPGAK”) that specifically interacts with mouse CD206 to target CD206/MRC1-expressing tumor-associated macrophages in mice. Here, we report studies on the binding of mUNO to human recombinant CD206 (hCD206) and on modeling the mUNO/hCD206 interaction by computational analysis. Fluorescence anisotropy analysis demonstrated that fluorophore-labeled mUNO interacts with hCD206. Microsecond time-scale molecular dynamics simulations and docking predictions showed that mUNO binds to a newly identified epitope between C-type lectin domains 1 and 2. The physical mechanisms that contribute to the docking interactions of mUNO include electrostatic interactions, aromatic interactions, and hydrogen bonds. We also demonstrate the selectivity of FAM-mUNO for CD206+-cultured human macrophages. The peptide mUNO appears to be the first ligand capable of interacting with this epitope of hCD206, for which no ligands have been reported. Our study has implications for targeting human M2-like tumor-associated macrophages, a subpopulation of immune cells with a major protumoral role.

Ligand binding dynamics and the concept of drug-target residence time are essential factors in the development of novel drugs. Conventional ligand binding assays, which usually collect end-point data, do not provide abundant information regarding the ligand binding kinetics. Therefore, novel methods that allow on-line monitoring of ligand binding processes have to be developed and implemented for drug discovery studies. In this study, we provide a short overview of novel possibilities to characterize ligand binding dynamics to different G protein-coupled receptors (GPCRs). Special attention has been paid to the ligand binding to melanocortin 4 receptors and to the development of a fluorescence anisotropy-based assay system using receptors in budded baculovirus particles. It has been shown that ligand binding to melanocortin 4 receptors occurs to tandemly arranged interconnecting ligand binding sites and that the conventional equilibrium usually cannot be achieved in this system. Therefore, the apparent potencies of the same ligand may differ by up to four orders of magnitude, depending on the experimental conditions and the reporter ligand used.

Quality of bioactive surface is crucial for achieving the required sensitivity and selectivity of biosensing systems. Numerous methods are available for the characterization of metal-coated surfaces, but only a few to test the efficacy of biomaterial immobilization and the level of non-specific binding. Herewith we propose to use total internal reflection fluorescence (TIRF) microscopy for the characterization of the surface analyte recognition capacity. Biomolecules were bound onto titanium/gold covered glass using three different self-assembled monolayers (SAM). The surfaces with attached antibodies were evaluated using the specific binding of fluorophore-labeled secondary antibodies and visualized with TIRF. Among studied SAMs, aminothiol layers with glutaraldehyde coupling demonstrated high binding capacity along with excellent homogeneity indicating their suitability for applications in biosensors.

A Forster resonance energy transfer (FRET)-based cyclic adenosine monophosphate (cAMP) biosensor was used to study the activation of the mutual receptor for human luteinizing hormone (hLH) and human chorionic gonadotropin (hCG) in living cells stably expressing the shared recombinant receptor for these hormones. This work demonstrates the applicability of a cAMP biosensor-based assay for the study of receptor-mediated signal transduction and the characterization of the biological activities of gonadotropins and their isoforms. The importance of this approach lies in the fact that only the active portion of the total hormone concentration elicits a measurable response. No significant differences in early receptor activation profiles were detected for either of the tested recombinant gonadotropins. However, the subsequent physiological regulations mediated by these hormones may be more complex. The ability to detect biologically active hormones at low concentrations forms the basis for the future quantifica...

Tumor-associated macrophages (TAMs) expressing the multi-ligand endocytic receptor mannose receptor (CD206/MRC1) contribute to tumor immunosuppression, angiogenesis, metastasis, and relapse. Here, we describe a peptide that selectively targets MRC1-expressing TAMs (MEMs). We performed in vivo peptide phage display screens in mice bearing 4T1 metastatic breast tumors to identify peptides that target peritoneal macrophages. Deep sequencing of the peptide-encoding inserts in the selected phage pool revealed enrichment of the peptide CSPGAKVRC (codenamed “UNO”). Intravenously injected FAM-labeled UNO (FAM-UNO) homed to tumor and sentinel lymph node MEMs in different cancer models: 4T1 and MCF-7 breast carcinoma, B16F10 melanoma, WT-GBM glioma and MKN45-P gastric carcinoma. Fluorescence anisotropy assay showed that FAM-UNO interacts with recombinant CD206 when subjected to reducing conditions. Interestingly, the GSPGAK motif is present in all CD206-binding collagens. FAM-UNO was able to transport drug-loaded nanoparticles into MEMs, whereas particles without the peptide were not taken up by MEMs. In ex vivo organ imaging, FAM-UNO showed significantly higher accumulation in sentinel lymph nodes than a control peptide. This study suggests applications for UNO peptide in diagnostic imaging and therapeutic targeting of MEMs in solid tumors.

Binding of fluorescent ligands (tracers) to their target receptors can be directly monitored over time, as the binding of a low-molecular-weight (LMW) tracer to a larger particle causes an increase of fluorescence anisotropy (FA). The combination of bright fluorophores, tracers with low nonspecific binding, and budded baculovirus particles (BVPs) for overexpression of G protein-coupled receptors (GPCRs) ensures a high signal-to-noise ratio in FA assays. The obtained data enable quantitative assessment of equilibrium binding and kinetic parameters for both the tracer and competing compounds as well as an estimation of the receptor concentration. FA assays have clear potential for implementation in drug screening systems, but also in studies of ligand-binding mechanisms for particular GPCRs.

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass

Implementation of fluorescence methods in studies of ligand binding to their receptors opens new possibilities to characterise these processes. One of the potential approaches is the detection of changes of the fluorescence anisotropy (FA) and/or total fluorescence intensity (TFI) signals upon binding reaction. However, to achieve significant changes in the FA/TFI signal, some requirements need to be met – the concentration of receptor binding sites as well as the dissociation constant of the interaction should be in the same order as the fluorescent ligand’s concentration. We have used FA assay to investigate ligand binding properties to Melanocortin 4 (MC4) receptor [1]. Implementation of budded baculoviruses (BBV) that display G protein-coupled receptors on their surfaces significantly increased sensitivity and temporal stability of this assay [2]. For the first time we demonstrate the applicability of BBV experimental setup to study Y1 receptor system. Here we used TAMRA-PYY, an Y1 receptor specific fluorescent peptide ligand, as a reporter ligand. Besides real-time monitoring of FA signal changes, up to 5 fold decrease in TFI signal was observed within TAMRA-PYY binding to the Y1 receptor. Pharmacological characterization of Y1 receptors with receptor-specific unlabelled ligands gave the rank order of potencies consistent with previously reported values. Additionally, allosteric heterogeneous interactions were revealed as koff values of TAMRA-PYY differed more than 7 times depending on the nature of dissociation initiated ligand. These observations provide evidence for similar allosteric receptor-ligand binding mechanism as previously shown for MC4 receptors [3].

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors. The change in cellular cAMP level has widely been used to estimate the biological activity of various GPCR-specific agents. Förster resonance energy transfer (FRET) biosensors have been around for almost 10 years and became increasingly popular for cAMP detection. Ratiometric sensitized emission assay of a FRET biosensor can easily be implemented on fluorescence plate reader platforms. For such assays a considerable amount of cells expressing the desired biosensor is needed. A method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system is the subject of this chapter.

Despite the availability of numerous conceptually different approaches for the characterization of ligand-receptor interactions, there remains a great requirement for complementary methods that are suitable for kinetic studies, especially for the characterization of membrane protein systems and G protein-coupled receptors (GPCRs) in particular. One of the potential approaches that inherently fits well for this purpose is fluorescence anisotropy (FA), a method that allows continuous monitoring of ligand binding processes and characterization of ligand binding dynamics. However, significant changes in FA signal of fluorescently labeled ligands can be detected only if the ratio of bound to free fluorescent ligand portions is altered, which means that receptor and ligand concentrations have to be comparable. As most of the GPCRs are normally present at relatively low concentrations in native tissues and conventional receptor preparations from overexpressed systems often generate high background levels due to significant autofluorescence, receptor preparations with sufficiently high receptor concentrations have become a critical requirement for successful FA assay performance. We propose that budded baculoviruses that display GPCRs on their surfaces can be used as a receptor source in FA assays. Here, we describe the experimental setup of this homogeneous budded baculovirus/FA-based assay system for investigation of receptor-ligand interactions and a novel strategy for FA kinetic data analysis that is taking into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the binding reaction. The developed budded baculovirus/FA-based assay system brings the experimental data to a level that could solve complex models of ligand-receptor interactions and become a valuable tool for the screening of pharmacologically active compounds. Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system.

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors. The change in cellular cAMP level has widely been used to estimate the biological activity of various GPCR-specific agents. Forster resonance energy transfer (FRET) biosensors have been around for almost 10 years and became increasingly popular for cAMP detection. Ratiometric sensitized emission assay of a FRET biosensor can easily be implemented on fluorescence plate reader platforms. For such assays a considerable amount of cells expressing the desired biosensor is needed. A method to achieve sufficient and reproducible level of cAMP biosensor protein expression with the means of BacMam transduction system is the subject of this chapter.

Summary 4-Cyclohexyl-4-[(1,2,4-triazol-1-yl)methyl]piperidine was introduced intostepwise peptide synthesis procedures using Boc chemistry and derivativesof d-4-iodophenylalanine and d-1,2,3,4-tetrahydroisoquinoline-3-carboxylicacid. A halogen replacement analogue (I-Mex2) of a known high affinitymelanocortin-4 receptor selective compound resulted. It showed a subnano-molar affinity when evaluated on the melanocortin-4 receptor in competi-tion with the a-MSH peptide analogue 125 I-NDP-MSH. By treatmentwith hexamethylditin and tetrakis(triphenylphosphine) palladium I-Mex2was converted to the corresponding trimethylstannyl derivative. In thenext step, Na 125 I was oxidized by an iodobead. Iododestannylationproceeded in the presence of 1M phosphate buffer, pH 2.5, and the radio-active derivative 125 I-Mex2 formed was separated by HPLC at 40%radiochemical yield. Preliminary investigation showed that 125 I-Mex2Copyright # 2003 John Wiley & Sons, Ltd. Received 20 January 2003Revised 22 May 2003Accepted 26 May 2003*Correspondence to: J.E.S. Wikberg, Department of Pharmaceutical Biosciences, Division ofPharmacology, Uppsala University, Box 591, Biomedicum, SE-751 24 Uppsala, Sweden. E-mail:jarl.wikberg@farmbio.uu.seContract/grant sponsor: Swedish Research Council; contract/grant number: 04X-05957 and 230-2000-291Contract/grant sponsor: Melacure Therapeutics AB

Bodipy-FL-NAN-190 was found to be well suited for characterization of ligand binding to 5-HT1A receptors expressed in budded baculovirus particles, as binding is accompanied by large increases in fluorescence intensity and anisotropy. This ligand appears to bind rapidly (t1/2,ass1 min), reversibly (t1/2,diss∼6 min) and has high affinity (Kd=0.30 ± 0.13 nM). This fluorescence anisotropy assay based on Bodipy-FL-NAN-190 binding to baculovirus particles was also a suitable assay system for the pharmacological characterization of non-labelled serotonergic ligands, as well as being sensitive to the presence of G-proteins and guanine nucleotides. Coexpression of αi subunits of human G-proteins in baculovirus particles resulted in the appearance of significantly greater proportion of nucleotide sensitive high affinity agonist binding sites. There were no significant differences between αi1 and αi3 subtypes, while ligand binding in the presence of αi2 had higher sensitivity to GDP and Mn(2+).

We present here the implementation of budded baculoviruses that display G protein-coupled receptors on their surfaces for the investigation of ligand–receptor interactions using fluorescence anisotropy (FA). Melanocortin 4 (MC4) receptors and the fluorescent ligand Cy3B-NDP-α-MSH were used as the model system. The real-time monitoring of reactions and the high assay quality allow the application of global data analysis with kinetic mechanistic models that take into account the effect of nonspecific interactions and the depletion of the fluorescent ligand during the reaction. The receptor concentration, affinity and kinetic parameters of fluorescent ligand binding as well as state anisotropies for different fluorescent ligand populations were determined. At low Cy3B-NDP-α-MSH concentrations, a one-site receptor–ligand binding model described the processes, whereas divergence from this model was observed at higher ligand concentrations, which indicated a more complex mechanism of interactions similar to those mechanisms that have been found in experiments with radioactive ligands. The information obtained from our kinetic experiments and the inherent flexibility of FA assays also allowed the estimation of binding parameters for several MC4 receptor-specific unlabelled compounds. In summary, the FA assay that was developed with budded baculoviruses led the experimental data to a level that would solve complex models of receptor–ligand interactions also for other receptor systems and would become as a valuable tool for the screening of pharmacologically active compounds.

G protein-coupled receptors (GPCR), also referred to as 7 transmembrane spanning receptors (7TM), constitute the largest superfamily of receptors in vertebrates and invertebrates. These receptors respond to a wide range of signaling compounds such as bioamines, lipids, peptides, hormones, odorants, and even photons. They mediate the majority of cell-to-cell communication in the human body and targets for nearly one third of the prescription drugs on the market.

Cyclic adenosine monophosphate (cAMP) is a second messenger of many G-protein-coupled receptors (GPCRs) and a useful readout molecule to estimate the biological activity of various GPCR-specific agents. Here we report the development and use of a Förster resonance energy transfer (FRET) biosensor for cAMP (Epac2-camps) combined with a baculovirus-based BacMam transduction system. The constructed BacMam-Epac2-camps viral transduction system is a simple and robust tool for ligand screening at the second-messenger level in a variety of mammalian cell lines. The level of biosensor protein expression can easily be adjusted in a dose-dependent manner depending on the multiplicity of viral infection. For setting up the assay, we used a B16F10 murine melanoma cell line with endogenous expression of melanocortin-1 receptor (MC(1)R). The receptor activation was characterized by a set of MC(1)R full and partial agonists. Bivalent ions Ca(2+) as well as Mg(2+) modulated ligand potencies, whereas the effect was ligand and ion specific. Results obtained for MC(1)R indicate that the BacMam-Epac2-camps system may also be applicable for studying the activation of other GPCRs and may be implemented in routine analysis as well as in high-throughput screening.

Recombinant heterotrimeric G-protein α i1 , α i2 and α i3 subunits were purified in GDP-depleting conditions by affinity chromatography using StrepII-tagged β 1 γ 2 subunits. Real-time monitoring of fluorescence anisotropy of Bodipy-FL-GTPγS was used for characterization of nucleotide binding properties and inactivation of the purified proteins. All GDP-depleted α i were unstable at room temperature and therefore nucleotide binding could be characterized only in a nonequilibrium state. In comparison to Mg 2+ , Mn 2+ inhibited nucleotide binding to all α i -heterotrimers studied and accelerated nucleotide release. Mn 2+ had stabilizing effect on the nucleotide free state of the α i1 subunit, whereas both Mn 2+ as well as G-protein activation by mastoparan destabilized the α i2 subunit.

Fluorescence anisotropy assay was implemented for characterization of ligand binding dynamics to melanocortin 4 (MC 4 ) receptors. This approach enables on-line monitoring of reactions that is essential for estimation of more correct binding parameters, understanding of ligand binding and its regulation mechanisms, and design of new drugs with desirable properties. Two different red-shifted fluorophore-labeled peptide ligands, Cy3B–NDP-α-MSH and TAMRA–NDP-α-MSH, were used and compared in assays that monitored their binding to MC 4 receptors in membranes of Sf9 insect cells. The Cy3B dye-labeled ligand exhibited improved performance in assays when compared with the TAMRA-labeled ligand, having higher photostability, insensitivity to buffer properties, and better signal/noise ratio. The binding of both ligands to membranes of Sf9 cells expressing MC 4 receptors was saturable and with high affinity. All studied MC 4 receptor-specific nonlabeled ligands displaced fluoroligands’ binding in a concentration-dependent manner with potencies in agreement with their pharmacological activities. On-line monitoring of the reactions revealed that equilibrium of peptide binding was not reached even after 3 h. Real-time monitoring of ligand binding dynamics enabled us to find optimal experimental conditions for each particular ligand and an improved estimate of their binding parameters.

The melanocortin 4 receptor (MC(4)R) binding of the peptide analogue of melanocyte stimulating hormone, [(125)I]NDP-MSH, and the low molecular weight radionucleid 1-(D-1,2,3,4-tetrahydroisoquinoline-3-carboxy-D-4-(125)iodophenylalanyl)-4-cyclohexyl-4-[(1,2,4-triazol-1-yl)methyl]piperidine trifluoroacetate ([(125)I]THIQ) were compared. Kinetic analysis indicated heterogeneity in the binding of both radioligands, the binding apparently proceeding to two tandemly arranged interconnected mutually dependent binding sites. Steric considerations and BRET analysis of Rluc and GFP tagged receptors proposed that these sites are located on different subunits of receptor dimers, which form receptor complexes. According to the minimal model proposed, ligand binding proceeds consecutively to the two binding sites of the dimer. After binding of the first ligand conformational transformations of the complex occur, which is followed by binding of the second ligand. When both receptor units have bound [(125)I]NDP-MSH, the radioligand can be released only from one unit. The [(125)I]NDP-MSH bound to the remaining unit stays practically irreversibly bound due to a very slow retransformation rate of the transformed complex. The considerably faster binding of [(125)I]THIQ did not allow accurate kinetic differentiation of the two binding sites. However, addition of NDP-MSH as well as a fragment of the human agouti protein, hAGRP(83-132) to the preformed [(125)I]THIQ-MC(4)R complex drastically retarded the release of [(125)I]THIQ from the complex, blocking conformational transformations in the complex by binding into the second binding site. The consecutive binding of ligands to the MC(4)R dimers has substantial impact on the apparent ligand potencies, when determined in competition with the two different radioligands applied herein; the apparent potencies of the same ligand differing up to three orders of magnitude when assayed in competition with [(125)I]NDP-MSH or [(125)I]THIQ.

This study evaluates the binding the melanocyte stimulating hormone peptide analogue [125I]NDP-MSH to melanocortin receptors MC1, MC3, MC4 and MC5 in insect cell membranes produced by baculovirus expression systems. The presence of Ca2+ was found to be mandatory to achieve specific [125I]NDP-MSH binding to the melanocortin receptors. Although association kinetics of [125I]NDP-MSH followed the regularities of simple bimolecular reactions, the dissociation of [125I]NDP-MSH from the melanocortin receptors was heterogeneous. Eleven linear and cyclic MSH peptides studied displaced the [125I]NDP-MSH binding to the studied melanocortin receptors, with the shapes of their competition curves varying from biphasic or shallow to super-steep (Hill coefficients ranging from 0.4 to 1.5). Notably the same peptide often gave highly different patterns on different melanocortin receptor subtypes; e.g. the MC4 receptor selective antagonist HS131 gave a Hill coefficient of 1.5 on the MC1 receptor but 0.5-0.7 on the MC(3-5) receptors. Adding a mask of one of the peptides to block its high affinity binding did not prevent other competing peptides to yield biphasic competition curves. The data indicate that the binding of MSH peptides to melanocortin receptors are governed by a complex dynamic homotropic co-operative regulations.

Background Heterotrimeric G-proteins relay extracellular signals to intracellular effector proteins. Multiple methods have been developed to monitor their activity; including labeled nucleotides and biosensors based on genetically engineered G-proteins. Here we describe a method for monitoring unlabeled nucleotide binding to endogenous G-proteins α-subunits in a homogeneous assay based on the interaction of 4′,5′-bis(1,2,3-dithioarsolan-2-yl)-2′,7′-difluorofluorescein (F2FlAsH) with G-protein α-subunits. Results The biarsenic fluorescent ligand F2FlAsH binds to various wild-type G-protein α-subunits (αi1, αi2, αi3, αslong, αsshort, αolf, αq, α13) via high affinity As-cysteine interactions. This allosteric label enables real time monitoring of the nucleotide bound states of α-subunits via changes in fluorescence anisotropy and intensity of their F2FlAsH-complexes. We have found that different α-subunits displayed different signal amplitudes when interacting with F2FlAsH, being more sensitive to nucleotide binding to αi, αs, αolf and αq than to α13. Addition of nucleotides to F2FlAsH-labeled α-subunits caused concentration-dependent effects on their fluorescence anisotropy. pEC50 values of studied nucleotides depended on the subtype of the α-subunit and were from 5.7 to 8.2 for GTPγS, from 5.4 to 8.1 for GppNHp and from 4.8 to 8.2 for GDP and lastly up to 5.9 for GMP. While GDP and GMP increased the fluorescence anisotropy of F2FlAsH complexes with αi-subunits, they had the opposite effect on the other αβγM complexes studied. Conclusions Biarsenical ligands interact allosterically with endogenous G-protein α-subunits in a nucleotide-sensitive manner, so the presence or absence of guanine nucleotides has an effect on the fluorescence anisotropy, intensity and lifetime of F2FlAsH-G-protein complexes.

The influence of various concentrations of 15 detergents at different temperatures on the fluidity of membranes was analysed by fluorescence anisotropy of inserted 1,6-diphenyl-1,3,5- hexatriene in rat cortical and Sf9 cell membranes. Comparison of the obtained data with the abilities of the detergents to solubilize muscarinic receptor subtypes indicated that not only the fluidity of the micelles formed but also the micelle formation process, the lipid/protein ratio, and presence of sterols have significant influence on the efficiency of the solubilization of mAChR. Digitonin seems to exploit efficiently all these factors by its biphasic modulatory effect on membrane fluidity during the receptor solubilization.

G protein coupled receptors (GPCR) comprise a large family of transmembrane proteins involved in the regulation of signal transduction through the cell membrane in response to various extracellular stimuli. GPCR have become important targets of many drugs for treatments of very different diseases. During the last decade several fluorescence-based methods have been implemented for the characterization of signal transduction via GPCRs, starting from ligand binding and including several steps leading up to a response on the level of gene regulation. We have proposed the fluorescence anisotropy (FA) and fluorescence intensity (FI) assay to investigate fluorescent ligand binding properties to different GPCRs (Veiksina et al., 2010). The implementations of budded baculoviruses that display G protein-coupled receptors on their surfaces have significantly increased sensitivity and applicability of these assays (Veiksina et al., 2014). The developed novel assay systems opened new possibilities for real-time monitoring of ligand binding to their receptors for understanding their particular kinetic properties. These assays are also compatible for homogenous HTS suitable fo ligand screening. There has been implemented assay systems for receptors of peptides like melanocortin (MC4R) and neuropeptide Y (NPY1R) as well as for receptors of monoamines like dopamine (D1DAR) and serotonin (5-HT1AR).

Melanocortin receptors (MCRs) are seven transmembrane G protein-coupled receptors that are known for their broad physiological relevance. The subtype 4 melanocortin receptor (MC4R) has emerged as a central element in the regulation of energy homeostasis, eating behavior and regulation of sexual functions. MCRs are governed by a complex dynamic homotropic regulation [1]. There is an increasing trend towards using fluorescence anisotropy (FA) for studying the aforementioned complex receptor-ligand interactions. FA allows the characterization of ligand binding dynamics [2]. The quality of the FA assay can be greatly increased with budded baculovirus particles that display the receptors of interest on their surfaces [3]. The use of a fluorescent ligand Cy3B-NDP-α-MSH has made it possible to study MC4Rs with higher precision and sampling rate [3]. However, this ligand has relatively slow kinetics. Modification of the structure of a MC4R antagonist revealed two new reporter ligands (UTBC101 and UTBC102) for fluorescence labeling. These new reporter ligands selectively bind to MC4Rs and exhibit improved kinetic properties. The association and dissociation rate constants of UTBC101 and UTBC102 are kon = ((2.0 ± 0.6) × 10^7 M^-1 min^-1), koff = ((4.6 ± 0.3) × 10^-3 min^-1) and kon = ((1.9 ± 0.5) × 10^7 M^-1 min^-1)), koff = ((1.0 ± 0.2) × 10^-1 min^-1), accordingly. UTBC101 and UTBC102 enable the characterization of both labelled and non-labelled ligand binding dynamics in regard to the MC4R. UTBC102 could be especially valuable for ligand screening, because of its very high dissociation rate, which makes it possible to achieve equilibrium conditions.