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1.
EBioMedicine ; 70: 103502, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34333234

RESUMO

BACKGROUND: Since 2020 SARS-CoV-2 spreads pandemically, infecting more than 119 million people, causing >2·6 million fatalities. Symptoms of SARS-CoV-2 infection vary greatly, ranging from asymptomatic to fatal. Different populations react differently to the disease, making it very hard to track the spread of the infection in a population. Measuring specific anti-SARS-CoV-2 antibodies is an important tool to assess the spread of the infection or successful vaccinations. To achieve sufficient sample numbers, alternatives to venous blood sampling are needed not requiring medical personnel or cold-chains. Dried-blood-spots (DBS) on filter-cards have been used for different studies, but not routinely for serology. METHODS: We developed a semi-automated protocol using self-sampled DBS for SARS-CoV-2 serology. It was validated in a cohort of matched DBS and venous-blood samples (n = 1710). Feasibility is demonstrated with two large serosurveys with 10247 company employees and a population cohort of 4465 participants. FINDINGS: Sensitivity and specificity reached 99·20% and 98·65%, respectively. Providing written instructions and video tutorials, 99·87% (4465/4471) of the unsupervised home sampling DBS cards could be analysed. INTERPRETATION: DBS-sampling is a valid and highly reliable tool for large scale serosurveys. We demonstrate feasibility and accuracy with a large validation cohort including unsupervised home sampling. This protocol might be of big importance for surveillance in resource-limited settings, providing low-cost highly accurate serology data. FUNDING: Provided by Bavarian State Ministry of Science and the Arts, LMU University-Hospital; Helmholtz-Centre-Munich, German Ministry for Education and Research (project01KI20271); University of Bonn; University of Bielefeld; the Medical Biodefense Research Program of Bundeswehr-Medical-Service; Euroimmun, RocheDiagnostics provided discounted kits and machines.


Assuntos
Anticorpos Antivirais/imunologia , Bioensaio/métodos , Teste Sorológico para COVID-19/métodos , COVID-19/sangue , COVID-19/imunologia , Teste em Amostras de Sangue Seco/métodos , SARS-CoV-2/imunologia , Infecções Assintomáticas , Estudos de Coortes , Humanos , Estudos Longitudinais , Sensibilidade e Especificidade , Manejo de Espécimes/métodos , Vacinação/métodos
2.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360555

RESUMO

Human cytosolic prolyl-tRNA synthetase (HcProRS) catalyses the formation of the prolyl-tRNAPro, playing an important role in protein synthesis. Inhibition of HcProRS activity has been shown to have potential benefits in the treatment of fibrosis, autoimmune diseases and cancer. Recently, potent pyrazinamide-based inhibitors were identified by a high-throughput screening (HTS) method, but no further elaboration was reported. The pyrazinamide core is a bioactive fragment found in numerous clinically validated drugs and has been subjected to various modifications. Therefore, we applied a virtual screening protocol to our in-house library of pyrazinamide-containing small molecules, searching for potential novel HcProRS inhibitors. We identified a series of 3-benzylaminopyrazine-2-carboxamide derivatives as positive hits. Five of them were confirmed by a thermal shift assay (TSA) with the best compounds 3b and 3c showing EC50 values of 3.77 and 7.34 µM, respectively, in the presence of 1 mM of proline (Pro) and 3.45 µM enzyme concentration. Co-crystal structures of HcProRS in complex with these compounds and Pro confirmed the initial docking studies and show how the Pro facilitates binding of the ligands that compete with ATP substrate. Modelling 3b into other human class II aminoacyl-tRNA synthetases (aaRSs) indicated that the subtle differences in the ATP binding site of these enzymes likely contribute to its potential selective binding of HcProRS. Taken together, this study successfully identified novel HcProRS binders from our anti-tuberculosis in-house compound library, displaying opportunities for repurposing old drug candidates for new applications such as therapeutics in HcProRS-related diseases.


Assuntos
Trifosfato de Adenosina/metabolismo , Aminoacil-tRNA Sintetases/antagonistas & inibidores , Bioensaio/métodos , Simulação por Computador , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Pirazinamida/química , Sítios de Ligação , Cristalografia por Raios X , Inibidores Enzimáticos/isolamento & purificação , Humanos , Ligantes , Modelos Moleculares , Conformação Proteica
3.
J Chem Ecol ; 47(7): 642-652, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34331170

RESUMO

Finding plant cultivars that are resistant or tolerant against lepidopteran pests, takes time, effort and is costly. We present here a high throughput leaf-disk consumption assay system, to screen plants for resistance or chemicals for their deterrence. A webcam capturing images at regular intervals can follow the feeding activities of 150 larvae placed into individual cages. We developed a computer program running under an open source image analysis program to analyze and measure the surface of each leaf disk over time. We further developed new statistical procedures to analyze the time course of the feeding activities of the larvae and to compare them between treatments. As a test case, we compared how European corn borer larvae respond to a commercial antifeedant containing azadirachtin, and to quinine, which is a bitter alkaloid for many organisms. As expected, increasing doses of azadirachtin reduced and delayed feeding. However, quinine was poorly effective at the range of concentrations tested (10-5 M to 10-2 M). The model cage, the camera holder, the plugins, and the R scripts are freely available, and can be modified according to the users' needs.


Assuntos
Bioensaio/métodos , Comportamento Alimentar , Lepidópteros/fisiologia , Animais , Análise por Conglomerados , Comportamento Alimentar/efeitos dos fármacos , Processamento de Imagem Assistida por Computador , Larva/crescimento & desenvolvimento , Larva/fisiologia , Lepidópteros/crescimento & desenvolvimento , Limoninas/farmacologia , Folhas de Planta/química , Folhas de Planta/metabolismo , Quinina/farmacologia , Zea mays/química , Zea mays/metabolismo
4.
Bioanalysis ; 13(15): 1195-1203, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34275327

RESUMO

Thousands of clinical trials all over the world were stopped, disrupted or delayed while countries grappled to contain the pandemic and research resources were redeployed. The long-term effects of the turbulence caused by the pandemic have yet to be fully understood, but it should already be clear that the increased focus on participant needs and on the logistical challenges of current models are not likely to fade away quickly. This disruption is opening doors for rethinking traditional approaches to clinical trial conduct - including decentralizing site visits, introducing new methods of sample collection, rethinking matrix selection, reducing sample volumes and collaborating on device development. These approaches reduce participant burden while improving critical trial data.


Assuntos
Bioensaio/métodos , COVID-19/epidemiologia , Ensaios Clínicos como Assunto , Humanos , Pandemias , SARS-CoV-2
5.
Bioanalysis ; 13(15): 1205-1211, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34275332

RESUMO

The COVID-19 pandemic challenged pharmaceutical and bioanalytical communities at large, in the development of vaccines and therapeutics as well as supporting ongoing drug development efforts. Existing processes were challenged to manage loss of staffing at facilities along with added workloads for COVID-19-related study support including conducting preclinical testing, initiating clinical trials, conducting bioanalysis and interactions with regulatory agencies, all in an ultra-rapid timeframes. A key factor of success was creative rethinking of processes and removing barriers - some of which hitherto had been considered immovable. This article describes how bioanalysis was crippled at the onset of the pandemic but how innovative and highly collaborative efforts across teams within and outside of both pharma, bioanalytical labs and regulatory agencies worked together remarkably well.


Assuntos
Bioensaio/métodos , COVID-19/epidemiologia , Desenvolvimento de Medicamentos/métodos , Humanos , Pandemias , SARS-CoV-2
6.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299143

RESUMO

Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum and are responsible for botulism, a fatal disorder of the nervous system mostly induced by food poisoning. Despite being one of the most potent families of poisonous substances, BoNTs are used for both aesthetic and therapeutic indications from cosmetic reduction of wrinkles to treatment of movement disorders. The increasing understanding of the biology of BoNTs and the availability of distinct toxin serotypes and subtypes offer the prospect of expanding the range of indications for these toxins. Engineering of BoNTs is considered to provide a new avenue for improving safety and clinical benefit from these neurotoxins. Robust, high-throughput, and cost-effective assays for BoNTs activity, yet highly relevant to the human physiology, have become indispensable for a successful translation of engineered BoNTs to the clinic. This review presents an emerging family of cell-based assays that take advantage of newly developed human pluripotent stem cells and neuronal function analyses technologies.


Assuntos
Bioensaio/métodos , Toxinas Botulínicas/farmacologia , Neurônios/citologia , Neurotoxinas/farmacologia , Células-Tronco Pluripotentes/citologia , Animais , Toxinas Botulínicas/classificação , Humanos , Neurônios/efeitos dos fármacos , Neurotoxinas/classificação , Células-Tronco Pluripotentes/efeitos dos fármacos
7.
Biosensors (Basel) ; 11(6)2021 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-34204930

RESUMO

The early detection of the human immunodeficiency virus (HIV) is of paramount importance to achieve efficient therapeutic treatment and limit the disease spreading. In this perspective, the assessment of biosensing assay for the HIV-1 p24 capsid protein plays a pivotal role in the timely and selective detection of HIV infections. In this study, multi-parameter-SPR has been used to develop a reliable and label-free detection method for HIV-1 p24 protein. Remarkably, both physical and chemical immobilization of mouse monoclonal antibodies against HIV-1 p24 on the SPR gold detecting surface have been characterized for the first time. The two immobilization techniques returned a capturing antibody surface coverage as high as (7.5 ± 0.3) × 1011 molecule/cm2 and (2.4 ± 0.6) × 1011 molecule/cm2, respectively. However, the covalent binding of the capturing antibodies through a mixed self-assembled monolayer (SAM) of alkanethiols led to a doubling of the p24 binding signal. Moreover, from the modeling of the dose-response curve, an equilibrium dissociation constant KD of 5.30 × 10-9 M was computed for the assay performed on the SAM modified surface compared to a much larger KD of 7.46 × 10-5 M extracted for the physisorbed antibodies. The chemically modified system was also characterized in terms of sensitivity and selectivity, reaching a limit of detection of (4.1 ± 0.5) nM and an unprecedented selectivity ratio of 0.02.


Assuntos
Bioensaio/métodos , Proteína do Núcleo p24 do HIV , Ressonância de Plasmônio de Superfície , Técnicas Biossensoriais , Ouro/química , HIV-1 , Limite de Detecção
8.
Molecules ; 26(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200415

RESUMO

Homovanillic acid (HVA) and vanillylmandelic acid (VMA) are end-stage metabolites of catecholamine and are clinical biomarkers for the diagnosis of neuroblastoma. For the first time in Korea, we implemented and validated a liquid chromatography tandem mass spectrometry (LC-MS/MS) assay to measure urinary concentrations of HVA and VMA according to Clinical and Laboratory Standards Institute guidelines. Our LC-MS/MS assay with minimal sample preparation was validated for linearity, lower limit of detection (LOD), lower limit of quantification (LLOQ), precision, accuracy, extraction recovery, carryover, matrix effect, and method comparison. A total of 1209 measurements was performed to measure HVA and VMA in spot urine between October 2019 and September 2020. The relationship between the two urinary markers, HVA and VMA, was analyzed and exhibited high agreement (89.1% agreement, kappa's k = 0.6) and a strong correlation (Pearson's r = 0.73). To our knowledge, this is the first study to utilize LC-MS/MS for simultaneous quantitation of spot urinary HVA and VMA and analyze the clinical application of both markers on a large scale for neuroblastoma patients.


Assuntos
Ácido Homovanílico/química , Neuroblastoma/diagnóstico , Neuroblastoma/metabolismo , Ácido Vanilmandélico/química , Bioensaio/métodos , Biomarcadores/metabolismo , Criança , Pré-Escolar , Cromatografia Líquida/métodos , Feminino , Seguimentos , Humanos , Lactente , Recém-Nascido , Limite de Detecção , Masculino , República da Coreia , Espectrometria de Massas em Tandem/métodos
9.
Methods Mol Biol ; 2320: 111-119, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302653

RESUMO

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs) have been shown to have great potential to play a key role in investigating cardiac diseases in vitro. Multielectrode array (MEA) system is sometimes preferable to patch-clamp in electrophysiological experiments in terms of several advantages. Here we show our protocol of electrophysiological examinations using MEA.


Assuntos
Bioensaio/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Análise Serial de Tecidos/métodos , Células Cultivadas , Fenômenos Eletrofisiológicos/fisiologia , Humanos , Técnicas de Patch-Clamp/métodos
10.
Methods Mol Biol ; 2320: 151-160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34302656

RESUMO

Human-induced pluripotent stem cell (iPSC) technology paves the way for next-generation drug-safety assessment. In particular, human iPSC-derived cardiomyocytes, which exhibit electrical activity, are useful as a human cell model for assessing QT-interval prolongation and the risk of the lethal arrhythmia Torsade de Pointes (TdP). In addition to proarrhythmia assay, contractile behavior has received increased attention in drug development. In this study, we developed a novel high-throughput in vitro assay system using motion vectors to evaluate the contractile activity of iPSC-derived cardiomyocytes as a physiologically relevant human platform. The methods presented here highlight the use of commercially available iPSC-derived cardiomyocytes, iCell cardiomyocytes, for contractility evaluation recorded by the motion vector system.


Assuntos
Bioensaio/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Arritmias Cardíacas/terapia , Células Cultivadas , Humanos , Síndrome do QT Longo/terapia , Torsades de Pointes/terapia
11.
Int J Mol Sci ; 22(11)2021 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-34200322

RESUMO

A novel bioluminescent Monoacylglycerol lipase (MAGL) substrate 6-O-arachidonoylluciferin, a D-luciferin derivative, was synthesized, physico-chemically characterized, and used as highly sensitive substrate for MAGL in an assay developed for this purpose. We present here a new method based on the enzymatic cleavage of arachidonic acid with luciferin release using human Monoacylglycerol lipase (hMAGL) followed by its reaction with a chimeric luciferase, PLG2, to produce bioluminescence. Enzymatic cleavage of the new substrate by MAGL was demonstrated, and kinetic constants Km and Vmax were determined. 6-O-arachidonoylluciferin has proved to be a highly sensitive substrate for MAGL. The bioluminescence assay (LOD 90 pM, LOQ 300 pM) is much more sensitive and should suffer fewer biological interferences in cells lysate applications than typical fluorometric methods. The assay was validated for the identification and characterization of MAGL modulators using the well-known MAGL inhibitor JZL184. The use of PLG2 displaying distinct bioluminescence color and kinetics may offer a highly desirable opportunity to extend the range of applications to cell-based assays.


Assuntos
Benzodioxóis/farmacologia , Benzotiazóis/metabolismo , Bioensaio/métodos , Luciferases/metabolismo , Luminescência , Monoacilglicerol Lipases/metabolismo , Piperidinas/farmacologia , Ansiolíticos/farmacologia , Inibidores Enzimáticos/farmacologia , Humanos , Monoacilglicerol Lipases/antagonistas & inibidores
12.
Molecules ; 26(13)2021 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206357

RESUMO

In the current work, a simple, economical, accurate, and precise HPLC method with UV detection was developed to quantify Favipiravir (FVIR) in spiked human plasma using acyclovir (ACVR) as an internal standard in the COVID-19 pandemic time. Both FVIR and ACVR were well separated and resolved on the C18 column using the mobile phase blend of methanol:acetonitrile:20 mM phosphate buffer (pH 3.1) in an isocratic mode flow rate of 1 mL/min with a proportion of 30:10:60 %, v/v/v. The detector wavelength was set at 242 nm. Maximum recovery of FVIR and ACVR from plasma was obtained with dichloromethane (DCM) as extracting solvent. The calibration curve was found to be linear in the range of 3.1-60.0 µg/mL with regression coefficient (r2) = 0.9976. However, with acceptable r2, the calibration data's heteroscedasticity was observed, which was further reduced using weighted linear regression with weighting factor 1/x. Finally, the method was validated concerning sensitivity, accuracy (Inter and Intraday's % RE and RSD were 0.28, 0.65 and 1.00, 0.12 respectively), precision, recovery (89.99%, 89.09%, and 90.81% for LQC, MQC, and HQC, respectively), stability (% RSD for 30-day were 3.04 and 1.71 for LQC and HQC, respectively at -20 °C), and carry-over US-FDA guidance for Bioanalytical Method Validation for researchers in the COVID-19 pandemic crisis. Furthermore, there was no significant difference for selectivity when evaluated at LLOQ concentration of 3 µg/mL of FVIR and relative to the blank.


Assuntos
Amidas/análise , Amidas/sangue , Antivirais/análise , Antivirais/sangue , Bioensaio/métodos , COVID-19/tratamento farmacológico , Cromatografia Líquida de Alta Pressão/métodos , Extração Líquido-Líquido/métodos , Pirazinas/análise , Pirazinas/sangue , Aciclovir/análise , Aciclovir/sangue , COVID-19/sangue , Calibragem , Estabilidade de Medicamentos , Congelamento , Humanos , Padrões de Referência , Reprodutibilidade dos Testes , Solventes/química
13.
Mutat Res Rev Mutat Res ; 787: 108363, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34083041

RESUMO

Dr. Bruce Ames turned 92 on December 16, 2020. He considers his most recent work linking adequate consumption of 30 known vitamins and minerals with successful aging to be his most important contribution. With the passage of time, it is not uncommon for the accomplishments of a well-known scientist to undergo a parsimonious reductionism in the public mind - Pasteur's vaccine, Mendel's peas, Pavlov's dogs, Ames' test. Those of us in the research generation subsequent to Dr. Ames' are undoubtedly affected by our own unconscious tendencies toward accepting the outstanding achievements of the past as commonplace. In doing so, seminal advances made by earlier investigators are often inadvertently subsumed into common knowledge. But having followed Ames' work since the mid-1970s, we are cognizant that the eponymous Ames Test is but a single chapter in a long and rich narrative. That narrative begins with Ames' classic studies on the histidine operon of Salmonella, for which he was elected to the National Academy of Sciences. A summary of the historical progression of the understanding of chemical carcinogenesis to which Ames and his colleagues contributed is provided. Any summary of a topic as expansive and complex as the ongoing unraveling of the mechanisms underlying chemical carcinogenesis will only touch upon some of the major conceptual advances to which Ames and his colleagues contributed. We hope that scientists of all ages familiar with Ames only through the eponymous Ames Test will further investigate the historical progression of the conceptualization of cancer caused by chemical exposure. As the field of chemical carcinogenesis gradually moves away from primary reliance on animal testing to alternative protocols under the rubric of New Approach Methodologies (NAM) an understanding of where we have been might help to guide where we should go.


Assuntos
Bioensaio/métodos , Animais , Bases de Dados de Ácidos Nucleicos , Humanos , Testes de Mutagenicidade , Mutação/genética
14.
PLoS Pathog ; 17(6): e1009683, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34166473

RESUMO

COVID-19 is a global crisis of unimagined dimensions. Currently, Remedesivir is only fully licensed FDA therapeutic. A major target of the vaccine effort is the SARS-CoV-2 spike-hACE2 interaction, and assessment of efficacy relies on time consuming neutralization assay. Here, we developed a cell fusion assay based upon spike-hACE2 interaction. The system was tested by transient co-transfection of 293T cells, which demonstrated good correlation with standard spike pseudotyping for inhibition by sera and biologics. Then established stable cell lines were very well behaved and gave even better correlation with pseudotyping results, after a short, overnight co-incubation. Results with the stable cell fusion assay also correlated well with those of a live virus assay. In summary we have established a rapid, reliable, and reproducible cell fusion assay that will serve to complement the other neutralization assays currently in use, is easy to implement in most laboratories, and may serve as the basis for high throughput screens to identify inhibitors of SARS-CoV-2 virus-cell binding and entry.


Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , Bioensaio/métodos , COVID-19/virologia , Receptores de Coronavírus/metabolismo , SARS-CoV-2/fisiologia , Glicoproteína da Espícula de Coronavírus/metabolismo , Enzima de Conversão de Angiotensina 2/genética , COVID-19/sangue , Fusão Celular , Células HEK293 , Humanos , Receptores de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/genética , Transfecção , Ligação Viral
15.
Methods Mol Biol ; 2276: 143-151, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060038

RESUMO

Deoxynucleoside 5'-triphosphates (dNTPs) are the molecular building blocks for DNA synthesis, and their balanced concentration in the cell is fundamental for health. dNTP imbalance can lead to genomic instability and other metabolic disturbances, resulting in devastating mitochondrial diseases.The accurate and efficient measurement of dNTPs from different biological samples and cellular compartments is vital to understand the mechanisms behind these diseases and develop and scrutinize their possible treatments. This chapter describes an update on the most recent development of the traditional radiolabeled polymerase extension method and its adaptation for the measurement of whole-cell and mitochondrial dNTP pools from cultured cells and tissue samples. The solid-phase reaction setting enables an increase in efficiency, accuracy, and measurement scale.


Assuntos
Bioensaio/métodos , Fracionamento Celular/métodos , Células/metabolismo , Desoxirribonucleotídeos/metabolismo , Mitocôndrias/metabolismo , Animais , Células Cultivadas , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Humanos , Camundongos , Mitocôndrias/genética
16.
Methods Mol Biol ; 2276: 409-423, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34060058

RESUMO

Platinum-based antitumor drugs play important roles in the clinical treatment of various tumors. Nevertheless, some deficiencies such as poor targeting ability, low bioavailability, in vivo deactivation, drug resistance, and side effects undermine the efficacy of these drugs. Mitochondria are important organelles which regulate the energy metabolism, physiological function, life span, and survival of the cells. Regulating or interfering with mitochondrial metabolism is of great significance in the prevention or treatment of cancers. Thus, a series of mitochondrion-targeted platinum complexes were prepared by modifying triphenylphosphine (TPP+) through chemical modifications, which endow traditional platinum drugs with new properties and mechanisms through interfering with mitochondrial DNA (mtDNA), mitochondrial membrane potential (MMP), mitochondrial morphology, mitochondrial bioenergetics, or production of reactive oxygen species (ROS), thereby opening a new path for the clinical application of platinum drugs. Here we introduce the synthesis of some TPP+-modified platinum (II, IV) complexes in details and the detection method of the activity parameters related to the mitochondrial functions.


Assuntos
Bioensaio/métodos , Mitocôndrias/metabolismo , Neoplasias/tratamento farmacológico , Compostos Organofosforados/química , Compostos Organoplatínicos/farmacologia , Antineoplásicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Metabolismo Energético , Humanos , Potencial da Membrana Mitocondrial , Mitocôndrias/efeitos dos fármacos , Neoplasias/química , Neoplasias/metabolismo , Espécies Reativas de Oxigênio/metabolismo
17.
Methods Mol Biol ; 2268: 119-136, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085265

RESUMO

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.


Assuntos
Baculoviridae/genética , Carbocianinas/química , Polarização de Fluorescência/métodos , Corantes Fluorescentes/química , Receptor Tipo 4 de Melanocortina/metabolismo , Proteínas Recombinantes/metabolismo , Animais , Ligação Competitiva , Bioensaio/métodos , Humanos , Cinética , Ligantes , Ligação Proteica , Receptor Tipo 4 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/genética , Células Sf9
18.
Methods Mol Biol ; 2268: 149-157, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085267

RESUMO

G protein-coupled receptors (GPCR) are one of the principal class of membrane proteins and around 30% of the currently marketed drugs act on one of them. The efficacious detection of ligands with the desired pharmacological profile remains a challenge of paramount importance in the GPCR drug discovery and pharmacological research. Recent evidences demonstrate that GPCR ligands can stabilize distinct receptor conformation and trigger various signaling pathways with different efficacies and/or potencies. This phenomenon called functional selectivity or biased signaling may lead to improved drugs with fewer side effects. Most receptors are promiscuous and can couple to more than one G protein family. To enable the discovery of biased ligands able to selectively trigger one G protein pathway over another, simple and efficient screening procedures are needed. The traditional assays aiming at detecting G protein activation monitor the generation of second messengers ([Ca2+]i, cAMP, IP1) or active G proteins (with GTP-g-S for instance). While these approaches have proven sensitive and robust, they are not suited for the detection of a single GPCR-G protein interaction. Here, we present in detail a method to assess directly the interaction between the receptor and the G protein. It permits the profiling of a receptor or a ligand toward G protein interactions and is compatible with high-throughput screening.


Assuntos
Bioensaio/métodos , Descoberta de Drogas/métodos , Proteínas de Ligação ao GTP/metabolismo , Luciferases/metabolismo , Nanotecnologia/métodos , Receptores Acoplados a Proteínas G/metabolismo , Células HEK293 , Humanos , Ligantes , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas
19.
Methods Mol Biol ; 2268: 193-205, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085270

RESUMO

Intracellular calcium mobilization can be measured using several methods varying in indicator dyes and devices used. In this chapter, we describe the fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for simultaneous concentration-dependent analysis of several receptor agonists and antagonists, useful in receptor characterization and drug discovery projects.


Assuntos
Bioensaio/métodos , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Descoberta de Drogas/métodos , Fluorometria/métodos , Ensaios de Triagem em Larga Escala/métodos , Receptores Acoplados a Proteínas G/metabolismo , Células Cultivadas , Humanos , Transdução de Sinais
20.
Methods Mol Biol ; 2268: 207-221, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34085271

RESUMO

GPCRs are responsible for activation of numerous downstream effectors. Live cell imaging of these effectors therefore provides a real-time readout of GPCR activity and allows for better understanding of temporal dynamics of GPCR-mediated signaling. Opsins, or optically activatable GPCRs, allow for these signaling pathways to be activated in a spatiotemporally precise and reversible manner. Here, we describe optogenetic methods for activating Gi, Gq, and Gs signaling pathways. Additionally, we present assays for detecting activation of these pathways in real time through live cell imaging of Gßγ translocation, PIP3 increase, PIP2 hydrolysis, cAMP production, and cell migration. These assays can be utilized for GPCR-targeted drug development, as well as for studies of a wide range of GPCR-mediated physiological processes.


Assuntos
Bioensaio/métodos , Transferência Ressonante de Energia de Fluorescência/métodos , Imagem Molecular/métodos , Opsinas/metabolismo , Optogenética/métodos , Receptores Acoplados a Proteínas G/metabolismo , Análise de Célula Única/métodos , Movimento Celular/fisiologia , Células Cultivadas , Humanos , Opsinas/genética , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais
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