עבודה זו נתמכה על ידי מכוני הבריאות הלאומיים גרנט GM 69829.

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	<li>Stephenson, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modification+of+receptor+theory.">A modification of receptor theory.</a> British Journal of Pharmacology. 11, 379-393 (1956).</li><li>Furchgott, R. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+b-haloalkylamines+in+the+differentiation+of+receptors+and+in+the+determination+of+dissociation+constants+of+receptor-agonist+complexes.">The use of b-haloalkylamines in the differentiation of receptors and in the determination of dissociation constants of receptor-agonist complexes.</a> Advances in Drug Research. 3, 21-55 (1966).</li><li>Ehlert, F. J., Suga, H., Griffin, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+agonism+and+inverse+agonism+in+functional+assays+with+constitutive+activity:+Estimation+of+orthosteric+ligand+affinity+constants+for+active+and+inactive+receptor+states.">Analysis of agonism and inverse agonism in functional assays with constitutive activity: Estimation of orthosteric ligand affinity constants for active and inactive receptor states.</a> J. Pharmacol. Exp. Ther. 33, 671-686 (2011).</li><li>Griffin, M. T., Figueroa, K. W., Liller, S., Ehlert, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+Agonist+Activity+at+G+Protein-Coupled+Receptors:+Analysis+of+M2+Muscarinic+Receptor+Signaling+through+Gi/o,Gs,+and+G15.">Estimation of Agonist Activity at G Protein-Coupled Receptors: Analysis of M2 Muscarinic Receptor Signaling through Gi/o,Gs, and G15.</a> J. Pharmacol. Exp. Ther. 321, 1193-1207 (2007).</li><li>Black, J. W., Leff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Operational+models+of+pharmacological+agonism.">Operational models of pharmacological agonism.</a> Proceedings of the Royal Society of LondonSeries B: Biological Sciences. 220, 141-162 (1983).</li><li>Tran, J. A., Chang, A., Matsui, M., Ehlert, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimation+of+relative+microscopic+affinity+constants+of+agonists+for+the+active+state+of+the+receptor+in+functional+studies+on+M2+and+M3+muscarinic+receptors.">Estimation of relative microscopic affinity constants of agonists for the active state of the receptor in functional studies on M2 and M3 muscarinic receptors.</a> Mol. Pharmacol. 75, 381-396 (2009).</li><li>Schultz, J., Hamprecht, B., Daly, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accumulation+of+adenosine+3':5'-cyclic+monophosphate+in+clonal+glial+cells:+labeling+of+intracellular+adenine+nucleotides+with+radioactive+adenine.">Accumulation of adenosine 3':5'-cyclic monophosphate in clonal glial cells: labeling of intracellular adenine nucleotides with radioactive adenine.</a> Proceedings of the National Academy of Sciences of the United States of America. 69, 1266-1270 (1972).</li><li>Berridge, M. J., Downes, C. P., Hanley, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lithium+amplifies+agonist-dependent+phosphatidylinositol+responses+in+brain+and+salivary+glands.">Lithium amplifies agonist-dependent phosphatidylinositol responses in brain and salivary glands.</a> Biochemical Journal. 206, 587-595 (1982).</li><li>Kendall, D. A., Hill, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods in Neurotransmitter Receptor Analysis. , 68-87 (1990).</li><li>Pulido-Rios, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vitro+Isolated+Tissue+Functional+Muscarinic+Receptor+Assays.">Vitro Isolated Tissue Functional Muscarinic Receptor Assays.</a> Current Protocols in Pharmacology. 48, 4-15 (2010).</li><li>Kenakin, T., Enna, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Current Protocols in Pharmacology. , (2001).</li><li>Ehlert, F. J., Griffin, M. T., Sawyer, G. W., Bailon, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=simple+method+for+estimation+of+agonist+activity+at+receptor+subtypes:+comparison+of+native+and+cloned+M3+muscarinic+receptors+in+guinea+pig+ileum+and+transfected+cells.">simple method for estimation of agonist activity at receptor subtypes: comparison of native and cloned M3 muscarinic receptors in guinea pig ileum and transfected cells.</a> Journal of Pharmacology and Experimental Therapeutics. 289, 981-992 (1999).</li><li>Burstein, E. S., Spalding, T. A., Brann, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacology+of+muscarinic+receptor+subtypes+constitutively+activated+by+G+proteins.">Pharmacology of muscarinic receptor subtypes constitutively activated by G proteins.</a> Mol. Pharmacol. 51, 312-319 (1997).</li></ol>

אין ניגודי אינטרסים הכריז.

בגלל השיטה שלנו להערכת RA אני (יחסית לערך K ב) רק דורש מדידת ריכוז בתגובה עקומות אגוניסט, הניתוח שלנו ניתן לעשות זאת בכל עת עקומות אלה נמדדים. אם יום יומית וריאציה בתגובה של הכנת הניסוי (למשל, תאים או רקמות) הוא גדול, ?...

quantifying agonist activity at g protein-coupled receptors

כאשר אגוניסט מפעיל אוכלוסייה של G-חלבון בשילוב קולטנים (GPCRs), זה מעורר מסלול איתות זה מגיע לשיאו בתגובה של התא או הרקמה. תהליך זה יכול להיות מנותח ברמה של קולטן יחיד, אוכלוסייה של קולטנים, או תגובה במורד הזרם. כאן אנו מתארים כיצד לנתח את התגובה במורד הזרם כדי לקבל אומדן של הזיקה אגוניסט מתמדת המדינה הפעילה של קולטנים יחיד. רצפטורים להתנהג כמו מתגים quantal כי חלופי בין מדינות פעיל פעיל (איור 1). המצב הפעיל אינטראקציה עם חלבונים ספציפיים G או שותפים איתות אחרים. בהעדר ligands, המדינה לא פעיל השולט. הכריכה של אגוניסט מעלה את ההסתברות כי קולטן יעבור למצב פעיל בגלל זיקה מתמדת למדינת פעיל (K ב) שלו הוא גדול בהרבה מזה של מדינת פעיל (K). סיכום שליציאות אקראיות של כל הקולטנים באוכלוסייה התשואות על רמה קבועה של הפעלת קולטן בזמן. הגומלין של ריכוז של אגוניסט לעורר חצי מקסימלי הפעלת קולטן שווה מתמיד את הזיקה שנצפו (K obs), וכן את החלק היחסי של אגוניסט קולטן מתחמי במצב פעיל מוגדר יעילות (ε) (איור 2). שיטות לניתוח התגובות במורד הזרם של GPCRs פותחו המאפשרים הערכה של K obs ואת היעילות היחסית של 1,2 אגוניסט. בדו&quot;ח זה, אנו מראים כיצד לשנות את הניתוח הזה כדי להעריך את ערך K אגוניסט b ביחס לזה של אגוניסט אחר. עבור מבחני כי התערוכה פעילות המכונן, אנו מראים כיצד להעריך K b ביחידות המוחלט של M -1. השיטה שלנו לניתוח ריכוז בתגובה אגוניסט עקומות 3,4 מורכברגרסיה ליניארית הגלובלית באמצעות מודל מבצעי 5. אנו מתארים הליך באמצעות יישום תוכנה, פריזמה (GraphPad תוכנה, Inc, סן דייגו, קליפורניה). ניתוח התשואות אומדן של המכפלה של K ו obs פרמטר יחסי היעילות (τ). אומדן τK obs של אגוניסט אחד, מחולק כי אחרת, היא מדד יחסי של K b (ע&quot;ר i) 6. במשך כל קולטן מפגין פעילות מכוננת, אפשר להעריך פרמטר יחסי היעילות של קומפלקס הקולטן חינם (τ sys). במקרה זה, הערך של K b אגוניסט שווה τK obs / τ sys 3. השיטה שלנו הוא שימושי לקביעת הסלקטיביות של אגוניסט עבור תת קולטן לכימות אגוניסט קולטן חלבוני איתות דרך שונה G.

Watch this Scientific Journal Video about Quantifying Agonist Activity at G Protein-coupled Receptors at JoVE.com

Quantifying Agonist Activity at G Protein-coupled Receptors

שיטה להערכת הזיקה המתמדת של אגוניסט למדינה פעיל (<em&gt; K<subB&gt;</sub</em&gt;) של קולטני G-חלבון בשילוב מתואר. הניתוח מספק אמצעים מוחלט או יחסי של<em&gt; K<subB&gt;</sub</em&gt; תלוי אם הפעלת קולטן המכונן הוא מדיד. השיטה שלנו חל במורד התגובות השונות של הפעלת הקולטן.

כימות פעילות אגוניסט ב G-חלבון יחד רצפטורים

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

quantifying-agonist-activity-at-g-protein-coupled-receptors

Research

JoVE Journal

Biology

18.3K Views.  University of California, Irvine. שיטה להערכת הזיקה המתמדת של אגוניסט למדינה פעיל ( K

Video: Quantifying Agonist Activity at G Protein-coupled Receptors

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a major pharmacological target for multiple indications, including analgesia, blood pressure regulation, and the treatment of psychiatric disease. Upon ligand binding, GPCRs catalyze the activation of intracellular G-proteins by stimulating the incorporation of guanosine triphosphate (GTP). Activated G-proteins then stimulate signaling pathways that elicit cellular responses. GPCR signaling can be monitored by measuring the incorporation of a radiolabeled and non-hydrolyzable form of GTP, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), into G-proteins. Unlike other methods that assess more downstream signaling processes, [35S]GTP&#947;S binding measures a proximal event in GPCR signaling and, importantly, can distinguish agonists, antagonists, and inverse agonists. The present protocol outlines a sensitive and specific method for studying GPCR signaling using crude membrane preparations of an archetypal GPCR, the &#181;-opioid receptor (MOR1). Although alternative approaches to fractionate cells and tissues exist, many are cost-prohibitive, tedious, and/or require non-standard laboratory equipment. The present method provides a simple procedure that enriches functional crude membranes. After isolating MOR1, various pharmacological properties of its agonist, [D-Ala, N-MePhe, Gly-ol]-enkephalin (DAMGO), and antagonist, naloxone, were determined.

Guanosine triphosphate (GTP) binding is one of the earliest events in G-Protein-Coupled Receptor (GPCR) activation. This protocol describes how to pharmacologically characterize specific GPCR-ligand interactions by monitoring the binding of the radio-labeled GTP analog, [35S]guanosine-5&#39;-O-(3-thio)triphosphate ([35S]GTP&#947;S), in response to a ligand of interest.

מדידת G- חלבון מצמידים איתות באמצעות רדיו שכותרתו GTP מחייב

G-Protein-Coupled Receptors (GPCRs) are a large family of transmembrane receptors that play critical roles in normal cellular physiology and constitute a ...

Biochemistry

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

רגישות סמים של adenylyl cyclase: התייעלות Assay ומזעור למולקולה קטנה ויישומים הקרנת siRNA

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Bioengineering

Stepwise Dosing Protocol for Increased Throughput in Label-Free Impedance-Based GPCR Assays

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach provides real-time cell monitoring with a device-dependent time resolution down to several tens of milliseconds and it is highly automated. However, when sample numbers get high (e.g., dose-response studies for various different ligands), the cost for the disposable electrode arrays as well as the available time resolution for sequential well-by-well recordings may become limiting. Therefore, we here present a serial agonist addition protocol which has the potential to significantly increase the output of label-free GPCR assays. Using the serial agonist addition protocol, a GPCR agonist is added sequentially in increasing concentrations to a single cell layer while continuously monitoring the sample&#39;s impedance (agonist mode). With this serial approach, it is now possible to establish a full dose-response curve for a GPCR agonist from just one single cell layer. The serial agonist addition protocol is applicable to different GPCR coupling types, Gq Gi/0 or Gs and it is compatible with recombinant and endogenous expression levels of the receptor under study. Receptor blocking by GPCR antagonists is assessable as well (antagonist mode).

This protocol demonstrates real-time recording of full dose-response relationships for agonist-induced GPCR activation from a single cell layer grown on a single microelectrode using label-free impedance measurements. The new dosing scheme significantly increases throughput without loss in time resolution.

פרוטוקול מינון סטפנסקי עבור תפוקה מוגברת ב-GPCR-מבוסס-תוויות חינם

Label-free impedance-based assays are increasingly used to non-invasively study ligand-induced GPCR activation in cell culture experiments. The approach ...

Parallel Interrogation of &#946;-Arrestin2 Recruitment for Ligand Screening on a GPCR-Wide Scale using PRESTO-Tango Assay

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent one of the most promising targets for the pharmaceutical industry. Ergo, the design, implementation, and optimization of GPCR ligand screening assays is crucial, as they represent remote-control tools for drug discovery and for manipulating GPCR pharmacology and outcomes. In the past, G-protein dependent assays typified this area of research, detecting ligand-induced events and quantifying the generation of secondary messengers. However, since the advent of functional selectivity, as well as an increased awareness of several other G protein-independent pathways and the limitations associated with G-protein dependent assays, there is a greater push towards the creation of alternative GPCR ligand screening assays. Towards this endeavor, we describe the application of one such resource, the PRESTO-Tango platform, a luciferase reporter-based system that enables the parallel and simultaneous interrogation of the human GPCR-ome, a feat which was previously considered technically and economically unfeasible. Based on a G-protein independent &#946;-arrestin2 recruitment assay, the universality of &#946;-arrestin2-mediated trafficking and signaling at GPCRs makes PRESTO-TANGO an apt tool for studying approximately 300 non-olfactory human GPCRs, including approximately 100 orphan receptors. PRESTO-Tango&#39;s sensitivity and robustness make it suitable for primary high-throughput screens using compound libraries, employed to uncover new GPCR targets for known drugs or to discover new ligands for orphan receptors.

Given that GPCRs are attractive druggable targets, GPCR ligand screening is thus indispensable for the identification of lead compounds and for deorphanization studies. Towards these efforts, we describe PRESTO-Tango, an open-source resource platform used for simultaneous profiling of transient &#946;-arrestin2 recruitment at approximately 300 GPCRs using a TEV-based reporter assay.

חקירה מקבילה של גיוס β-Arrestin2 עבור ליגיון הקרנה בקנה מידה מוקטן של הסרט באמצעות שיטת הטנגו-פרסטו

As the largest and most versatile gene superfamily and mediators of a gamut of cellular signaling pathways, G-protein-coupled receptors (GPCRs) represent ...

A "Dual-Addition" Calcium Fluorescence Assay for the High-Throughput Screening of Recombinant G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening (HTS) of random small molecule libraries against GPCRs is used by the pharmaceutical industry for target-specific drug discovery. In this study, an HTS was employed to identify novel small-molecule ligands of invertebrate-specific neuropeptide GPCRs as probes for physiological studies of vectors of deadly human and veterinary pathogens.
The invertebrate-specific kinin receptor was chosen as a target because it regulates many important physiological processes in invertebrates, including diuresis, feeding, and digestion. Furthermore, the pharmacology of many invertebrate GPCRs is poorly characterized or not characterized at all; therefore, the differential pharmacology of these groups of receptors with respect to the related GPCRs in other metazoans, especially humans, adds knowledge to the structure-activity relationships of GPCRs as a superfamily. An HTS assay was developed for cells in 384-well plates for the discovery of ligands of the kinin receptor from the cattle fever tick, or southern cattle tick, Rhipicephalus microplus. The tick kinin receptor was stably expressed in CHO-K1 cells.
The kinin receptor, when activated by endogenous kinin neuropeptides or other small molecule agonists, triggers Ca2+ release from calcium stores into the cytoplasm. This calcium fluorescence assay combined with a &#34;dual-addition&#34; approach can detect functional agonist and antagonist &#34;hit&#34; molecules in the same assay plate. Each assay was conducted using drug plates carrying an array of 320 random small molecules. A reliable Z&#39; factor of 0.7 was obtained, and three agonist and two antagonist hit molecules were identified when the HTS was at a 2 &#181;M final concentration. The calcium fluorescence assay reported here can be adapted to screen other GPCRs that activate the Ca2+ signaling cascade.

In this work, a high-throughput, intracellular calcium fluorescence assay for 384-well plates to screen small molecule libraries on recombinant G protein-coupled receptors (GPCRs) is described. The target, the kinin receptor from the cattle fever tick, Rhipicephalus microplus, is expressed in CHO-K1 cells. This assay identifies agonists and antagonists using the same cells in one "dual-addition" assay.

בדיקת סידן פלואורסצנטית "דו-תוספת" לבדיקת תפוקה גבוהה של קולטנים מצומדים לחלבון G רקומביננטי

G protein-coupled receptors (GPCRs) represent the largest superfamily of receptors and are the targets of numerous human drugs. High-throughput screening ...

<meta charset="utf8"></head><body>חקר ליגנדות קולטן המופעלות על ידי פרוטאז בתאי אנדותל

Characterizing Modulators of Protease-Activated Receptors with a Calcium Mobilization Assay Using a Plate Reader

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding dyes and imaging instruments that can measure fluorescent emissions from up to 1,536 wells simultaneously. However, these instruments are much more expensive and can be challenging to maintain relative to widely available plate readers that scan wells individually. Described here is an optimized plate reader assay for use with an endothelial cell line (EA.hy926) to measure the protease-activated receptor (PAR)-driven activation of G&#945;q signaling and subsequent calcium mobilization using the calcium-binding dye Fluo-4. This assay has been used to characterize a range of PAR ligands, including the allosteric PAR1-targeting anti-inflammatory "parmodulin" ligands identified in the Dockendorff lab. This protocol obviates the need for an automated liquid handler and permits the medium-throughput screening of PAR ligands in 96-well plates and should be applicable to the study of other receptors that initiate calcium mobilization.

<meta charset="utf8"></head><body>Author Spotlight: פיתוח פרמודולינים כדי להתמקד בקולטנים המופעלים על ידי פרוטאז לשליטה בדלקת

An improved protocol for a calcium mobilization assay with endothelial cells, used to identify ligands of protease-activated receptors (PARs), has been developed. The new protocol reduces total assay time by 90-120 min and yields reproducible concentration-response curves.

אפיון אפנן קולטנים המופעלים על ידי פרוטאז עם בדיקת ניוד סידן באמצעות קורא צלחות

Changes in calcium concentration in cells are rapidly monitored in a high-throughput fashion with the use of intracellular, fluorescent, calcium-binding ...

Methods for the Discovery of Novel Compounds Modulating a Gamma-Aminobutyric Acid Receptor Type A Neurotransmission

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the identification of novel molecules active in preclinical assays from an in vitro recombinant receptor to their pharmacological effects at native receptors in rodent brain slices. For compounds binding at the benzodiazepine site of the receptor, the first step is to set up a primary screen that consists of developing radioligand binding assays on cell membranes expressing the major GABAA subtypes. Then, taking advantage of the heterologous expression of rodent and human GABAA receptors in Xenopus oocytes or HEK 293 cells, it is possible to explore, in electrophysiological assays, the physiological properties of the different receptor subtypes and the pharmacological properties of the identified compounds. The Xenopus oocyte system will be presented here, starting with the isolation of the oocytes and their microinjection with different mRNAs, up to the pharmacological characterization using two-electrode voltage clamps. Finally, recordings conducted in rodent brain slices will be described that are used as a secondary physiological test to assess the activity of molecules at their native receptors in a well-defined neuronal circuit. Extracellular recordings using population responses of multiple neurons are demonstrated together with the drug application.

Here, we present protocols to discover compounds active at GABAA receptors, from the binding to the physiology and pharmacology.

שיטות הגילוי של תרכובות הרומן להתכוונן קולטן גאמא אמינו בוטירית הקלד עצבית

This manuscript presents a step-by-step protocol for screening compounds at gamma-aminobutyric acid type A (GABAA) receptors and its use towards the ...

Neuroscience

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

G protein-coupled receptors (GPCRs) are of great importance to the pharmaceutical industry as they are involved in many human diseases and include well-validated targets for therapeutic intervention. Discovery of lead compounds, including small synthetic molecules, that specifically inhibit the receptor&#39;s function, is an important initial step in drug development and relies on sensitive, specific, and robust cell-based assays. Here, we describe a kinetic cellular assay with a fluorescent readout primarily designed to identify receptor-specific antagonists that inhibit the intracellular Ca2+ release evoked upon the activation of the CXC chemokine receptor 4 (CXCR4) by its endogenous ligand, the CXC chemokine ligand 12 (CXCL12). A key advantage of this method is that it also enables screening of compounds endowed with intrinsic agonistic properties (i.e., compounds eliciting an increase in intracellular Ca2+ concentration in the absence of CXCL12) or compounds modulating the receptor&#39;s function via interaction with allosteric binding sites (i.e., positive and negative allosteric modulators (PAMs and NAMs, respectively)). On the down side, autofluorescent compounds might interfere with the assay&#39;s readout, thereby hampering reliable data interpretation. Most likely this assay can be implemented, with minimal adaptations, as a generic drug discovery assay for many other GPCRs of which the activation leads to a release of intracellular Ca2+.

A Kinetic Fluorescence-based Ca2+ Mobilization Assay to Identify G Protein-coupled Receptor Agonists, Antagonists, and Allosteric Modulators

The described cellular assay is designed for the identification of CXC chemokine receptor 4 (CXCR4)-interacting agents that inhibit or stimulate, either competitively or allosterically, the intracellular Ca2+ release initiated by CXCR4 activation.

כימות פעילות אגוניסט ב G-חלבון יחד רצפטורים

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אפיון אפנן קולטנים המופעלים על ידי פרוטאז עם בדיקת ניוד סידן באמצעות קורא צלחות

שיטות הגילוי של תרכובות הרומן להתכוונן קולטן גאמא אמינו בוטירית הקלד עצבית

קינטי מבוסס על-ידי קרינה פלואורסצנטית Ca^{2 +} גיוס Assay לזהות את G-חלבון בשילוב קולטן אגוניסטים היריבים, מאפננים Allosteric

כימות פעילות אגוניסט ב G-חלבון יחד רצפטורים

Summary

Explore More Videos

Chapters in this video

מדידת G- חלבון מצמידים איתות באמצעות רדיו שכותרתו GTP מחייב

רגישות סמים של adenylyl cyclase: התייעלות Assay ומזעור למולקולה קטנה ויישומים הקרנת siRNA

פרוטוקול מינון סטפנסקי עבור תפוקה מוגברת ב-GPCR-מבוסס-תוויות חינם

חקירה מקבילה של גיוס β-Arrestin2 עבור ליגיון הקרנה בקנה מידה מוקטן של הסרט באמצעות שיטת הטנגו-פרסטו

בדיקת סידן פלואורסצנטית "דו-תוספת" לבדיקת תפוקה גבוהה של קולטנים מצומדים לחלבון G רקומביננטי

אפיון אפנן קולטנים המופעלים על ידי פרוטאז עם בדיקת ניוד סידן באמצעות קורא צלחות

אפיון אפנן קולטנים המופעלים על ידי פרוטאז עם בדיקת ניוד סידן באמצעות קורא צלחות

אפיון אפנן קולטנים המופעלים על ידי פרוטאז עם בדיקת ניוד סידן באמצעות קורא צלחות

שיטות הגילוי של תרכובות הרומן להתכוונן קולטן גאמא אמינו בוטירית הקלד עצבית

קינטי מבוסס על-ידי קרינה פלואורסצנטית Ca2 + גיוס Assay לזהות את G-חלבון בשילוב קולטן אגוניסטים היריבים, מאפננים Allosteric

קינטי מבוסס על-ידי קרינה פלואורסצנטית Ca^{2 +} גיוס Assay לזהות את G-חלבון בשילוב קולטן אגוניסטים היריבים, מאפננים Allosteric