LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMOSTRATION. Financed by the program

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1 TÁMOP C-13/1/KONV projekt Az élettudományi-klinikai felsőoktatás gyakorlatorientált és hallgatóbarát korszerűsítése a vidéki képzőhelyek nemzetközi versenyképességének erősítésére program keretében finanszírozott LABORATÓRIUMI GYAKORLAT SILLABUSZ SYLLABUS OF A PRACTICAL DEMOSTRATION Financed by the program Practice-oriented, student-friendly modernization of the biomedical education for strengthening the international competitiveness of the rural Hungarian universities Dátum / Date: NOVEMBER 2. / NOVEMBER 2, 2017 Helyszín / Place: LAB # 135 Gyakorlati foglalkozás címe / Title of the practical demonstration: Measuring the binding affinity and agonist activity of opioid compounds with in vitro binding assays Gyakorlatvezető / Demonstrator: Edina Szűcs, Ferenc Zádor Biological Research Centre Address: H-6726 Szeged, Temesvári krt. 62. Mail: H-6701 Szeged, POB

2 Module # 1 Principles of in vitro binding assays The basis of the assays is to add a radioactive compound to a protein homogenate, membrane fraction, cell culture etc. and then incubate them together in certain conditions until the equilibrium binding is reached. The experimental conditions of the equilibrium binding are always defined by the applied radiochemical. Afterwards the bound and free radioactive ligands can be separated from each other; therefore, the amount of bound radioactive ligand can be measured. In this practice we will demonstrate two binding assays, radioligand competition and [ 35 S]GTP S binding assay to examine ligand binding affinity and agonist activity, respectively. page 2

3 Module # 2 Radioligand competition binding assays: Background The radioligand competition binding experiment is a type of binding assay, where we apply the radioactive ligand (in most cases tritium labeled) in fixed concentrations in the presence of increasing concentrations of an unlabeled ligand. If the unlabeled ligand has specificity towards the receptor as the radioligand, they will compete with each other for the same binding site and the unlabeled ligand will displace the radioligand in a concentration dependent manner. The displacement will be indicated by the reduced detected radioactivity in the sample. This way we can receive binding affinity information about the applied unlabeled ligand. see presentation! page 3

4 Module # 3 Functional [ 35 S]GTP S binding assay: Background During [ 35 S]GTPγS binding assays we monitor the target receptor mediated G-protein activation, namely the GDP GTP exchange of G, in the presence of an agonist ligand in increasing concentration. The nucleotide exchange is detected by a non-hydrolysable, radioactive GTP analogue called [ 35 S]GTPγS, which contains a sulphur 35 isotope ( 35 S) in the γ phosphate group instead of an oxygen atom. Because of the γ-thiophosphate bound the [ 35 S]GTPγS withstand the GTPase activity of G, thus it cannot hydrolyze to GDP and the G-protein cannot reassociate to a heterotrimer. As a consequence the G bound with [ 35 S]GTPγS accumulates and we can measure the incorporated 35 S radioactivity in the samples. The higher the incorporated 35 S the higher is the agonist activity of the examined compound, since it stimulated the nucleotide exchange in a greater extent. see presentation! page 4

5 Module # 4 Opioid receptor source for binding assays homogenates of membrane fractions (see simplified figure) are applied (since opioid receptors are transmembrane receptor proteins) usually rat (see figure) or mice brain/spinal cord samples are used for κ type opioid receptor studies guinea pig brain or spinal cord samples are used because rats and mice express κ opioid receptors in very low quantities samples are processed by multiple centrifugation and homogenization steps to obtain membrane fractions for the assays the samples are processed in Tris-HCl buffer (ph 7.4) to maintain the protein structure for [ 35 S]GTPγS assays Tris-HCl, EDTA, MgCl 2 buffer is used during preparation to allow the nucleotide exchange process during equilibrium binding page 5

6 Module # 5 Experimental design Purpose of the experiments: to compare the affinity and agonist activity of two opioid ligands, morphine and a structurally modified morphine (14-O-methylmorphine) Experimental setup for competition binding assays: morphine and the morphine analogue in increasing concentrations 10 concentration points for both ligands (0.1 nm 10 µm) 1 point for total specific binding (without ligands) and 1 point for non-specific binding (10 µm naloxone antagonist) each point in 2 parallels (24 tubes in total/ligand) see demonstration! [ 3 H]DAMGO in fix concentration (~ 1 nm) ~0.5 mg/ml/tube membrane fraction 1 ml final volume for each tube assays are performed in Tris-HCl (ph. 7.4) Experimental setup for [ 35 S]GTPγS assays: similar setup, but 6 concentration points for each ligand with similar concentration range for non-specific binding 10 µm unlabeled [ 35 S]GTPγS is used each point in 3 parallels (24 tubes in total/ligand) see demonstration! [ 35 S]GTPγS in fixed concentrations (~0.05 nm) 30 µm GDP to maintain the nucleotide exchange ~ 10 µg/ml/tube membrane fraction 1 ml final volume for each tube assays are performed in Tris-HCl, EDTA, MgCl 2 buffer (ph. 7.4) page 6

7 Module # 6 Applied radiochemicals: [ 3 H]DAMGO and [ 35 S]GTPγS [ 3 H]DAMGO: [ 3 H][D-Ala 2, N-MePhe 4, Gly-ol]-enkephalin synthetic opioid peptide high µ opioid receptor specificity more stable than normal endogenous opioids tritium labeled (red asterisks) incubation time and temperature: 45 min. 35 o C [ 35 S]GTPγS: sulphur 35 isotope ( 35 S) in the γ phosphate group instead of an oxygen atom (red asterisk) non-hydrolysable binds to G α incubation time and temperature: 60 min. 30 o C page 7

8 Module # 7 Separating the bound and free radiochemicals and sample measurements Separation: samples are filtered by rapid vacuum filtration through fiberglass filters using a cell harvester (see figure) see demonstration! washed with Tris buffer (ph 7.4) 3 times the thickness of the fiberglass filter is defined by the radiochemical ([ 3 H]DAMGO thinner, [ 35 S]GTPγS thicker) Measuring the samples: the radioactivity of each sample is detected in a liquid scintillator (see figure) using a scintillator cocktail see demonstration! page 8

9 Module # 8 Data analysis non-specific binding is subtracted from total binding to obtain specific binding total specific binding in both binding assays is normalized to 100% the radioactivity of the specifically bound radiochemicals are presented in the function of the applied ligand concentration in a logarithmic scale data points are fitted using non-linear regression to determine the parameters with GraphPad Prism 5 curve fitting program see demonstration! logic 50 value (binding affinity) of the ligands in competition binding, the lower the value the higher the affinity E max value (maximum efficacy) and logec 50 value (ligand potency) describes the agonist activity of the ligands (lower logec 50 means higher potency, similar to logic 50 ) page 9

10 Module # 9 Interpreting the results [ 3 H]DAMGO specific binding (%) Total IC 50 : 0.8 nm Morphine 14-O-methylmorphine IC 50 : 4.5 nm Level of total specific binding log[ligand] (M) the structure modification increased the µ opioid receptor affinity, because the analogue needs lower concentrations to reach 50% inhibition of radioligand specific binding also it increased the maximum ability (efficacy) to stimulate the G-protein and needs lower concentrations to achieved this compared to morphine [ 35 S]GTP S specific binding (%) Morphine 14-O-methylmorphine EC 50 : 77.8 nm EC 50 : nm Level of basal activity E max : 173 % E max : % Basal log[ligand] (M) page 10