receptor characterization

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different methods of characterizing receptors


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RECEPTOR CHARACTERIZATION METHODS PRESENTED BY: M.SAI PRIYA Ms.pharm Department of Regulatory toxicology NIPER H yderabad 1


Introduction Characterization of receptors involve either assessment of their ability to bind ligands or measure receptor activation as a result of agonist or inverse agonist interactions Multitude of techniques are available for measuring receptor activity in cells and membranes, prior to solubilization The detection methods involve electrochemical or fluorescence measurments These provides both quantitative & qualitative measurements (presence and extent of ligand binding, receptor activation ,function and their binding kinetics R.uddin, J.simms, D.poyner, Functional characterisation of receptors, 2018; 1046-2023(17 ) 2 2/10/2019

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Radio immunoassay Fluorescence polarisation Fluorescence resonance energy transfer Surface plasmon resonance 3 2/10/2019 DETECTION METHODS De Jong LA, Uges DR, Franke JP, Bischoff R. Receptor–ligand binding assays: technologies and applications. Journal of Chromatography . 2005 Dec 27;829(1-2):1-25.

Radio immunoassay (RIA):

Radio immunoassay (RIA) RIA was developed in 1959 by Rosalyn Yalow and Solomon Berson for measurement of insulin in plasma and received Noble prize in 1977 Radio immunoassay is based upon the competition between labeled and unlabeled antigen for specific antibody sites, forming antigen-antibody complexes Radio ligands are used to measure the ligand binding to receptors and have high affinity, high specific activity to detect low receptor densities, and receptor specificity It is a very sensitive technique and measures about (10 -12 ) concentration of antigen 4 2/10/2019 Kumar V, Gill KD. To Perform Radioimmunoassay. In Basic Concepts in Clinical Biochemistry: A Practical Guide 2018 (pp. 151-152).

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Ranjan K, Minakshi P, Prasad G. Application of molecular and serological diagnostics in veterinary parasitology. J. Adv. Parasitol. 2015;2(4):80-99. 5 2/10/2019


Applications Characterization of enkephalins (opiate like penta peptides) Characterisation of human interferon αβ receptor Binding of 125I– Human growth harmone to specific receptors in human cultured lymphocytes Expression of C-type natriuretic peptide and its specific guanylyl cyclase-coupled receptor characterization of signal transduction pathways in α 1C-adrenergic receptor 6 2/10/2019 Kim SM, Kim SH, Cho KW, Young S. Expression of C-type Natriuretic Peptide and its Specific Guanylyl Cyclase-Coupled Receptor in Pig Ovarian Granulosa Cells.


Limitations Lack of direct linear relationship between analyte concentration and signal response Difficulty of automation Prolonged reaction time as a consequence of highly diluted reagent is used Lengthy counting time Possible health hazards due to handling of radioisotopes All the reagents must be added precisely Short shelf-life of radio labeled compounds 7 2/10/2019

Fluorescence polarisation (FP):

Fluorescence polarisation (FP) Fluorescence polarisation is a homogenous, versatile solution-based technique that has been widely used to study molecular interactions It was first theoretically described by PERRIN in 1926 The principle of assay is that a fluorescent dye attached to (an antigen or an antibody fragment) can be exited by plane polarised light at the appropriate wavelength The change in polarisation is proportional to the amount of antigen in sample, and is measured by an analyser 8 2/10/2019 Klein C, Batz HG, Draeger B, Guder HJ, Herrmann R, Josel HP, Nägele U, Schenk R, Vogt B. Fluorescence Polarisation Immunoassay. InFluorescence Spectroscopy 2013 (pp. 245-258).

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9 2/10/2019 Klein C,, Fluorescence Polarisation Immunoassay. InFluorescence Spectroscopy 1993 (pp. 245-258).


APPLICATIONS Identification and characterization of a selective peroxisome proliferative activated receptor β / δ (NR1C2) antagonist Characterization of Fluorescent-Labeled Selective Adenosine A 2B Receptor Verification of in Vitro Folding of the Y2 G Protein-Coupled Receptor Functional characterisation of GPCR 10 Schmidt P,,Improved in Vitro Folding of the Y2 G Protein-Coupled Receptor into Bicelles. Frontiers in molecular biosciences. 2018 Jan 17;4:100. 2/10/2019

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Leigh A stoddart, fluorescence and bioluminiscence based approaches to study GPCR ligand binding 2015 Nov 5; 173(20):3028-3037. 11 2/10/2019

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Advantages: Homogenous assay format Reduced assay time and cost Suitable for short inter/intra molecular distances It is a non- radiative process Disadvantages: Requires multiple labels Sensitivity to inner-filter and auto-fluorescence Limited to short distances to obtain high signal changes 12 2/10/2019

Fluorescence resonance energy transfer (FRET):

Fluorescence resonance energy transfer (FRET) FRET involves the transfer of excitation energy of a donor fluorophore in a non-radiative fashion through dipole—dipole interactions to an acceptor fluorophore The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor This makes FRET extremely sensitive to small changes in distance When donor and acceptor are in proximity (1-10nm) due to interactions of two molecules, the acceptor emission will increase because of intermolecular FRET from donor to acceptor FRET can be measured by the quenching of donor fluorescence and a reduction of the fluorescence life time, accompanied by increase in acceptor fluorescence emission 13 2/10/2019 Klein C, et. al, Fluorescence Polarisation Immunoassay. InFluorescence Spectroscopy 1993 (pp. 245-258).

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O.K.Castell, single molecule fluorescence for membrane proteins, methods. 2018 May 23; S1046-2023(17)30433-4 . 15 2/10/2019


Applications Visualization of the activation of the histamine H3 receptor (H3R) Detection of β2-adrenergic receptor dimerization in living cells Enhanced Fluorescence Resonance Energy Transfer in characterization of GPCR α4β3δ GABA Receptors Characterization by Fluorescence Resonance Energy Transfer Characterization of Serotonin Receptors in Response to Ligand Binding 16 2/10/2019 Schreiber B,, Boost Fluorescence Resonance Energy Transfer (FRET) Studies of Membrane Receptors in Live Cells. Biophysical Journal. 2018 Feb 2;114(3):165a.

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Advantages: Simple, predictive Insensitive to inner-filter effects Suitable for small ( < 10 k Da ) ligands It does not require washing and separation steps Disadvantages: Suffer from auto-fluorescence Local motion effects Limited by lifetime of dye, ligand size, charge, molecular weight 17 2/10/2019

Surface Plasmon resonance (SPR):

Surface Plasmon resonance (SPR) SPR is the surface sensitive spectroscopic method which measures the changes in refractive index of the medium directly in contact with the sensor surface As the molecules bind and dissociate from the sensor surface, the resulting changes in resonance signal creates a sensor gram Sensor gram describe the changes in SPR signal versus time The method allows monitoring of many label-free molecular interactions and their kinetic rates and binding affinities 18 2/10/2019 Willets KA, Van Duyne RP. Localized surface Plasmon resonance spectroscopy and sensing. Annu. Rev. Phys. Chem.. 2007 May 5;58:267-97.

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19 2/10/2019 Homola J, Yee SS, Gauglitz G. Surface Plasmon resonance sensors. Sensors and Actuators B: Chemical. 2010 Jan 25;54(1-2):3-15.

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20 2/10/2019 Green RJ, Frazier RA, Shakesheff KM, Davies MC, Roberts CJ, Tendler SJ. Surface Plasmon resonance analysis of dynamic biological interactions with biomaterials. Biomaterials. 2000 Sep 1;21(18):1823-35.

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Patching SG. Surface Plasmon resonance spectroscopy for characterisation of membrane protein–ligand interactions and its potential for drug discovery. Biochimica et Biophysica Acta (BBA)-Biomembranes. 2014 Jan 1;1838(1):43-55. 21 2/10/2019


Applications Direct analysis of GPCR agonist interaction by surface Plasmon resonance Characterizing the signaling potential of GPCR by selectively acting on β 2 adrenoreceptors Characterization of murine IL-13 receptor α 2 Allosteric coupling of drug binding and intracellular signaling in A2A adenosine receptor 22 2/10/2019 Winter G, Vogt A, Glatting G, Kletting P, Beer A. Characterization of the receptor binding kinetics of PSMA-specific peptides by Surface Plasmon Resonance Spectroscopy. Journal of Nuclear Medicine. 2018 May 1;59(supplement 1):1128


Limitations SPR detection decreases exponentially with distance from surface Estimated detection limit is ~200nm SPR detection relies on mass changes Refractive index is temperature dependent Non-specific interactions also results in SPR signal 23 2/10/2019

Advanced techniques:

Advanced techniques BRET(Bioluminiscence resonance energy transfer) TR-FRET(Time resolved resonance energy transfer) SCAM(Saturation cystein scanning mutagenesis) Isothermal titration calorimetry 24 2/10/2019 Wild KD, Porreca F, Yamamura HI, Raffa RB. Differentiation of receptor subtypes by thermodynamic analysis: application to opioid delta receptors. 2004 Dec 6;91(25):12018-21 .


Summary Receptor characterization assess the extent of binding of ligand with receptor or measures receptor activation, binding kinetics and affinity Many detection methods such as fluorescence, thermal and electrochemical methods are available Recent advances includes sensors and detectors which show more sensitivity and specificity Identification of receptors which in turn are used to detect the drug targets 25 2/10/2019

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