Measuring Receptor Target Coverage: A Radioligand Competition Binding Protocol for Assessing the Association and Dissociation Rates of Unlabeled Compounds

David A. Sykes1, Mark R. Dowling1, Steven J. Charlton1

1 Novartis Institutes for Biomedical Research, Horsham, West Sussex, United Kingdom
Publication Name:  Current Protocols in Pharmacology
Unit Number:  Unit 9.14
DOI:  10.1002/0471141755.ph0914s50
Online Posting Date:  September, 2010
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Abstract

The kinetics of the ligand‐receptor interaction is an important feature in lead optimization for new drug candidates. The protocol described in this unit is a kinetic radioligand competition binding assay that makes possible the determination of both the association and dissociation rates of unlabeled receptor ligands. Curr. Protoc. Pharmacol. 50:9.14.1‐9.14.30. © 2010 by John Wiley & Sons, Inc.

Keywords: kinetics; radioligand binding; receptor; association rate; dissociation rate

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Determination of Bmax and Kd Values by Radioligand Saturation
  • Basic Protocol 2: Determination of the Dissociation Rate (koff) of a Radiolabeled Ligand from a Receptor
  • Basic Protocol 3: Determination of the Observed Association Rate (kob) of the Radiolabel
  • Basic Protocol 4: Competition Kinetics Between [3H]NMS and Unlabeled Ligands
  • Support Protocol 1: Preparation of Receptor Membranes from Cultured Cells
  • Support Protocol 2: Determination of Unlabeled Competitor Ki Values
  • Support Protocol 3: Calculation of Kd and Bmax from Saturation Experiments
  • Support Protocol 4: Calculation of Unlabeled Competitor Ki Values from Competition Experiments
  • Support Protocol 5: Calculation of the Dissociation Rate (koff) of [3H]NMS
  • Support Protocol 6: Calculation of the Association Rate (kon) of [3H]NMS
  • Support Protocol 7: Calculation of the Kinetic Rate Constants of Unlabeled Ligands from Competition Binding Experiments
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Determination of Bmax and Kd Values by Radioligand Saturation

  Materials
  • Radioligand: e.g., 12 µM 3H‐labeled N‐methyl scopolamine (80 to 82 Ci/mmol; PerkinElmer)
  • Assay buffer (see recipe)
  • 10 µM (10×) atropine stock solution (Sigma‐Aldrich)
  • CHO‐M 3 membrane preparation (see protocol 5 and other units in Chapter 1 for detailed methods on preparing membranes containing various receptor populations)
  • 0.5% (w/v) polyethyleneimine (see recipe)
  • Wash buffer (see recipe), ice cold
  • Scintillation cocktail (e.g., PerkinElmer Microscint 20 or Ultima‐Flo AP)
  • 96‐well deep‐well polypropylene plates (Fisher Scientific)
  • Multichannel pipettor and reservoir (e.g., Matrix)
  • 8‐ml and 30‐ml polypropylene screw‐top bottles (Fisher Scientific)
  • TopSeal‐A plate sealers (PerkinElmer)
  • Filtration cell harvester (e.g., FilterMate, PerkinElmer)
  • 96‐well unifilter GF/B filter plates with filter backs (Receptor Technologies, http://www.receptortechnologies.co.uk/)
  • Temperature controlled laboratory oven
  • Scintillation counters (TopCount from PerkinElmer, plus Beckman LS 6500 scintillation counter)
  • 5‐ml scintillation vials

Basic Protocol 2: Determination of the Dissociation Rate (koff) of a Radiolabeled Ligand from a Receptor

  Materials
  • Radioligand: e.g., 12 µM 3H‐labeled N‐methyl scopolamine (80 to 82 Ci/mmol; PerkinElmer)
  • Receptor preparation of interest (e.g., CHO‐M 3 membranes, see protocol 5)
  • Assay buffer (see recipe)
  • 1.33 µM (1.33×) atropine stock solution (Sigma‐Aldrich)
  • 0.5% (w/v) polyethyleneimine (see recipe)
  • Wash buffer (see recipe), ice cold
  • Scintillation cocktail (e.g., PerkinElmer Microscint 20 or Ultima‐Flo AP)
  • 30‐ml polypropylene screw‐top bottles (Fisher Scientific)
  • Multichannel pipettor and reservoirs (e.g., Matrix)
  • 96‐well deep‐well polypropylene plates (Fisher Scientific)
  • TopSeal‐A plate sealers (PerkinElmer)
  • Electronic timer
  • Filtration cell harvester (e.g., FilterMate, PerkinElmer)
  • 96‐well unifilter GF/B filter plates with filter backs (Receptor Technologies, http://www.receptortechnologies.co.uk/)
  • Temperature controlled laboratory oven
  • Scintillation counters (TopCount from PerkinElmer, plus Beckman LS 6500 scintillation counter)
  • 5‐ml scintillation vials

Basic Protocol 3: Determination of the Observed Association Rate (kob) of the Radiolabel

  Materials
  • Assay buffer (see recipe)
  • 5 µM (5×) atropine stock solution (Sigma‐Aldrich)
  • Radioligand: e.g., 12 µM 3H‐labeled N‐methyl scopolamine (80 to 82 Ci/mmol; PerkinElmer)
  • Receptor preparation of interest (e.g., CHO‐M 3 membranes see protocol 5)
  • 0.5% (w/v) polyethyleneimine (see recipe)
  • Wash buffer (see recipe), ice cold
  • Scintillation cocktail (e.g., PerkinElmer Microscint 20 or Ultima‐Flo AP)
  • 96‐well deep‐well polypropylene plates (Fisher Scientific)
  • Multichannel pipettor and reservoirs (e.g., Matrix)
  • 8‐ and 30‐ml polypropylene screw‐top bottles (Fisher Scientific)
  • Electronic timer
  • Temperature controlled laboratory oven
  • TopSeal‐A plate sealers (PerkinElmer)
  • Filtration cell harvester (e.g., FilterMate, PerkinElmer)
  • 96‐well unifilter GF/B filter plates with filter backs (Receptor Technologies, http://www.receptortechnologies.co.uk/)
  • Scintillation counters (TopCount from PerkinElmer, plus Beckman LS 6500 scintillation counter)
  • 5‐ml scintillation vials

Basic Protocol 4: Competition Kinetics Between [3H]NMS and Unlabeled Ligands

  Materials
  • Unlabeled competitor
  • Assay buffer (see recipe)
  • Radioligand: e.g., 12 µM 3H‐labeled N‐methyl scopolamine (80 to 82 Ci/mmol; PerkinElmer)
  • Receptor preparation of interest (e.g., CHO‐M 3 membranes see protocol 5)
  • 0.5% (w/v) polyethyleneimine (see recipe)
  • Wash buffer (see recipe), ice cold
  • 96‐well deep‐well polypropylene plates (Fisher Scientific)
  • Multichannel pipettor and reservoirs (e.g., Matrix)
  • Electronic timer
  • 60‐ml polypropylene screw top bottle (Fisher Scientific)
  • TopSeal‐A plate sealers (PerkinElmer)
  • Shaker
  • Temperature‐controlled laboratory oven
  • Filtration cell harvester (e.g., FilterMate, PerkinElmer)
  • 96‐well unifilter GF/B filter plates with filter backs (Receptor Technologies, http://www.receptortechnologies.co.uk/)
  • Scintillation counters (TopCount from PerkinElmer, plus Beckman LS 6500 scintillation counter)
  • 5‐ml scintillation vials

Support Protocol 1: Preparation of Receptor Membranes from Cultured Cells

  Materials
  • CHO cells expressing receptor of interest (e.g., CHO‐M 3; PerkinElmer), and culture medium
  • Membrane preparation buffers A, B, and C (see recipes)
  • 500‐cm2 cell‐culture plates
  • Cell culture incubator
  • Cell scrapers
  • 50‐ml conical polypropylene centrifuge tubes (Falcon)
  • Centrifuge (e.g., Beckman Ultracentrifuge)
  • Polytron tissue homogenizer (e.g., Werke, Ultra Turrax)
  • Ultracentrifuge tubes
  • 0.5‐ml microcentrifuge tubes
  • Additional reagents and equipment for protein assay ( appendix 3A)

Support Protocol 2: Determination of Unlabeled Competitor Ki Values

  • Receptor preparation of interest (e.g., CHO‐M 3 membranes; see protocol 5)
  • Competitor ligands (10 mM stock solutions)
  • 96‐well polypropylene plates (Corning, Fisher Scientific)
  • Electronic timer
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Figures

Videos

Literature Cited

Literature Cited
   Barlow, R.B., Birdsall, N.J., and Hulme, E.C. 1979. Temperature coefficients of affinity constants for the binding of antagonists to muscarinic receptors in the rat cerebral cortex. Br. J. Pharmacol. 66:587‐590.
   Barnes, P.J., Belvisi, M.G., Mak, J.C., Haddad, E.B., and O'Connor, B. 1995. Tiotropium bromide (Ba 679 BR), a novel long‐acting muscarinic antagonist for the treatment of obstructive airways disease. Life Sci. 56:853‐859.
   Birdsall, N.J., Burgen, A.S., Hulme, E.C., and Wells, J.W. 1979. The effects of ions on the binding of agonists and antagonists to muscarinic receptors. Br. J. Pharmacol. 67:371‐377.
   Carter, C.M.S., Leighton‐Davies, J.R., and Charlton, S.J. 2007. Miniaturized receptor binding assays: Complications arising from ligand depletion. J. Biomolec. Screen. 12:255‐266.
   Cheng, Y. and Prusoff, W.H. 1973. Relationship between inhibition constant (K1) and concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic‐reaction. Biochem. Pharmacol. 22:3099‐3108.
   Colquhoun, D. 1968. The rate of equilibration in a competitive n drug system and the auto‐inhibitory equations of enzyme kinetics: Some properties of simple models for passive sensitization. Proc. R. Soc. Lond. B Biol. Sci. 170:135‐154.
   Copeland, R.A., Pompliano, D.L., and Meek, T.D. 2007. Drug‐target residence time and its implications for lead optimization. Nat. Rev. Drug. Discov. 5:730‐739.
   Disse, B., Speck, G.A., Rominger, K.L., Witek, T.J. Jr., and Hammer, R. 1999. Tiotropium (Spiriva): Mechanistical considerations and clinical profile in obstructive lung disease. Life Sci. 64:457‐464.
   Dowling, M.R. and Charlton, S.J. 2006. Quantifying the association and dissociation rates of unlabelled antagonists at the muscarinic M3 receptor. Br. J. Pharmacol. 148:927‐937.
   Haddad, E.B., Mak, J.C., and Barnes, P.J. 1994. Characterization of [3H] Ba 679 BR, a slowly dissociating muscarinic antagonist, in human lung: Radioligand binding and autoradiographic mapping. Mol. Pharmacol. 45:899‐907.
   Hulme, E.C. and Birdsall, N.J.M. 1992. Strategy and tactics in receptor‐binding studies. In Receptor‐Ligand Interactions. A Practical Approach (E.C. Hulme, ed.) pp. 63‐176. Oxford University Press, New York.
   Motulsky, H. and Christopolous, A. 2003. Fitting models to biological data using linear and nonlinear regression: A practical guide to curve fitting. Graphpad Software, San Diego, Calif.
   Motulsky, H.J. and Mahan, L.C. 1984. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25:1‐9.
   Mersmann, H.J. and McNell, R.L. 1992. Ligand binding to the porcine adipose tissue β‐adrenergic receptor. J. Anim. Sci. 70:787‐797.
   Rang, H.P. 1966. The kinetics of action of acetylcholine antagonists in smooth muscle. Proc. R. Soc. Lond. B Biol. Sci. 164:488‐510.
   Smith, D.A., Jones, B.C., and Walker, D.K. 1996. Design of drugs involving the concepts and theories of drug metabolism and pharmacokinetics. Med. Res. Rev. 16:243‐266.
   Sykes, D.A., Dowling, M.R., and Charlton, S.J. 2009. Exploring the mechanism of agonist efficacy: A relationship between efficacy and agonist dissociation rate at the muscarinic M3 receptor. Mol. Pharmacol. 76:543‐551.
   Summerhill, S., Stroud, T., Nagendra, R., Perros‐Huguet, C., and Trevethick, M. 2008. A cell‐based assay to assess the persistence of action of agonists acting at recombinant human β(2) adrenoceptors. J. Pharmacol. Toxicol. Methods 58:189‐197.
   Takahashi, T., Belvisi, M.G., Patel, H., Ward, J.K., Tadjkarimi, S., Yacoub, M.H., and Barnes, P.J. 1994. Effect of Ba 679 BR, a novel long‐acting anticholinergic agent, on cholinergic neurotransmission in guinea pig and human airways. Am. J. Respir. Crit. Care Med. 150:1640‐1645.
   Vauquelin, G. and Van Liefde, I. 2006. Slow antagonist dissociation and long‐lasting in vivo receptor protection. Trends Pharmacol. Sci. 27:356‐359.
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