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Receptor Binding Assay for NO-Independent Activators of Soluble Guanylate Cyclase

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Guanylate Cyclase and Cyclic GMP

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1020))

Abstract

The characterization of the interaction between a ligand and its receptor is crucial for a broad variety of applications in academia as well as in the pharmaceutical industry. Although various sophisticated high-throughput technologies have been established to investigate the binding of ligands to their receptors, classical filtration-based receptor binding assays still have some advantages when smaller number of samples need to be tested. Here we describe a technically easy, cheap, and reliable receptor binding assay that was successfully applied to determine the binding constant of the NO-independent activator of soluble guanylate cyclase, cinaciguat, and the impact of other small molecules on its interaction with the enzyme.

Conflict of Interest

Peter M. Schmidt was employed from 2000 to 2003 by Bayer Pharma AG and is currently a full-time employee of CSL. Johannes-Peter Stasch is currently a full-time employee of Bayer Pharma AG.

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References

  1. Eglen RM, Reisine T, Roby P, Rouleau N, Illy C, Bosse R, Bielefeld M (2008) The use of AlphaScreen technology in HTS: current status. Curr Chem Genomics 1:2–10

    Article  PubMed  CAS  Google Scholar 

  2. Ullman EF, Kirakossian H, Singh S, Wu ZP, Irvin BR, Pease JS, Switchenko AC, Irvine JD, Dafforn A, Skold CN et al (1994) Luminescent oxygen channeling immunoassay: measurement of particle binding kinetics by chemiluminescence. Proc Natl Acad Sci USA 91(12):5426–5430

    Article  PubMed  CAS  Google Scholar 

  3. Bazin H, Trinquet E, Mathis G (2002) Time resolved amplification of cryptate emission: a versatile technology to trace biomolecular interactions. J Biotechnol 82(3):233–250

    PubMed  CAS  Google Scholar 

  4. Degorce F, Card A, Soh S, Trinquet E, Knapik GP, Xie B (2009) HTRF: a technology tailored for drug discovery—a review of theoretical aspects and recent applications. Curr Chem Genomics 3:22–32

    Article  PubMed  CAS  Google Scholar 

  5. Olson KR, Eglen RM (2007) Beta galactosidase complementation: a cell-based luminescent assay platform for drug discovery. Assay Drug Dev Technol 5(1):137–144

    Article  PubMed  CAS  Google Scholar 

  6. Rothkegel C, Schmidt PM, Atkins DJ, Hoffmann LS, Schmidt HH, Schroder H, Stasch JP (2007) Dimerization region of soluble guanylate cyclase characterized by bimolecular fluorescence complementation in vivo. Mol Pharmacol 72(5):1181–1190

    Article  PubMed  CAS  Google Scholar 

  7. Jason-Moller L, Murphy M, Bruno J (2006) Overview of Biacore systems and their applications. Current protocols in protein science Chapter 19:Unit 19.14. doi: 10.1002/0471142301.ps1914s45

  8. Navratilova I, Hopkins AL (2011) Emerging role of surface plasmon resonance in fragment-based drug discovery. Future Med Chem 3(14):1809–1820

    Article  PubMed  CAS  Google Scholar 

  9. Glickman JF, Schmid A, Ferrand S (2008) Scintillation proximity assays in high-throughput screening. Assay Drug Dev Technol 6(3):433–455

    Article  PubMed  CAS  Google Scholar 

  10. O’Neill MA, Gaisford S (2011) Application and use of isothermal calorimetry in pharmaceutical development. Int J Pharm 417(1–2):83–93

    Article  PubMed  Google Scholar 

  11. Jerabek-Willemsen M, Wienken CJ, Braun D, Baaske P, Duhr S (2011) Molecular interaction studies using microscale thermophoresis. Assay Drug Dev Technol 9(4):342–353

    Article  PubMed  CAS  Google Scholar 

  12. Schmidt PM (2009) Biochemical detection of cGMP from past to present: an overview. Handb Exp Pharmacol 191:195–228

    Article  PubMed  CAS  Google Scholar 

  13. Schmidt P, Schramm M, Schroder H, Stasch JP (2003) Receptor binding assay for nitric oxide- and heme-independent activators of soluble guanylate cyclase. Anal Biochem 314(1):162–165

    Article  PubMed  CAS  Google Scholar 

  14. Stasch JP, Schmidt P, Alonso-Alija C, Apeler H, Dembowsky K, Haerter M, Heil M, Minuth T, Perzborn E, Pleiss U, Schramm M, Schroeder W, Schroder H, Stahl E, Steinke W, Wunder F (2002) NO- and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle. Br J Pharmacol 136(5):773–783

    Article  PubMed  CAS  Google Scholar 

  15. Stasch JP, Schmidt PM, Nedvetsky PI, Nedvetskaya TY, Kumar HSA, Meurer S, Deile M, Taye A, Knorr A, Lapp H, Muller H, Turgay Y, Rothkegel C, Tersteegen A, Kemp-Harper B, Muller-Esterl W, Schmidt HH (2006) Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels. J Clin Invest 116(9):2552–2561

    Article  PubMed  CAS  Google Scholar 

  16. Hoffmann LS, Schmidt PM, Keim Y, Schaefer S, Schmidt HH, Stasch JP (2009) Distinct molecular requirements for activation or stabilization of soluble guanylyl cyclase upon haem oxidation-induced degradation. Br J Pharmacol 157(5):781–795

    Article  PubMed  CAS  Google Scholar 

  17. Evgenov OV, Pacher P, Schmidt PM, Hasko G, Schmidt HH, Stasch JP (2006) NO-independent stimulators and activators of soluble guanylate cyclase: discovery and therapeutic potential. Nat Rev 5(9):755–768

    Article  CAS  Google Scholar 

  18. Schmidt HH, Schmidt PM, Stasch JP (2009) NO- and haem-independent soluble guanylate cyclase activators. Handb Exp Pharmacol 191:309–339

    Article  PubMed  CAS  Google Scholar 

  19. Wang Y, Liu H, McKenzie G, Witting PK, Stasch JP, Hahn M, Changsirivathanathamrong D, Wu BJ, Ball HJ, Thomas SR, Kapoor V, Celermajer DS, Mellor AL, Keaney JF Jr, Hunt NH, Stocker R (2010) Kynurenine is an endothelium-derived relaxing factor produced during inflammation. Nat Med 16(3):279–285

    Article  PubMed  CAS  Google Scholar 

  20. Hahn MG, Alonso-Alija C, Stoll F, Heil M, Mittendorf M, Schlemmer KH, Wunder F, Stasch JP (2007) Design and synthesis of the first NO- and haem-independent sGC activator BAY 58-2667 for the treatment of acute decompensated heart failure. BMC Pharmacol 7:P25

    Article  Google Scholar 

  21. Seidel D, Pleiss U (2010) Labelling of the guanylate cyclase activator cinaciguat (BAY 58-2667) with carbon-14, tritium and stable isotopes. J Label Compd Radiopharm 53:130–139

    Article  CAS  Google Scholar 

  22. Scott CW, Gomes BC, Hubbs SJ, Koenigbauer HC (1995) A filtration-based assay to quantitate granulocyte-macrophage colony-stimulating factor binding. Anal Biochem 228(1):150–154

    Article  PubMed  CAS  Google Scholar 

  23. Demoliou-Mason CD, Barnard EA (1984) Solubilization in high yield of opioid receptors retaining high-affinity delta, mu and kappa binding sites. FEBS Lett 170(2):378–382

    Article  PubMed  CAS  Google Scholar 

  24. Atha DH, Ingham KC (1981) Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume. J Biol Chem 256(23):12108–12117

    PubMed  CAS  Google Scholar 

  25. Bhat R, Timasheff SN (1992) Steric exclusion is the principal source of the preferential hydration of proteins in the presence of polyethylene glycols. Protein Sci 1(9):1133–1143

    Article  PubMed  CAS  Google Scholar 

  26. Schmidt PM, Schramm M, Schroder H, Wunder F, Stasch JP (2004) Identification of residues crucially involved in the binding of the heme moiety of soluble guanylate cyclase. J Biol Chem 279(4):3025–3032

    Article  PubMed  CAS  Google Scholar 

  27. Foerster J, Harteneck C, Malkewitz J, Schultz G, Koesling D (1996) A functional heme-binding site of soluble guanylyl cyclase requires intact N-termini of alpha 1 and beta 1 subunits. Eur J Biochem 240(2):380–386

    Article  PubMed  CAS  Google Scholar 

  28. Schmidt P, Schramm M, Schroder H, Stasch JP (2003) Preparation of heme-free soluble guanylate cyclase. Protein Expr Purif 31(1):42–46

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. CSL Limited, BIO21 Institute, Parkville, VIC, Australia

    Peter M. Schmidt

  2. Cardiovascular Research, Bayer Pharma AG, Wuppertal, Germany

    Johannes-Peter Stasch

Authors
  1. Peter M. Schmidt
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  2. Johannes-Peter Stasch
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Editor information

Editors and Affiliations

  1. Addenbrooke’s Hospital, Department of Medicine, University of Cambridge, Hills Road, Cambridge, CB2 2QQ, United Kingdom

    Thomas Krieg

  2. Toxicology and Clinical Pharmacy, Department of Pharmacology, Universität Tübingen, Auf der Morgenstelle 8, Tübingen, 72076, Germany

    Robert Lukowski

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Schmidt, P.M., Stasch, JP. (2013). Receptor Binding Assay for NO-Independent Activators of Soluble Guanylate Cyclase. In: Krieg, T., Lukowski, R. (eds) Guanylate Cyclase and Cyclic GMP. Methods in Molecular Biology, vol 1020. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-459-3_13

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  • DOI: https://doi.org/10.1007/978-1-62703-459-3_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-458-6

  • Online ISBN: 978-1-62703-459-3

  • eBook Packages: Springer Protocols

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