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Journal of Biomolecular NMR

, Volume 69, Issue 4, pp 229–235 | Cite as

Label-free NMR-based dissociation kinetics determination

  • Pablo Trigo-Mouriño
  • Christian Griesinger
  • Donghan LeeEmail author
Article

Abstract

Understanding the dissociation of molecules is the basis to modulate interactions of biomedical interest. Optimizing drugs for dissociation rates is found to be important for their efficacy, selectivity, and safety. Here, we show an application of the high-power relaxation dispersion (RD) method to the determination of the dissociation rates of weak binding ligands from receptors. The experiment probes proton RD on the ligand and, therefore, avoids the need for any isotopic labeling. The large ligand excess eases the detection significantly. Importantly, the use of large spin-lock fields allows the detection of faster dissociation rates than other relaxation approaches. Moreover, this experimental approach allows to access directly the off-rate of the binding process without the need for analyzing a series of samples with increasing ligand saturation. The validity of the method is shown with small molecule interactions using two macromolecules, bovine serum albumin and tubulin heterodimers.

Keywords

NMR Ligand binding Relaxation dispersion 

Notes

Funding

This work was supported by funds from the James Graham Brown Foundation, the National Center for Research Resources CoBRE (1P30GM106396), the Max Planck Society, and the EU (ERC Grant Agreement Number 233227 to C.G.). P.T.M. acknowledges the Humboldt Foundation for a postdoctoral research fellowship.

Supplementary material

10858_2017_150_MOESM1_ESM.docx (1.2 mb)
Supplementary material 1 (DOCX 1194 KB)

References

  1. Agafonov RV, Wilson C, Otten R, Buosi V, Kern D (2014) Energetic dissection of Gleevec’s selectivity toward human tyrosine kinases. Nat Struct Mol Biol 21:848–853CrossRefGoogle Scholar
  2. Alberty RA, Hammes GG (1958) Application of the theory of diffusion-controlled reactions to enzyme kinetics. J Phys Chem 62:154–159CrossRefGoogle Scholar
  3. Andersen OA, Nathubhai A, Dixon MJ, Eggleston IM, van Aalten DM (2008) Structure-based dissection of the natural product cyclopentapeptide chitinase inhibitor argifin. Chem Biol 15:295–301CrossRefGoogle Scholar
  4. Ban D, Gossert AD, Giller K, Becker S, Griesinger C, Lee D (2012) Exceeding the limit of dynamics studies on biomolecules using high spin-lock field strengths with a cryogenically cooled probehead. J Magn Reson 221:1–4ADSCrossRefGoogle Scholar
  5. Ban D, Sabo TM, Griesinger C, Lee D (2013) Measuring dynamic and kinetic information in the previously inaccessible supra-tc window of nanoseconds to microseconds by solution NMR spectroscopy. Molecules 18:11904–11937CrossRefGoogle Scholar
  6. Braunschweiler L, Ernst RR (1983) Coherence transfer by isotropic mixing: application to proton correlation spectroscopy. J Magn Reson (1969) 53:521–528CrossRefGoogle Scholar
  7. Canales A, Nieto L, Rodriguez-Salarichs J, Sanchez-Murcia PA, Coderch C, Cortes-Cabrera A, Paterson I, Carlomagno T, Gago F, Andreu JM, Altmann KH, Jimenez-Barbero J, Diaz JF (2014) Molecular recognition of epothilones by microtubules and tubulin dimers revealed by biochemical and NMR approaches. ACS Chem Biol 9:1033–1043CrossRefGoogle Scholar
  8. Carr HY, Purcell EM (1954) Effects of diffusion on free precession in nuclear magnetic resonance experiments. Phys Rev 94:630–638ADSCrossRefGoogle Scholar
  9. Copeland RA (2016) The drug-target residence time model: a 10-year retrospective. Nat Rev Drug Discov 15:87–95CrossRefGoogle Scholar
  10. Copeland RA, Pompliano DL, Meek TD (2006) Drug-target residence time and its implications for lead optimization. Nat Rev Drug Discov 5:730–739CrossRefGoogle Scholar
  11. Dalvit C (2007) Ligand- and substrate-based 19F NMR screening: principles and applications to drug discovery. Progr Nucl Magn Reson Spectrosc 51:243–271CrossRefGoogle Scholar
  12. Davis DG, Perlman ME, London RE (1994) Direct measurements of the dissociation-rate constant for inhibitor-enzyme complexes via the T 1r and T 2 (CPMG) methods. J Magn Reson B 104:266–275CrossRefGoogle Scholar
  13. Delaglio F, Grzesiek S, Vuister GW, Zhu G, Pfeifer J, Bax A (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J Biomol NMR 6:277–293CrossRefGoogle Scholar
  14. Desvaux H, Berthault P, Birlirakis N, Goldman M (1994) Off-resonance ROESY for the study of dynamic processes. J Magn Reson A 108:219–229ADSCrossRefGoogle Scholar
  15. Desvaux H, Berthault P, Birlirakis N, Goldman M, Piotto M (1995) Improved versions of off-resonance ROESY. J Magn Reson A 113:47–52ADSCrossRefGoogle Scholar
  16. Eichmuller C, Skrynnikov NR (2005) A new amide proton R 1r experiment permits accurate characterization of microsecond time-scale conformational exchange. J Biomol NMR 32:281–293CrossRefGoogle Scholar
  17. Eigen M, Hammes GG (2006) Elementary steps in enzyme reactions (as studied by relaxation spectrometry). In: Nord FF (ed) Advances in enzymology and related areas of molecular biology. Wiley, New York, pp 1–38Google Scholar
  18. Fielding L, Rutherford S, Fletcher D (2005) Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems. Magn Reson Chem 43:463–470CrossRefGoogle Scholar
  19. Furtig B, Nozinovic S, Reining A, Schwalbe H (2015) Multiple conformational states of riboswitches fine-tune gene regulation. Curr Opin Struct Biol 30:112–124CrossRefGoogle Scholar
  20. Guedich S, Puffer-Enders B, Baltzinger M, Hoffmann G, Da Veiga C, Jossinet F, Thore S, Bec G, Ennifar E, Burnouf D, Dumas P (2016) Quantitative and predictive model of kinetic regulation by E. coli TPP riboswitches. RNA Biol 13:373–390CrossRefGoogle Scholar
  21. Guo D, Mulder-Krieger T, IJzerman AP, Heitman LH (2012) Functional efficacy of adenosine A(2)A receptor agonists is positively correlated to their receptor residence time. Br J Pharmacol 166:1846–1859CrossRefGoogle Scholar
  22. Guo D, Heitman LH, IJzerman AP (2015) The role of target binding kinetics in drug discovery. ChemMedChem 10:1793–1796CrossRefGoogle Scholar
  23. Hajduk PJ (2006) Fragment-based drug design: how big is too big? J Med Chem 49:6972–6976CrossRefGoogle Scholar
  24. Hajduk PJ, Greer J (2007) A decade of fragment-based drug design: strategic advances and lessons learned. Nat Rev Drug Discov 6:211–219CrossRefGoogle Scholar
  25. Hothersall JD, Guo D, Sarda S, Sheppard RJ, Chen H, Keur W, Waring MJ, IJzerman AP, Hill SJ, Dale IL, Rawlins PB (2017) Structure-activity relationships of the sustained effects of adenosine A2A receptor agonists driven by slow dissociation kinetics. Mol Pharmacol 91:25–38CrossRefGoogle Scholar
  26. Ishima R (2014) CPMG relaxation dispersion. In: Livesay DR (ed) protein dynamics: methods and protocols. Humana Press, Totowa, pp 29–49CrossRefGoogle Scholar
  27. Ishima R, Torchia DA (2003) Extending the range of amide proton relaxation dispersion experiments in proteins using a constant-time relaxation-compensated CPMG approach. J Biomol NMR 25:243–248CrossRefGoogle Scholar
  28. Jones JA, Hodgkinson P, Barker AL, Hore PJ (1996) Optimal sampling strategies for the measurement of spin–spin relaxation times. J Magn Reson B 113:25–34CrossRefGoogle Scholar
  29. Keighley W (2011) The need for high throughput kinetics early in the drug discovery process. Drug Disc World Summer 2011:39–45Google Scholar
  30. Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta 1814:942–968CrossRefGoogle Scholar
  31. Kozakov D, Hall DR, Jehle S, Luo L, Ochiana SO, Jones EV, Pollastri M, Allen KN, Whitty A, Vajda S (2015) Ligand deconstruction: why some fragment binding positions are conserved and others are not. Proc Natl Acad Sci USA 112:E2585-2594ADSCrossRefGoogle Scholar
  32. Lin PC (2015) Assessment of chemical exchange in tryptophan-albumin solution through 19F multicomponent transverse relaxation dispersion analysis. J Biomol NMR 62:121–127CrossRefGoogle Scholar
  33. Lipton SA (2004) Turning down, but not off. Nature 428:473–473ADSCrossRefGoogle Scholar
  34. Lu H, Tonge PJ (2010) Drug-target residence time: critical information for lead optimization. Curr Opin Chem Biol 14:467–474CrossRefGoogle Scholar
  35. Meiboom S, Gill D (1958) Modified spin echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691ADSCrossRefGoogle Scholar
  36. Moschen T, Wunderlich CH, Spitzer R, Levic J, Micura R, Tollinger M, Kreutz C (2015) Ligand-detected relaxation dispersion NMR spectroscopy: dynamics of preQ1-RNA binding. Angew Chem Int Ed Engl 54:560–563Google Scholar
  37. Moschen T, Grutsch S, Juen MA, Wunderlich CH, Kreutz C, Tollinger M (2016) Measurement of ligand-target residence times by 1H relaxation dispersion NMR spectroscopy. J Med Chem 59:10788–10793CrossRefGoogle Scholar
  38. Namanja AT, Wang XJ, Xu B, Mercedes-Camacho AY, Wilson BD, Wilson KA, Etzkorn FA, Peng JW (2010) Toward flexibility-activity relationships by NMR spectroscopy: dynamics of Pin1 ligands. J Am Chem Soc 132:5607–5609CrossRefGoogle Scholar
  39. Neu A, Neu U, Fuchs AL, Schlager B, Sprangers R (2015) An excess of catalytically required motions inhibits the scavenger decapping enzyme. Nat Chem Biol 11:697–704CrossRefGoogle Scholar
  40. Ohlson S (2008) Designing transient binding drugs: a new concept for drug discovery. Drug Discov Today 13:433–439CrossRefGoogle Scholar
  41. Palmer AG III (2004) NMR characterization of the dynamics of biomacromolecules. Chem Rev 104:3623–3640CrossRefGoogle Scholar
  42. Pan AC, Borhani DW, Dror RO, Shaw DE (2013) Molecular determinants of drug-receptor binding kinetics. Drug Discov Today 18:667–673CrossRefGoogle Scholar
  43. Peng JW, Wilson BD, Namanja AT (2009) Mapping the dynamics of ligand reorganization via 13CH3 and 13CH2 relaxation dispersion at natural abundance. J Biomol NMR 45:171–183CrossRefGoogle Scholar
  44. Peuker S, Cukkemane A, Held M, Noe F, Kaupp UB, Seifert R (2013) Kinetics of ligand-receptor interaction reveals an induced-fit mode of binding in a cyclic nucleotide-activated protein. Biophys J 104:63–74CrossRefGoogle Scholar
  45. Seeman P (2014) Clozapine, a fast-off-D2 antipsychotic. ACS Chem Neurosci 5:24–29CrossRefGoogle Scholar
  46. Shimizu Y, Ishii T, Ogawa K, Sasaki S, Matsui H, Nakayama M (2015) Biochemical characterization of smoothened receptor antagonists by binding kinetics against drug-resistant mutant. Eur J Pharmacol 764:220–227CrossRefGoogle Scholar
  47. Smith CA, Ban D, Pratihar S, Giller K, Schwiegk C, de Groot BL, Becker S, Griesinger C, Lee D (2015) Population shuffling of protein conformations. Angew Chem Int Ed Engl 54:207–210CrossRefGoogle Scholar
  48. Sugase K, Dyson HJ, Wright PE (2007) Mechanism of coupled folding and binding of an intrinsically disordered protein. Nature 447:1021–1025ADSCrossRefGoogle Scholar
  49. Swinney DC (2009) The role of binding kinetics in therapeutically useful drug action. Curr Opin Drug Discov Dev 12:31–39Google Scholar
  50. Tolkatchev D, Xu P, Ni F (2003) Probing the kinetic landscape of transient peptide-protein interactions by use of peptide 15N NMR relaxation dispersion spectroscopy: binding of an antithrombin peptide to human prothrombin. J Am Chem Soc 125:12432–12442CrossRefGoogle Scholar
  51. Tummino PJ, Copeland RA (2008) Residence time of receptor-ligand complexes and its effect on biological function. BioChemistry 47:5481–5492CrossRefGoogle Scholar
  52. Vauquelin G, Bostoen S, Vanderheyden P, Seeman P (2012) Clozapine, atypical antipsychotics, and the benefits of fast-off D2 dopamine receptor antagonism. Naunyn Schmiedebergs Arch Pharmacol 385:337–372CrossRefGoogle Scholar
  53. Vilums M, Zweemer AJ, Yu Z, de Vries H, Hillger JM, Wapenaar H, Bollen IA, Barmare F, Gross R, Clemens J, Krenitsky P, Brussee J, Stamos D, Saunders J, Heitman LH, Ijzerman AP (2013) Structure-kinetic relationships—an overlooked parameter in hit-to-lead optimization: a case of cyclopentylamines as chemokine receptor 2 antagonists. J Med Chem 56:7706–7714CrossRefGoogle Scholar
  54. Zintsmaster JS, Wilson BD, Peng JW (2008) Dynamics of ligand binding from 13C NMR relaxation dispersion at natural abundance. J Am Chem Soc 130:14060–14061CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2017

Authors and Affiliations

  • Pablo Trigo-Mouriño
    • 1
  • Christian Griesinger
    • 1
  • Donghan Lee
    • 2
    Email author
  1. 1.Department of NMR-Based Structural BiologyMax-Planck Institute for Biophysical ChemistryGöttingenGermany
  2. 2.James Graham Brown Cancer CenterUniversity of LouisvilleLouisvilleUSA

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