Label-free NMR-based dissociation kinetics determination
- 789 Downloads
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.
KeywordsNMR Ligand binding Relaxation dispersion
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.
- 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
- 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
- Keighley W (2011) The need for high throughput kinetics early in the drug discovery process. Drug Disc World Summer 2011:39–45Google Scholar
- 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
- Swinney DC (2009) The role of binding kinetics in therapeutically useful drug action. Curr Opin Drug Discov Dev 12:31–39Google Scholar
- 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