Trigo-Mouriño Pablo, Griesinger Christian, Lee Donghan
Department of NMR-Based Structural Biology, Max-Planck Institute for Biophysical Chemistry, Göttingen, Germany.
James Graham Brown Cancer Center, University of Louisville, Louisville, KY, 40202, USA.
J Biomol NMR. 2017 Dec;69(4):229-235. doi: 10.1007/s10858-017-0150-5. Epub 2017 Nov 16.
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.
理解分子解离是调节具有生物医学意义的相互作用的基础。发现优化药物的解离速率对其疗效、选择性和安全性很重要。在此，我们展示了高功率弛豫色散（RD）方法在测定弱结合配体与受体解离速率方面的应用。该实验探测配体上的质子RD，因此无需任何同位素标记。大量过量的配体显著简化了检测过程。重要的是，使用大的自旋锁定场能够检测到比其他弛豫方法更快的解离速率。此外，这种实验方法无需分析一系列配体饱和度增加的样品，就可以直接获得结合过程的解离速率。使用两种大分子——牛血清白蛋白和微管蛋白异二聚体的小分子相互作用证明了该方法的有效性。
