Development of Platform for Ligand Screening using Hyperpolarized NMR

描述（由申请人提供）：蛋白质-配体相互作用的筛选代表了现代药物发现过程中的初始关键步骤。存在用于此目的的各种技术，每种技术具有不同的选择性和吞吐量水平。核磁共振（NMR）已经是一种流行的筛选技术，因为化学位移赋予的结构特异性，主要的NMR可观察参数。NMR在特异性区分信号与配体结合形式的能力方面也是独特的。然而，NMR的灵敏度有限。与其他技术相比，筛选应用中的通量降低。此外，对样品质量和浓度规定了严格的条件。例如，基于NMR的筛选通常涉及使用相对于靶蛋白大量过量的配体，在这种情况下，对游离配体进行观察。因此，可能错过具有低解离速率的最期望的配体，从而降低了该方法的特异性。然而，使用新出现的超极化技术，最重要的溶解动态核极化（DNP），可以大大提高液态NMR实验的灵敏度。虽然这种技术最近已被证明可以消除许多上述限制，但不存在能够利用其优点进行高通量筛选的NMR仪器。在这里，我们将开发一个多路复用NMR探针和专用光谱仪，允许使用超极化配体进行平行筛选。探针和光谱仪将被设计用于氟原子上超极化的配体，并具有检测质子信号的附加能力。超极化的氟是一个特别有吸引力的目标，因为这个核允许获得无背景光谱，同时当配体与目标结合时，化学位移和弛豫行为会发生相对较大的变化。这些性质允许通过单扫描NMR测量快速检测结合。探测作为次级核的质子的能力将允许对样品质量进行在线评估。氟在药物中是一种丰富的原子核;大约20%的市售药物分子含有氟原子。此外，其他药物分子可以通过与氟化报告配体一起竞争性结合到靶位点来鉴定。因此，酶标化检测可以相当普遍地用于同时观察多个不同配体与靶的结合，用于通过配体浓度滴定来测定解离常数，以及用于靶/反靶筛选。在最后一步中，将开发用于这些应用的NMR方法，并使用药物相关目标在新硬件上进行专门演示。这些发展将允许通过NMR灵敏和快速地检测配体结合，改善这种多功能光谱技术用于筛选应用的益处。

DESCRIPTION (provided by applicant): The screening for protein-ligand interactions represents an initial key step in the modern drug discovery process. Various techniques exist for this purpose, each of which with a varying level of selectivity and throughput. Nuclear magnetic resonance (NMR) already is a popular screening technique because of the structural specificity imparted by chemical shift, the primary NMR observable parameter. NMR is also unique in the ability to specifically distinguish signals from the bound form of the ligand. NMR, however, suffers from limited sensitivity. The throughput in screening applications is reduced compared to other techniques. Furthermore, stringent conditions on sample quality and concentration are imposed. For example, NMR based screening often involves the use of a large excess of ligand over target protein, in which case observation is performed on the free ligand. As a result, the most desirable ligands with low off-rates may be missed, reducing the specificity of the method. Sensitivity in a liquid state NMR experiment can however be greatly enhanced using newly emerging hyperpolarization techniques, foremost dissolution dynamic nuclear polarization (DNP). While this technique has recently been demonstrated to remove many of the above mentioned limitations, NMR instrumentation that enables utilization of its benefits for high-throughput screening does not exist. Here, we will develop a multiplexed NMR probe and dedicated spectrometer that allows for parallel screening using hyperpolarized ligands. The probe and spectrometer will be designed for ligands hyperpolarized on fluorine atoms, with the added capability of detection of proton signals. Hyperpolarized fluorine is a particularly attractie target because this nucleus allows for the acquisition of background free spectra and at the same time is subject to comparatively large changes in chemical shift and relaxation behavior when a ligand binds to a target. These properties allow for the rapid detection of binding via single scan NMR measurement. The capability for detection of proton as a secondary nucleus will allow for an on-line assessment of sample quality. Fluorine is an abundant nucleus in pharmaceuticals; approximately 20% of marketed drug molecules contain a fluorine atom. Furthermore, other drug molecules can be identified by competitive binding to a target site together with a fluorinated reporter ligand. Parallelized detection can therefore be quite generally used for simultaneous observation of binding of multiple different ligands to a target, for determination of dissociation constants by titration of ligand concentration, and for target/anti-target screens. In a final step, the NMR methods for these applications will be developed and demonstrated specifically on the new hardware, using pharmaceutically relevant targets. These developments will allow for the sensitive and rapid detection of ligand binding by NMR, improving access to the benefits of this versatile spectroscopic technique for screening applications.