Development of Platform for Ligand Screening using Hyperpolarized NMR

描述(由申请人提供)：筛选蛋白质-配体相互作用是现代药物发现过程中的第一个关键步骤。为此目的存在各种技术，每种技术都具有不同水平的选择性和吞吐量。核磁共振已经是一种流行的筛选技术，因为化学位移所赋予的结构特异性是核磁共振的主要观察参数。核磁共振也是独一无二的，能够专门区分信号和配体的结合形式。然而，核磁共振的灵敏度有限。与其他技术相比，筛选应用中的吞吐量降低。此外，对样品的质量和浓度也提出了严格的条件。例如，基于核磁共振的筛选通常涉及使用大量过量的配体而不是目标蛋白，在这种情况下，观察是在自由配体上进行的。因此，可能会错过最理想的低失配率的配体，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，首先是溶解动态核极化(DNP)，可以大大提高液态核磁共振实验的灵敏度。虽然这项技术最近已被证明消除了上述许多限制，但能够利用其优势进行高通量筛选的核磁共振仪器尚不存在。在这里，我们将开发一种多路核磁共振探针和专用光谱仪，允许使用超极化配体进行并行筛选。探测器和光谱仪将设计用于氟原子上的超极化配体，并增加探测质子信号的能力。超极化的氟是一个特别有吸引力的靶，因为这个核允许获得背景自由光谱，同时当配体与靶结合时，它的化学位移和弛豫行为会发生较大的变化。这些特性允许通过单次扫描核磁共振测量来快速检测结合。探测作为二次核的质子的能力将允许在线评估样品质量。氟在药物中是一种丰富的原子核；大约20%的上市药物分子含有一个氟原子。此外，其他药物分子可以通过与目标位点的竞争结合以及氟化的报告配体来识别。因此，并行检测可以非常普遍地用于同时观察多个不同配体与靶的结合，通过滴定配体浓度来确定解离常数，以及用于靶/反靶筛选。在最后一步中，这些应用的核磁共振方法将专门在新硬件上开发和演示，使用与药物相关的靶标。这些发展将允许通过核磁共振灵敏和快速地检测配体结合，改善获得这一多功能光谱技术用于筛选应用的好处。

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.