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是现代药物发现过程中最初的关键步骤。为此目的存在各种各样的技术，每种技术都具有不同程度的选择性和吞吐量。由于化学位移（核磁共振的主要观测参数）所赋予的结构特异性，核磁共振（NMR）已经成为一种流行的筛选技术。核磁共振也独特的能力，具体区分信号从结合形式的配体。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了。此外，对样品质量和浓度施加了严格的条件。例如，基于核磁共振的筛选通常涉及在靶蛋白上使用大量过量的配体，在这种情况下，对自由配体进行观察。因此，最理想的配体与低脱失率可能会错过，降低了该方法的特异性。然而，利用新兴的超极化技术，尤其是溶解动态核极化技术（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经被证明可以消除上述许多限制，但目前还不存在能够利用其优势进行高通量筛选的核磁共振仪器。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平行筛选。探针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。超极化氟是一个特别有吸引力的目标，因为这个核允许获得背景自由光谱，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。这些特性允许通过单扫描核磁共振测量快速检测结合。质子作为次级核的检测能力将允许对样品质量进行在线评估。氟是药物中丰富的原子核；市场上大约20%的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与靶位点竞争结合来识别。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标的结合，通过滴定配体浓度来确定解离常数，以及靶标/反靶标筛选。在最后一步，这些应用程序的核磁共振方法将开发和演示具体在新的硬件上，使用药学相关的目标。这些发展将允许通过核磁共振对配体结合进行灵敏和快速的检测，从而提高这种多功能光谱技术在筛选应用中的优势。

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.