Nanoscale Encapsulation for Fragment Based Drug Discovery

 描述（由申请人提供）：在过去的二十年中，蛋白质-配体相互作用的高通量筛选已经发展成为药物发现过程中的核心工具。非常高通量的体外筛选（HTS）策略通常基于使用高灵敏度光谱技术（例如荧光和最近的质谱）检测配体结合。这些类型的方法通常不能揭示所检测的蛋白质-配体复合物的结构背景。出于这个原因，基于NMR的筛选，虽然通量较低，但已经成为一个重要的贡献者，特别是对于HTS命中的事后验证。“最近，筛选库的设计 变得更加复杂。这对于过去十年左右开发的基于片段的药物发现库尤其如此。这些库的设计是为了利用组合化学的进步，目标是扩大采取“击中”的灵活性 一种“铅”化合物。不幸的是，片段型配体本质上是弱结合剂。这引入了相当大的材料成本（为了达到检测结合所需的浓度）和通过非特异性相互作用的假阳性结合的可能性增加。这项提案将制定一项战略，在很大程度上消除这两个问题。它是基于反胶束的纳米级特性。利用反胶束作为受限空间，我们预计能够1）有效地检测弱特异性结合; 2）减少或消除非特异性结合; 3）显着减少所需的配体和蛋白质的量;最重要的是，4）进入化学空间的一个区域，该区域是高度期望的，但使用现有的筛选策略（即弱亲水性结合剂）难以进入。这些进展依赖于我们最近开发的能力，以创建封装在反胶束的纳米级水核心内的单个蛋白质分子的解决方案。我们将开发高分辨率溶液NMR光谱以及在高温超导背景下使用荧光这种筛选策略。初步结果表明，这种方法的可行性。几种生物医学感兴趣的蛋白质将用于开发和测试纳米级封装的使用，以增强从片段库中发现低亲和力结合剂。二氢叶酸还原酶，一个历史上的药物靶点，将用于开发许多策略。人类醛酮还原酶（AKR 1c 3），目前前列腺癌的目标，也将被筛选，以证明该方法的大可溶性蛋白质的效用。肉豆蔻酰化HIV基质蛋白将在其脂质挤出膜锚定状态下进行检查，并且将代表一类迄今为止通常无法使用更标准方法进行满意筛选的蛋白质。总之，本文提出的研究将证明使用纳米级封装筛选片段库的显著增强，并可能导致发现针对与重要人类疾病相关的这些靶点的初始化合物。

 DESCRIPTION (provided by applicant): Over the past two decades high-throughput screening of protein-ligand interactions has evolved to become a central tool in the process of drug discovery. The very high-throughput in vitro screening (HTS) strategies are generally based on detection of ligand-binding using highly sensitive spectroscopic techniques such as fluorescence and, more recently, mass spectrometry. These types of approaches typically fail to reveal structural context for the protein-ligand complexes that are detected. For this reason, NMR-based screening, though of lower throughput, has emerged as an important contributor especially for post facto validation of HTS "hits." Recently, the design of screening libraries has become more sophisticated. This is particularly true with the fragment-based drug discovery libraries developed over the past decade or so. These libraries are designed to take advantage of advances in combinatorial chemistry with the goal of expanding the flexibility of taking a "hit" to a "lead" compound. Unfortunately, fragment-type ligands are inherently weak binders. This introduces a considerable cost in material (in order to reach concentrations necessary to detect binding) and an increased likelihood of false positive binding through non-specific interactions. This proposal will develop a strategy that will largely eliminate both of these concerns. It is based on the nanoscale properties of the reverse micelle. Utilizing the reverse micelle as a confined space, we anticipate being able to 1) efficiently detect weak specific binding; 2) reduce or eliminate non-specific binding; 3) significantly reduce the amount of ligand and protein required; and, most importantly, 4) enter a region of chemical space that is highly desired but difficult to access using existing screening strategies (i.e. weak hydrophilic binders). These advances rely on our recently developed ability to create solutions of individual protein molecules encapsulated within the nanoscale water core of reverse micelles. We will develop this screening strategy for high-resolution solution NMR spectroscopy as well as in a HTS context using fluorescence. Preliminary results indicate the feasibility of this approach. Several proteins of biomedical interest will be used to develop and test the use of nanoscale encapsulation to enhance the discovery of low affinity binders from a fragment library. Dihydrofolate reductase, a historical drug target, will be used to develop many of the strategies. A human aldoketoreductase enzyme (AKR1c3), currently a target for prostate cancer, will also be screened to demonstrate the utility of the approach for large soluble proteins. The myristoylated HIV matrix protein will be examined in its lipid extruded membrane-anchored state and will be representative of a class of proteins that have thus far generally eluded satisfactory screening using more standard methods. In summary, the studies proposed here will demonstrate a significant enhancement in screening of fragment libraries using nanoscale encapsulation and may lead to the discovery of initial compounds directed at these targets related to important human diseases.