Nanoscale Encapsulation for Fragment Based Drug Discovery

用于基于片段的药物发现的纳米级封装

基本信息

批准号：
9241998
负责人：
A. JOSHUA WAND
金额：
$ 17.51万
依托单位：
UNIVERSITY OF PENNSYLVANIA
依托单位国家：
美国
项目类别：
财政年份：
2016
资助国家：
美国
起止时间：
2016-03-10 至 2018-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9241998
关键词：
Affinity Alkanes Behavior Binding Chemicals Complex Confined Spaces Crystallization Crystallography Data Detection Dihydrofolate Reductase Dimensions Drug Targeting Encapsulated Enzymes Face Fluorescence Goals HIV Heteronuclear NMR Human In Vitro Individual Inferior Lead Libraries Ligand Binding Ligands Lipids Liquid substance Malignant neoplasm of prostate Mass Spectrum Analysis Membrane Methodology Methods Micelles Modification Morphologic artifacts Muramidase NMR Spectroscopy Nucleic Acids Performance Pharmaceutical Preparations Process Property Proteins Resistance Resolution Resources Sampling Solvents System Techniques Technology Testing Time Translations Validation Viscosity Water antimicrobial aqueous base biophysical techniques combinatorial chemistry comparative cost design drug discovery experimental study flexibility high throughput screening human disease hydrophilicity improved innovation interest lipophilicity member mutant nanoscale particle public health relevance screening small molecule tool

项目摘要

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.

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发展成为药物发现过程中的核心工具，通常基于极高通量体外筛选（HTS）策略。使用灵敏的高光谱技术（例如荧光法和最近的质谱法）检测配体结合，这些类型的方法通常无法揭示所检测到的蛋白质-配体复合物的结构背景。 ,尽管通量较低，但已成为重要的贡献者，特别是对于 HTS“命中”的事后验证而言，最近，筛选文库的设计已成为重要的贡献者。对于过去十年左右开发的基于片段的药物发现库来说尤其如此，这些库旨在利用组合化学的进步，以扩大“成功”的灵活性。不幸的是，片段型配体本质上是弱结合剂，这导致了相当大的材料成本（为了达到检测结合所需的浓度）并且通过非特异性相互作用增加了假阳性结合的可能性。该提案将开发一种策略，在很大程度上消除这两个问题。它基于反胶束的纳米级特性，利用反胶束作为有限空间，我们预计能够 1) 有效地检测弱特异性结合； ) 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) 进入所需但使用现有筛选策略难以进入的化学空间区域（即弱亲水性结合剂依赖于进步）。基于我们最近开发的将单个蛋白质分子封装在反胶束纳米级水核内的解决方案，我们将开发这种用于高分辨率溶液核磁共振光谱以及使用荧光的高温超导筛选策略。初步结果表明这种方法的可行性。将使用几种具有生物医学意义的蛋白质来开发和测试纳米级封装的使用，以增强从片段库（历史上的药物靶标）中发现低亲和力结合物。开发许多策略，目前前列腺癌的靶标人类醛酮还原酶（AKR1c3）也将被筛选，以证明该方法对大可溶性蛋白质的实用性。 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使用更标准方法通常无法令人满意的筛查的蛋白质。总之，这里提出的研究将证明筛查的显着增强。使用纳米级封装构建片段库，可能会发现针对与重要人类疾病相关的这些靶点的初始化合物。