A Specific Mechanism for Non-Specific Inhibition

描述（由申请人提供）：假阳性，混杂配体主导着hit list，是药物发现筛选中的关键问题。在这里，我们考虑一个单一的机制，可能会统一我们对大多数混杂命中的理解：化合物聚集之后的受体隔离。许多分子，包括撞击物、先导物、试剂，甚至一些药物，都可以聚集，当它们聚集时，它们的性质会发生巨大的变化，通常是有害的，但有时是有益的。我们调查了聚合器在生物学相关文库中的流行程度，确定了它们是如何起作用的，并开发了快速筛选来检测它们。具体目标是：1。探讨聚集物在筛选中的流行程度及其对生物检测的影响。我们从简单但重要的问题开始：筛选库形成混杂聚合的百分比是多少？所有假阳性的百分比是由聚合引起的？它们会抑制哪些受体？我们将使用上一阶段开发的一种检测方法，筛选由数十万个分子组成的大型药物筛选文库，用于聚合器。我们将详细描述每一类假阳性，而不仅仅是聚合。除了酶外，我们将探讨聚集是否影响膜结合受体。一系列更复杂的问题调查了对下游生物分析的影响。聚集体破坏膜和溶血，是适当的怀疑，他们可能会影响毒理学分析。我们还将研究聚集体是否可以在口服给药药物中持续存在，并在其分布中发挥作用，正如其他人所建议的那样。2. 探讨以聚集为基础的抑制机制。基于聚集的抑制的确切机制尚不清楚。例如，我们不明白为什么聚合关联会导致酶抑制。是聚合体使酶变性，还是作用更微妙，可能影响动态运动？聚集体的结构是什么？它们像囊泡还是固体胶体？它们对蛋白质的亲和力是什么？简单的技术，如离心、凝胶电泳和酶学，以及更高分辨率的技术，如电子显微镜和氘交换质谱，将被用来研究这些问题。公共卫生相关性：高通量筛选是发现新药线索最广泛使用的技术，但不幸的是，这些筛选主要是错误的命中。大多数错误的命中都可以解释为许多有机分子在溶液中聚集的趋势，从而破坏了分析。通过理解和控制聚合，这个项目试图解决这个问题，从而改善药物发现

DESCRIPTION (provided by applicant): False positive, promiscuous ligands dominate hit lists and are a key problem in drug discovery screens. Here we consider a single mechanism that might unite our understanding of most promiscuous hits: compound aggregation followed by receptor sequestration. Many molecules, including hits, leads, reagents, and even some drugs, can aggregate, and when they do so their properties change dramatically, often perniciously but sometimes beneficially. We investigate the prevalence of aggregators in biologically relevant libraries, determine how they act, and develop rapid screens to detect them. The specific aims are: 1. To explore the prevalence of aggregators in screening and their effect on biological assays. We begin with simple-but-essential questions: what percentage of screening libraries form promiscuous aggregates? What percentage of all false-positives owe to aggregation? What range of receptors will they inhibit? We will screen large, drug-like screening libraries composed of hundreds-of-thousands of molecules for aggregators, using an assay developed in the last period. We will characterize every class of false positive, not only aggregates, in detail. We will explore whether aggregation affects membrane-bound receptors, in addition to enzymes. A more complex series of questions investigates effects on downstream biological assays. Aggregates disrupt membranes and are hemolytic, and it is appropriate to wonder how they might affect toxicological assays. We will also investigate whether aggregates can persist among orally-dosed drugs, playing a role in their distribution, as has been suggested by others. 2. To investigate the mechanism of aggregation-based inhibition. The exact mechanism of aggregation-based inhibition remains unclear. For instance, we do not understand why aggregate association leads to enzyme inhibition. Does the aggregate denature the enzyme, or is the effect more subtle, perhaps affecting dynamic motion? What is the structure of aggregates; do they resemble vesicles or solid colloids? What is their affinity for protein? A combination of simple techniques, such as centrifugation, gel electrophoresis, and enzymology, and higher resolution techniques, such as electron microscopy and deuterium-exchange mass spectroscopy, will be used to investigate these questions. PUBLIC HEALTH RELEVANCE: High throughput screening is the most widely used technique to discover new drug leads, but unfortunately, these screens are dominated by false hits. Most of these false hits are explained by the tendency of many organic molecules to aggregate in solution, and so disrupt assays. By understanding and controlling aggregation, this project attempts to address this problem, and so improve drug discovery