A Specific Mechanism for Non-Specific Inhibition

描述（由申请人提供）：假阳性、混杂配体在命中列表中占主导地位，并且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，可能会统一我们的理解最混杂的命中：复合聚集受体螯合。许多分子，包括命中，铅，试剂，甚至一些药物，可以聚集，当他们这样做时，他们的性质发生了巨大的变化，通常是有害的，但有时是有益的。我们调查了生物学相关图书馆中聚合器的流行情况，确定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.探讨聚集剂在筛查中的流行情况及其对生物测定的影响。我们开始与简单但基本的问题：有多少百分比的筛选库形成混杂的聚集体？所有假阳性中有多少百分比是由于聚合造成的？它们会抑制哪些受体？我们将使用上一时期开发的分析方法，筛选由数十万个分子组成的大型药物样筛选文库。我们将详细描述每一类误报，而不仅仅是聚合。我们将探讨聚集是否影响膜结合受体，除了酶。一系列更复杂的问题调查了对下游生物测定的影响。聚集体破坏细胞膜并具有溶血性，因此有必要考虑它们如何影响毒理学测定。我们还将研究聚集体是否可以在口服药物中持续存在，在其分布中发挥作用，正如其他人所建议的那样。2.探讨基于聚集的抑制机制。基于聚集的抑制的确切机制仍不清楚。例如，我们不明白为什么聚集体缔合会导致酶抑制。聚集体是否使酶变性，或者这种作用更微妙，可能影响动态运动？聚集体的结构是什么？它们类似于囊泡还是固体胶体？它们对蛋白质的亲和力如何？一个简单的技术相结合，如离心，凝胶电泳，酶学，和更高的分辨率技术，如电子显微镜和氘交换质谱，将被用来调查这些问题。公共卫生相关性：高通量筛选是发现新药先导化合物的最广泛使用的技术，但不幸的是，这些筛选被假命中所主导。大多数这些错误的命中是由许多有机分子在溶液中聚集的趋势解释的，因此破坏了测定。通过理解和控制聚集，该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