Identification and Characterization of Mitochondrial Toxins

描述(由申请人提供)：许多环境、药物和工业化合物通过对线粒体功能的有害影响而对人类健康产生负面影响。线粒体毒性被定义为线粒体功能和/或数量的减少，导致呼吸和能量产生减少。线粒体功能的严重丧失可导致重要器官因细胞损伤、细胞凋亡或坏死而受损。目前还没有导致线粒体功能障碍的化学相关化合物的数据库，也没有可靠的方法来预测线粒体毒性。为了建立线粒体毒物数据库，利用原代培养的兔肾近端小管细胞(RPTC)和海马生物科学细胞外通量(XF)分析仪进行了1760种不同化合物的呼吸测定。这一筛选的结果确定了22种减少非偶联线粒体呼吸的化合物，这是对线粒体功能的压力测试。从这22个化合物中，根据三个或三个以上化合物之间的分子相似性确定了五个化学簇。其中一个簇已经被比对，以确定与线粒体毒性有关的一组初步化学结构，定义为毒物。我们假设，由不同的化学实体定义的离散毒素载体可以识别以前未知的和未来的线粒体毒物。我们将从三个具体目标来检验这一假说。具体目标1是利用化学信息学分析来阐明已识别的五种线粒体毒物簇的毒素分子。具体目标2是通过对一个由50,000个小分子组成的不同文库进行电子筛选，识别与已阐明的毒素载体相关的线粒体毒物。所得化合物将使用RPTC和96孔XF仪器测试它们对线粒体呼吸的影响。已确认的线粒体毒物将被包括在化学信息学模型中，并用于提纯毒素载体。具体目的3是通过检测代表性化合物对解偶联氧化磷酸化、破坏电子传递链和/或改变线粒体通透性转换孔(MPT)的影响，分析已鉴定的毒物对线粒体损伤的机制。这项研究的长期目标是设计能够可靠地预测线粒体毒性的毒素载体，用于化学发现和风险评估。通过完成概述的提案，我将获得的培训将增强我的研究专业知识，并为我将创新概念应用于毒理学领域做好准备。 公共卫生相关性：我们假设，由不同的化学实体定义的离散毒素基团可以识别以前未知的和未来的线粒体毒物。对兔肾近端小管细胞原代培养中的不同化学库进行高通量呼吸测定筛选，鉴定出五类分子上相似的线粒体毒物。识别出的簇将被用来定义毒素载体，这些毒素载体可以可靠地预测线粒体毒性，用于化学设计、药物发现和人类健康风险评估。

DESCRIPTION (provided by applicant): Many environmental, pharmaceutical, and industrial compounds negatively affect human health by deleterious effects on mitochondrial function. Mitochondrial toxicity is defined as a decrease in the function and /or number of mitochondria, leading to decreased respiration and energy production. Severe loss of mitochondrial function can result in damage to vital organs due to cellular injury, apoptosis, or necrosis. Currently there is no database of chemically related compounds that cause mitochondrial dysfunction, and there are no reliable methods for predicting mitochondrial toxicity. To develop a database of mitochondrial toxicants, a novel respirometric assay of 1760 diverse compounds was conducted using primary cultures of rabbit renal proximal tubule cells (RPTC) and the Seahorse Biosciences Extracellular Flux (XF) analyzer. The results of this screen identified 22 compounds that diminish uncoupled mitochondrial respiration, a stress test for mitochondrial function. From these 22 compounds, five chemical clusters were identified based on molecular similarity between three or more compounds. One of these clusters has been aligned to identify a preliminary group of chemical structures related to mitochondrial toxicity, defined as a toxicophore. We hypothesize that discrete toxicophores defined by distinct chemical entities can identify previously unknown and future mitochondrial toxicants. We will examine this hypothesis in three Specific Aims. Specific Aim 1 is to elucidate the toxicophores for the five identified clusters of mitochondrial toxicants using cheminformatic analysis. Specific Aim 2 is to identify mitochondrial toxicants related to the elucidated toxicophores through an in silico screen of a diverse library of 50,000 small molecules. The resulting compounds will be examined for their effects on mitochondrial respiration using RPTC and a 96-well XF instrument. Confirmed mitochondrial toxicants will be included into the cheminformatic models and used to refine the toxicophores. Specific Aim 3 is to analyze the mechanism of mitochondrial damage for the identified toxicophores by examining the effects of representative compounds on uncoupling oxidative phosphorylation, disruption of the electron transport chain, and/or altering the mitochondrial permeability transition pore (MPT). The long-term goal of this research is to design toxicophores that can reliably predict mitochondrial toxicity for both chemical discovery and risk assessment. The training that I will acquire by completing the outlined proposal will enhance my research expertise, and will prepare me to apply innovative concepts to the field of toxicology. PUBLIC HEALTH RELEVANCE: We hypothesize that discrete toxicophores defined by distinct chemical entities can identify previously unknown and future mitochondrial toxicants. A high-throughput respirometric screen of a diverse chemical library in primary cultures of rabbit renal proximal tubule cells identified five clusters of molecularly similar mitochondrial toxicants. The identified clusters will be used to define toxicophores that can reliably predict mitochondrial toxicity for chemical design, drug discovery, and human health risk assessment.