Toxicology in the 21st Century Program (Tox21) - Systems Toxicology

Tox 21计划的联邦合作伙伴包括环境保护局（EPA）、食品和药物管理局（FDA）和NIH，并由NCATS和国家环境健康科学研究所（NIEHS）的国家毒理学计划（NTP）领导。这些机构共同努力推进体外毒理学测试。Tox 21计划由三个NCATS团队组成：系统毒理学，基因组毒理学和计算毒理学。 系统毒理学团队已确定、开发、优化和/或筛选了超过19种检测方法。重点包括进行5次在线筛选，包括CHO和HepG 2细胞中的半胱天冬酶3/7测定，一式三份的人胆固醇X受体，以及针对Tox 21 10 K化合物收集的基于乙酰胆碱酯酶（AChE）酶的测定（有或无代谢能力）和5次在线验证测定，包括基于酶的AChE测定（有或无肝微粒体测定），与Tox 21机器人系统上的LOPAC数据库进行比对所有这些测定在转移到机器人在线验证和在线筛选之前都进行了优化和评价。 乙酰胆碱酯酶是体内主要的胆碱酯酶，代谢一种关键的神经递质乙酰胆碱。 AChE活性的抑制可导致神经毒性，已知的抑制剂包括有机磷农药、化学战剂、药物和各种植物化学物质。在与CFSAN/FDA的合作中，系统毒理学团队已经筛选了两种基于细胞的AChE检测方法，其荧光和颜色度量读数以均匀的格式与Tox 21 10 K化合物集合进行比较。 此外，已经开发了基于酶的AChE测定法（有或没有肝微粒体），并针对一组已知的AChE抑制剂进行了验证，这些AChE抑制剂需要代谢才能成为活性化合物。此外，该团队还与NTP，EPA和FDA合作进行线粒体项目，并在后续试验中测试了初步筛选中确定的34种化合物，包括活性氧（ROS），p53和Nrf 2/ARE（抗氧化反应元件），线粒体耗氧量，Parkin易位以及C.优雅已知的线粒体复合物抑制剂（例如，鱼藤酮）和解偶联剂（例如，溴虫腈）以及潜在的新型复合物抑制剂和解偶联剂。该团队还通过在高含量成像仪中筛选接触2816种化合物的药物集合，优化和验证了1536孔板形式的体外共培养血管生成测定。从筛选中，鉴定出许多已知的血管生成抑制剂，如托泊替康、多西他赛和硼替佐米。本研究还鉴定了几种潜在的新型血管生成抑制剂，包括硫柳汞和podofilox。在这些抑制剂中，一些化合物被证明参与缺氧诱导因子-1-α（HIF-1）和核因子-κ B（NF-B）途径。 雌激素相关受体α（ERR）是一种孤儿核受体（NR），在能量稳态中发挥作用，并控制线粒体氧化呼吸。在与NIEHS的合作中，我们基于化合物结构相似性分析鉴定了包括他汀类药物簇在内的五种以上的激动剂簇（例如，他汀类药物）初步筛选后。我们还进行了后续测定（例如，siRNA敲除），我们发现了可能通过影响ERR信号传导而充当内分泌干扰物的化合物。此外，该团队还从初步筛选中确定了一组CAR激活剂和灭活剂。已经进行了几项后续研究，包括人肝细胞中的CAR易位，以确认具有CAR活性的化合物。

The Tox21 programs federal partners include the Environmental Protection Agency (EPA), the Food and Drug Administration (FDA) and NIH, with leadership from NCATS and the National Toxicology Program (NTP) at the National Institute of Environmental Health Sciences (NIEHS). These agencies work together to advance in vitro toxicological testing. The Tox21 Program is comprised of three NCATS teams: Systems Toxicology, Genomic Toxicology, and Computational Toxicology. The Systems Toxicology team has identified, developed, optimized, and/or screened more than 19 assays. Highlights range from performing 5 online screens, including caspase 3/7 assays in CHO and HepG2 cells, human pregnane X receptor in triplicate and acetylcholinesterase (AChE) enzyme-based assay with or without metabolic capability in single against the Tox21 10K compound collection and 5 online validation assays, including enzyme-based AChE assay with or without liver microsomes assays, against the LOPAC collection on the Tox21 robotic system. All of these assays were optimized and evaluated before moving to robotic online validation and online screening. AChE is the primary cholinesterase in the body that metabolizes a key neurotransmitter, acetylcholine. Inhibition of AChE activity can lead to neurotoxicity and known inhibitors include organophosphorus pesticides, chemical warfare agents, drugs, and various phytochemicals. In collaboration with the CFSAN/FDA, the Systems Toxicology team has screened two cell-based AChE assays with fluorescence and color metric readouts in a homogenous format against the Tox21 10K compound collection. In addition, enzyme-based AChE assays with or without liver microsomes have been developed and validated against a group of known AChE inhibitors that need metabolism to become active compounds. In addition, the team has worked with NTP, EPA and FDA on the mitochondria project and tested 34 compounds identified from the primary screening in the follow-up assays including reactive oxygen species (ROS), p53 and Nrf2/ARE (antioxidant response element), mitochondrial oxygen consumption, Parkin translocation, and larval development and ATP status in the C. elegans. Known mitochondrial complex inhibitors (e.g., rotenone) and un-couplers (e.g., chlorfenapyr), as well as potential novel complex inhibitors and un-couplers, were detected. The team has also optimized and validated in vitro co-culture angiogenesis assay in a 1536-well plate format by screening a drug collection contacting 2816 compounds in a high content imager. From the screen, many known angiogenesis inhibitors were identified such as topotecan, docetaxel, and bortezomib. Several potential novel angiogenesis inhibitors were also identified from this study including thimerosal and podofilox. Among the inhibitors, some compounds were proved to be involved in the hypoxia inducible factor-1-alpha (HIF-1) and the nuclear factor-kappa B (NF-B) pathways. The estrogen-related receptor alpha (ERR) is an orphan nuclear receptor (NR) that plays a role in energy homeostasis and controls mitochondrial oxidative respiration. In collaboration with NIEHS, we identified more than five agonist clusters including statin cluster based on compound structural similarity analysis (e.g., statins) after primary screening. We also performed follow-up assays (e.g., siRNA knockdown) we identified compounds that might act as endocrine disrupters through effects on ERR signaling. In addition, the team has identified a group of CAR activators and deactivators from the primary screening. Several follow-up studies including CAR translocation in human hepatocytes have been performed to confirm the compounds with CAR activity.