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

21 世纪毒理学计划 (Tox21) - 系统毒理学

• 批准号: 10683008
• 负责人: Menghang Xia
• 金额: $ 60.05万
• 依托单位: NATIONAL CENTER FOR ADVANCING TRANSLATIONAL SCIENCES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10683008
• 关键词: 2019-nCoV; 3-Dimensional; ACE2; Acetylcholine; Ache; Active Sites; Adherent Culture; Advocate; Agonist; Animal Testing; Attenuated; Benchmarking; Binding; Biochemical Pathway; Biological; Biological Assay; Biological Models; Biomedical Engineering; COVID-19; COVID-19 pandemic; COVID-19 treatment; CYP2D6 gene; CYP3A4 gene; Cell Survival; Cells; Cellular Assay; Chemical Warfare Agents; Chemicals; Chlorpromazine; Cholinergic Receptors; Cholinesterases; Collaborations; Consumption; Cosmetics; Curcumin; Cysteine; DNA Repair; Data; Data Set; Disease Outbreaks; Docking; Dose; Elements; Emetine; Engineered skin; Enzymes; Epithelial; Evaluation; Event; Genomics; Goals; Gonadotropin-Releasing Hormone Receptor; Half-Life; Health; High Pressure Liquid Chromatography; Histone Deacetylase; Human; Human Cell Line; IL8 gene; In Vitro; KISS1 gene; Lead; Leadership; Libraries; Literature; Liver Microsomes; Lopinavir/Ritonavir; Lysine; Mass Spectrum Analysis; Measures; Metabolic; Metabolic Activation; Metabolic Biotransformation; Metabolism; Methods; Microsomes; Mission; Modeling; Molecular; Muscarinic M1 Receptor; Muscarinics; NADP; National Center for Advancing Translational Sciences; National Institute of Environmental Health Sciences; National Toxicology Program; Neurons; Neurotransmitters; Nuclear Receptors; Parents; Pathogenicity; Pathway interactions; Peptides; Pesticides; Pharmaceutical Preparations; Physiological; Phytochemical; Plasma; Rattus; Reporter Genes; Reporting; Research Personnel; Role; SARS-CoV-2 infection; Safety; Signal Pathway; Signal Transduction; Skin; Specificity; System; TMPRSS2 gene; TP53 gene; Testing; Therapeutic Agents; Thick; Tight Junctions; Time; Topical application; Toxic effect; Toxicology; United States Environmental Protection Agency; United States Food and Drug Administration; United States National Institutes of Health; Viral; Work; Xenobiotic Metabolism; antagonist; base; beta-Lactamase; biological systems; cell dimension; cellular targeting; computational toxicology; consumer product; cytokine; cytotoxic; cytotoxicity; design; drug candidate; drug market; drug repurposing; endoplasmic reticulum stress; environmental chemical; environmental toxicology; genotoxicity; hazard; high throughput screening; in vitro Assay; in vitro Model; in vivo; inhibitor; irritation; monolayer; neurotoxicity; nitazoxanide; predictive modeling; programs; receptor; response; robotic system; screening; side effect; stem cells

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 10 assays. Highlights range from performing 4 online screenings, including gonadotropin-releasing hormone receptor, Muscarinic Ach receptor M1, Kisspeptin receptor, and 5Hydroxytryptamine receptor 2A assays in both agonist and antagonist modes against the Tox21 10K compound library on the Tox21 robotic system. The US Tox2Program has utilized a quantitative high throughput screening (qHTS) approach to profile thousands of environmental chemicals using a battery of in vitro cell-based assays. The limitation of these assays, particularly those that measure events associated with DNA damage and repair (i.e., genotoxicity), is the absence of a xenobiotic metabolism capability. To overcome this limitation, we investigated methods to incorporate a metabolic component (e.g., liver microsomes) into existing Tox21 assays. We used a p53 beta-lactamase reporter gene assay (p53-bla) as a model system to incorporate metabolic capability into qHTS assays. We screened the Tox21 10K compound library using a p53-bla assay alone or with rat liver microsomes (RLM) or human liver microsomes (HLM) supplemented with NADPH, to identify compounds that induce p53 signaling after biotransformation. Two hundred seventy-eight compounds were identified as active under any of these three conditions. Of these 278 compounds, 73 gave more potent responses in the p53-bla plus RLM assay, and 2 were more potent in the p53-bla plus HLM assay compared with the responses they generated in the p53-bla assay without microsomes. To confirm the role of metabolism in the differential responses, we retested these 75 compounds in the absence of NADPH or with heat-attenuated microsomes. Forty-four compounds treated with RLM, but none with HLM, became less potent under these conditions, confirming the role of RLM in metabolic activation. Further evidence of biotransformation was obtained by measuring the half-life of the parent compounds in the presence of microsomes. Taken together, our study indicates that this approach can identify compounds that are not be detected by standard screening methods that lack metabolic capability. Assessing irritation and sensitization potential is a key element in the safety evaluation of topical drugs and other consumer products such as cosmetics. To evaluate the compounds for their irritation and sensitization potential, we tested about 500 topically applied compounds by using monolayer skin cells and three-dimensional culture models including reconstructed human epithelial and full-thickness skin models by measuring tight junctions, cell viability, and cytokine secretions for assessing chemical irritation and sensitization. This study represents the first step in advocating for replacement of current animal tests with bio-engineered skin models. To develop an HTS comparable method of direct peptide reactivity assay (DPRA) that has been used for assessing compound sensitization potential, we modified DPRA assay measuring the amount of free cysteine or lysine peptide from traditionally using a high-performance liquid chromatography platform to a high throughput tandem mass spectrum system, which greatly increases the screening throughput. Recently, we have validated and screened KeratinoSens Nrf2-ARE-Luc assay against the Tox21 10K compound library to identify the compounds with sensitization potential. After primary screening, we identified a group of Nrf2/ARE activators and further evaluated them in a battery of in vitro assays including DPRA, IL-8, and human cell line activation test (hCLAT). 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. To identify environmental chemicals that inhibit AChE activity using in vitro models, we have used enzyme- and cell-based AChE inhibition assay to screen the Tox21 10K compound library. From the primary screening, over 100 AChE inhibitors have been identified. These compounds were further studied their AChE inhibition in SH-SY5Y and human neuron stem cells (hNSC) in monolayer culture vs spheroid culture. Some AChE inhibitors showed more potency in hNSC spheroid culture compared to monolayer culture, while higher expression levels of CYP3A4 and CYP2D6 were found in spheroids than in monolayer culture, suggesting CYP enzymes were involved in bioactivation. To explore the interactions between AChE and their inhibitors, we used a molecular docking analysis to study the binding mode of these AChE inhibitors and found that all the AChE inhibitors were predicted to bind the active sites of the AChE. Using this approach, we identified a group of known AChE inhibitors and many previously not reported AChE inhibitors including some AChE inhibitors that need metabolic activation. Many of these AChE inhibitors were marketed drugs, withdrawn drugs, or unapproved drug candidates. They compared IC50s of these AChE inhibitors with human plasma concentrations (Cmax) from the literature and calculated the ratios of IC50 over Cmax. A common benchmark indicating physiological relevance is IC50/Cmax < 10. Using these ratio values can prioritize AChE inhibitors for further in-depth study. Since the outbreak of a global pandemic coronavirus disease-19 (COVID-19), various potential therapeutic agents for COVID-19 are being investigated worldwide mainly through the drug repurposing approach. Many drugs employed as anti-viral may exert unwanted side effects (i.e., toxicity) via unknown mechanisms. To quickly assess these drugs for their potential toxicological effects and mechanisms, we used the Tox21 in vitro assay datasets generated from screening 10,000 compounds consisting of approved drugs and environmental chemicals against multiple cellular targets and pathways. Many these drugs were shown to be active modulators in our nuclear receptor signaling pathway assays. Several signaling pathways including AR, Nrf2/ARE, ER, FXR, ER stress, and targets like HDAC were reported for their role in regulating ACE2 and TMPRSS2 expression, which are the main host cell factors that aid in SARS-CoV-2 pathogenicity. Some of the drugs in our study were shown to be cytotoxic against a wide range of cells and they include chlorpromazine, curcumin, emetine, lopinavir, ritonavir, niclosamide, and nitazoxanide. The cytotoxicity of these compounds might be due to their target promiscuity as shown by their activities in multiple Tox21 in vitro assays. Although in vitro cytotoxicity assays were used to screen chemicals/drugs for their relative toxicities at micro-molar concentrations, these assays can identify a potential hazard due to the multiple uses or high doses of the drugs when administered in humans. These qHTS data are publicly available and can provide valuable information on drug activities and their off-target effects, which can be further investigated for their potential uses in treating COVID-19 infection.

Tox21 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。 Tox21 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。 系统毒理学团队已鉴定、开发、优化和/或筛选了 10 多种检测方法。亮点包括进行 4 项在线筛选，包括针对 Tox21 机器人系统上的 Tox21 10K 化合物库以激动剂和拮抗剂模式进行促性腺激素释放激素受体、毒蕈碱 Ach 受体 M1、Kisspeptin 受体和 5-羟色胺受体 2A 检测。 美国 Tox2Program 采用定量高通量筛选 (qHTS) 方法，通过一系列体外细胞检测来分析数千种环境化学物质。这些检测的局限性，特别是那些测量与 DNA 损伤和修复（即基因毒性）相关的事件的检测，是缺乏外源代谢能力。为了克服这一限制，我们研究了将代谢成分（例如肝微粒体）纳入现有 Tox21 检测的方法。我们使用 p53 β-内酰胺酶报告基因检测 (p53-bla) 作为模型系统，将代谢能力纳入 qHTS 检测中。我们单独使用 p53-bla 测定或与补充 NADPH 的大鼠肝微粒体 (RLM) 或人肝微粒体 (HLM) 一起筛选 Tox21 10K 化合物库，以鉴定在生物转化后诱导 p53 信号传导的化合物。在这三种条件下，有 278 种化合物被鉴定为具有活性。在这 278 种化合物中，73 种在 p53-bla 加 RLM 测定中给出了更有效的反应，2 种在 p53-bla 加 HLM 测定中比它们在没有微粒体的 p53-bla 测定中产生的反应更有效。为了确认代谢在差异反应中的作用，我们在不存在 NADPH 或使用热衰减微粒体的情况下重新测试了这 75 种化合物。在这些条件下，用 RLM 处理的 44 种化合物（但没有用 HLM 处理的化合物）的效力减弱，证实了 RLM 在代谢激活中的作用。通过测量微粒体存在下母体化合物的半衰期，获得了生物转化的进一步证据。综上所述，我们的研究表明，这种方法可以识别缺乏代谢能力的标准筛选方法无法检测到的化合物。 评估刺激和致敏潜力是外用药物和化妆品等其他消费品安全性评估的关键要素。为了评估这些化合物的刺激性和致敏潜力，我们通过测量紧密连接、细胞活力和细胞因子分泌，使用单层皮肤细胞和三维培养模型（包括重建的人类上皮和全层皮肤模型）测试了约 500 种局部应用的化合物用于评估化学刺激性和致敏性。这项研究代表了倡导用生物工程皮肤模型取代当前动物测试的第一步。为了开发一种可用于评估化合物致敏潜力的直接肽反应性测定 (DPRA) 的 HTS 类似方法，我们将测量游离半胱氨酸或赖氨酸肽量的 DPRA 测定法从传统使用高效液相色谱平台修改为高效液相色谱平台。通量串联质谱系统，大大提高了筛选通量。最近，我们针对 Tox21 10K 化合物库验证和筛选了 KeratinoSens Nrf2-ARE-Luc 检测，以识别具有致敏潜力的化合物。经过初步筛选后，我们鉴定了一组 Nrf2/ARE 激活剂，并通过一系列体外测定（包括 DPRA、IL-8 和人类细胞系激活测试 (hCLAT)）进一步评估它们。 AChE 是体内主要的胆碱酯酶，负责代谢关键的神经递质乙酰胆碱。抑制 AChE 活性可导致神经毒性，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。为了使用体外模型识别抑制 AChE 活性的环境化学物质，我们使用基于酶和细胞的 AChE 抑制测定来筛选 Tox21 10K 化合物库。通过初步筛选，已鉴定出 100 多种 AChE 抑制剂。 进一步研究了这些化合物在单层培养与球体培养中的 SH-SY5Y 和人神经元干细胞 (hNSC) 中的 AChE 抑制作用。与单层培养相比，一些 AChE 抑制剂在 hNSC 球体培养中表现出更强的效力，而在球体中发现 CYP3A4 和 CYP2D6 的表达水平比在单层培养中更高，表明 CYP 酶参与生物激活。为了探索AChE与其抑制剂之间的相互作用，我们使用分子对接分析来研究这些AChE抑制剂的结合模式，发现所有AChE抑制剂都预计与AChE的活性位点结合。使用这种方法，我们鉴定了一组已知的 AChE 抑制剂和许多以前未报道的 AChE 抑制剂，包括一些需要代谢激活的 AChE 抑制剂。这些乙酰胆碱酯酶抑制剂中有许多是上市药物、撤回药物或未经批准的候选药物。他们将这些 AChE 抑制剂的 IC50 与文献中的人血浆浓度 (Cmax) 进行了比较，并计算了 IC50 与 Cmax 的比率。指示生理相关性的常见基准是 IC50/Cmax < 10。使用这些比率值可以优先考虑 AChE 抑制剂以进行进一步深入研究。 自全球大流行冠状病毒病（COVID-19）爆发以来，世界范围内主要通过药物再利用方法研究各种潜在的 COVID-19 治疗药物。许多用作抗病毒药物可能会通过未知机制产生不良副作用（即毒性）。为了快速评估这些药物的潜在毒理学作用和机制，我们使用了 Tox21 体外测定数据集，这些数据集是通过筛选 10,000 种化合物而生成的，这些化合物由已批准的药物和针对多个细胞靶点和途径的环境化学品组成。在我们的核受体信号通路测定中，许多这些药物被证明是活性调节剂。据报道，包括 AR、Nrf2/ARE、ER、FXR、ER 应激和 HDAC 等靶标在内的多种信号通路在调节 ACE2 和 TMPRSS2 表达中发挥作用，ACE2 和 TMPRSS2 是有助于 SARS-CoV-2 致病性的主要宿主细胞因子。我们研究中的一些药物被证明对多种细胞具有细胞毒性，包括氯丙嗪、姜黄素、依米丁、洛匹那韦、利托那韦、氯硝柳胺和硝唑尼特。这些化合物的细胞毒性可能是由于它们的靶标混杂性，如它们在多个 Tox21 体外测定中的活性所示。尽管体外细胞毒性测定用于筛选化学物质/药物在微摩尔浓度下的相对毒性，但这些测定可以识别由于药物在人体中多次使用或高剂量而产生的潜在危险。这些 qHTS 数据是公开的，可以提供有关药物活性及其脱靶效应的有价值的信息，可以进一步研究它们在治疗 COVID-19 感染方面的潜在用途。