Phenotypic Assay Design and Development for Rare and Neglected Diseases

罕见和被忽视疾病的表型测定设计和开发

• 批准号: 10469250
• 负责人: James Inglese
• 金额: $ 51.34万
• 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/10469250
• 关键词: AMP Deaminase; Adrenal Cortex Hormones; Adult; Affect; Age related macular degeneration; Aging; Alleles; Animal Model; Anti-Infective Agents; Antineoplastic Agents; Antiparasitic Agents; Applications Grants; Autoimmune; Autophagocytosis; Basic Science; Biological; Biological Assay; Biological Models; Biology; Blood; Blood Platelets; Bone Marrow Transplantation; CD86 gene; Cancer Model; Carbon; Cell Aging; Cell Differentiation process; Cell model; Cells; Cellular Assay; Chemicals; Chemistry; Chromosome abnormality; Chromosomes; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communicable Diseases; Communities; Congenital Disorders; Cytometry; DNA Sequence Alteration; Deamination; Defect; Development; Diamond-Blackfan anemia; Disease; Disease model; Drug Compounding; Embryo; Engineering; Enzymes; Erinaceidae; Erythropoiesis; Ethnic group; Evaluation; Extramural Activities; Failure; Fission Yeast; Frequencies; Gametogenesis; Gene Mutation; Gene Silencing; Genes; Genetic Screening; Genomics; Geography; Glucose; Growth; Half-Life; Human; Immunity; Individual; Infection; Inflammatory; Inherited; Interferons; Investigation; Iron Chelation; Knowledge; Laboratories; Lasers; Leukocytes; Libraries; Life; Link; Live Birth; Malaria; Malignant Neoplasms; Measures; Mediating; Membrane; Messenger RNA; Metabolic; Methodology; Microbe; Microscopy; Minority; Modeling; Molecular Biology; Morphology; Multienzyme Complexes; Mus; Muscle Development; Muscle Weakness; Myositis; Natural Products; Nematoda; Nonsense Codon; Organism; Palliative Care; Parasites; Parents; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pharmacology; Phenocopy; Phenotype; Plasmodium falciparum; Process; Production; Progressive Disease; Protein Isoforms; Proteins; Quality Control; Quantitative Microscopy; RNA Interference; Recombinants; Reporter; Research Project Grants; Residual state; Reticulocytes; Retina; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rodent; Role; Scientist; Siblings; Signal Transduction; Skeletal Muscle; Source; System; T-Cell Activation; TP53 gene; Technology; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transfusion; Translational Research; Translations; Ubiquitination; Uniparental Disomy; Work; Yeasts; assay development; base; blastomere structure; curative treatments; design; drug discovery; early screening; gene product; genome editing; genome-wide; glucose metabolism; high throughput screening; human disease; immunoregulation; inhibitor/antagonist; interest; loss of function; mRNA Decay; malaria infection; method development; muscle metabolism; neglect; novel; peripheral blood; pharmacophore; programs; prototype; public-private partnership; repository; research and development; response; screening; sensor; sight restoration; small molecule; small molecule libraries; smoothened signaling pathway; transcription activator-like effector nucleases; ubiquitin-protein ligase

This project includes the development of cell- and model organism-based assays to target signaling, metabolic and proliferative pathways, and to phenocopy inherited genetic mutations leveraging disease knowledge from basic and clinical research programs. The assay designs make use of recent advances in molecular biology and novel reporter systems that enable efficient compound library screening. The assay designs are considered in the context of analysis and progression strategies for evaluation of approved drugs and investigational agents using quantitative high throughput screening (qHTS) technologies. The lab has a strong emphasis on methods development research to advance assay and screening efficiency in drug discovery and chemical genomics. AMP deaminase (AMPD) deficiency. ADST is developing a coincidence reporter assay to aid in the discovery of compounds that increase expression of the enzyme AMP deaminase implicated in idiopathic inflammatory myositis (IIM), a rare autoimmune progressive disease that afflicts the skeletal muscle of patients, comprised of errors in both immune regulation and intrinsic muscle metabolism. AMP deaminase (AMPD) catalyzes the deamination of AMP to IMP. In humans AMPD activity is encoded by at least three genes. AMPD isoforms have tissue specific expression patterns in adults and stage specific expression patterns during the muscle development. Drugs which stimulate the expression of AMPD1 are hypothesized to reverse muscle weakness. Assays for the identification of nonsense-mediated mRNA decay (NMD) modulators. Genetic mutations resulting in premature stop codon-containing mRNAs are subjected to nonsense-mediated mRNA decay (NMD) quality control. Residual function of a truncated gene product could mitigate a pathophysiologic phenotype NMD suppression may provide a therapeutic strategy. Based on a prior NMD assay used in gene-silencing studies, we developed an NMD-sensitive loss-of-function, gain-of-signal assay using our coincidence-reporter technology to examine chemical libraries for discovery of novel NMD-modulating chemotypes. Secondary phenotypic assays will measure the impact of modulating NMD in the context of allele-specific disease gene mutations. Inhibition of gametogenic genes in S. pombe as a model for Uniparental Disomy: Uniparental disomy (UPD) is a chromosomal abnormality where an individual inherits a pair of homologous chromosomes originating from only one parent and is often linked to cancer and other congenital disorders. Gametogenesis in fission yeast has been used as a model system for defects in the RNA interference (RNAi) machinery of higher organisms. ADST is reengineering a medium throughput yeast assay to a higher throughput system capable of aHTS for the evaluation of a large-scale chemical libraries. MARCH1 E3 Ligase attenuators to enhance antimalaria immunity. The E3 ubiquitin ligase Membrane Associated RING-CH (MARCH) 1 promotes ubiquitination and degradation of MHC II and/or CD86, while inhibiting type-1 interferon response, thereby serving as a novel target for antimalaria immunity. A previous genome-wide genetic screen of early P. yoellii infection in mice identified MARCH1 as a host gene of interest that was shown to interact with STING and MAVS to regulate type 1 interferon signaling, T cell activation and IFN- production during malaria infection. A coincidence reporter-based cellular assay is being employed to identify inhibitors of the MARCH1 pathway aiming to identify chemotypes that may serve as the basis for novel E3 ubiquitin ligase pathway modulators. Inhibition of Hedgehog autoprocessing and cholesteroylation. The Hedgehog (Hh) signaling pathway is essential to embryonic cell differentiation. Hh protein concentration gradients are critical to proper embryonic morphologic development, where pathway malfunction can result in cancer. We are developing, optimizing, and validating cell-based qHTS assays for the identification of Hedgehog (Hh) autoprocessing inhibitors. Inhibitors of this process may have potential as anticancer agents. Diamond Blackfan Anemia (DBA): ADST is developing assay strategies as part of a translational research project for DBA a rare, congenital disease seen in all ethnic groups with a frequency of 7 cases per million live births. DBA patients have no reticulocytes in peripheral blood indicating a failure of erythropoiesis whereas production of the white cells and platelets are unaffected. For a minority of patients, bone marrow transplantation from a healthy sibling is a curative therapy, otherwise there are a variety of palliative therapies that can prolong life into the third decade. Current treatments include corticosteroid therapy, which results in reduced growth and other complications, or lifelong transfusion and iron chelation. Glucose-regulating multienzyme compartment high throughput quantitative microscopy-based assay. ADST is developing a 1536-well plate format microscopy-based qHTS assay for discovery of compounds that modulate the formation of a dynamically assembled multienzyme complex regulating glucose-derived carbon flux in cells. The pilot screening of pharmacologically active small molecules for or against the assembly will assist to understand the biological significance of the assembly in the cell. Novel pharmacophores, which promote or disrupt the assembly in human disease models, will be found through HTS for therapeutic intervention in the treatment of glucose metabolism-associated human diseases. Controlling cellular senescence with small molecule compounds modulating the 133p53 isoform: ADST is developing a laser-cytometry based HTS assay for discovery of compounds that modulate the cellular distribution and half-life of a specific p53 isoform in cells. The functional and regulatory role served by 133p53 between cellular senescence and autophagy is relevant to both aging and cancer. Compounds that modulate cellular phenotypes based on expression of a GFP- 133p53 sensor will be explored for their pharmacological effect in relevant aging and cancer model systems. Discovery of Drugs for inherited rare blinding retinal degenerations: ADST has entered a public-private partnership aimed at alleviating and curing blinding retinal degenerative diseases, which utilizes ADSTs expertise in assay development, chemical biology, and qHTS to develop assays and chemical library testing paradigms to identify drugs and compounds that may save and restore vision for people affected by retinal diseases such as retinitis pigmentosa and age-related macular degeneration.The use of primary rodent retinal cells benefits from the low-volume assay technology employed at ADST. Novel sources of chemical libraries evaluated by model organisms of infectious disease: To probe the biological activity of chemistry methodology-enabled libraries, privileged-chemotype collections, isolated natural products (NPs), and pre-fractionated culturable NP extracts (NPEs), ADST is testing the anti-infective and anti-parasitic potential of these chemical repositories in model organism microbes and parasites. These infectious disease assays use qHTS to provide a pharmacological assessment of library activity. Previously, we exposed five P. falciparum lines of distinct geographic origin to chemical libraries using qHTS to evaluate intraerythrocytic viability as a model for the blood-borne propagation stage of malaria. ADST is developing bacterial, fungal, and nematode recombinant organisms to approximate the essentiality and vulnerability of unique target enzymes in parasitic species.

该项目包括开发基于细胞和模型生物的检测方法，以靶向信号传导、代谢和增殖途径，并利用基础和临床研究项目的疾病知识对遗传性基因突变进行表型复制。 该检测设计利用了分子生物学的最新进展和新颖的报告系统，可实现高效的化合物库筛选。分析设计是在使用定量高通量筛选（qHTS）技术评估已批准药物和研究药物的分析和进展策略的背景下考虑的。该实验室非常重视方法开发研究，以提高药物发现和化学基因组学的测定和筛选效率。 AMP 脱氨酶 (AMPD) 缺乏。 ADST 正在开发一种巧合报告基因检测，以帮助发现可增加与特发性炎症性肌炎 (IIM) 相关的 AMP 脱氨酶表达的化合物，IIM 是一种罕见的自身免疫性进行性疾病，影响患者的骨骼肌，由免疫和免疫系统错误组成。调节和内在肌肉代谢。 AMP 脱氨酶 (AMPD) 催化 AMP 脱氨基生成 IMP。 在人类中，AMPD 活性至少由三个基因编码。 AMPD 亚型在成人中具有组织特异性表达模式，在肌肉发育过程中具有阶段特异性表达模式。 刺激 AMPD1 表达的药物被认为可以逆转肌肉无力。 用于鉴定无义介导的 mRNA 衰减 (NMD) 调节剂的测定。导致过早终止密码子的 mRNA 的基因突变受到无义介导的 mRNA 衰减 (NMD) 质量控制。截短基因产物的残余功能可以减轻病理生理表型，NMD 抑制可以提供一种治疗策略。 基于之前用于基因沉默研究的 NMD 测定，我们开发了一种 NMD 敏感的功能丧失、信号增益测定，使用我们的符合报告技术来检查化学库，以发现新型 NMD 调节化学型。二次表型测定将测量在等位基因特异性疾病基因突变的背景下调节 NMD 的影响。 粟酒裂殖酵母中配子发生基因的抑制作为单亲二体性的模型：单亲二体性（UPD）是一种染色体异常，其中个体继承了仅源自父母一方的一对同源染色体，并且通常与癌症和其他先天性疾病有关。 裂殖酵母的配子发生已被用作高等生物 RNA 干扰 (RNAi) 机制缺陷的模型系统。 ADST 正在将中等通量酵母测定重新设计为能够进行 aHTS 的更高通量系统，用于评估大规模化学库。 MARCH1 E3 连接酶衰减剂可增强抗疟疾免疫力。 E3 泛素连接酶膜相关 RING-CH (MARCH) 1 促进 MHC II 和/或 CD86 的泛素化和降解，同时抑制 1 型干扰素反应，从而成为抗疟疾免疫的新靶点。 先前对小鼠早期约氏疟原虫感染进行的全基因组遗传筛选将 MARCH1 确定为感兴趣的宿主基因，该基因与 STING 和 MAVS 相互作用，在疟疾感染期间调节 1 型干扰素信号传导、T 细胞激活和 IFN-产生。基于巧合报告基因的细胞测定被用来鉴定 MARCH1 通路的抑制剂，旨在鉴定可作为新型 E3 泛素连接酶通路调节剂基础的化学型。 抑制 Hedgehog 自动加工和胆固醇化。 Hedgehog (Hh) 信号通路对于胚胎细胞分化至关重要。 Hh 蛋白浓度梯度对于正确的胚胎形态发育至关重要，其中途径故障可能导致癌症。 我们正在开发、优化和验证基于细胞的 qHTS 测定法，用于鉴定 Hedgehog (Hh) 自动加工抑制剂。 该过程的抑制剂可能具有作为抗癌剂的潜力。 钻石黑扇贫血症 (DBA)：ADST 正在开发检测策略，作为 DBA 转化研究项目的一部分。DBA 是一种罕见的先天性疾病，见于所有种族群体，发病率为每百万活产儿 7 例。 DBA患者外周血中没有网织红细胞，表明红细胞生成失败，而白细胞和血小板的产生不受影响。 对于少数患者来说，来自健康兄弟姐妹的骨髓移植是一种治疗方法，否则有多种姑息疗法可以将生命延长到三十岁。 目前的治疗方法包括皮质类固醇治疗，这会导致生长减缓和其他并发症，或终身输血和铁螯合。 葡萄糖调节多酶室高通量定量显微镜检测。 ADST 正在开发一种基于显微镜的 1536 孔板格式 qHTS 测定，用于发现调节细胞中葡萄糖衍生碳通量的动态组装多酶复合物形成的化合物。 对支持或反对该组装的药理学活性小分子进行初步筛选将有助于了解细胞中组装的生物学意义。通过 HTS 可以发现促进或破坏人类疾病模型中组装的新型药效团，用于治疗与葡萄糖代谢相关的人类疾病。 用调节 133p53 同工型的小分子化合物控制细胞衰老：ADST 正在开发一种基于激光细胞术的 HTS 测定法，以发现调节细胞中特定 p53 同工型的细胞分布和半衰期的化合物。 133p53 在细胞衰老和自噬之间发挥的功能和调节作用与衰老和癌症相关。 将探索基于 GFP-133p53 传感器表达调节细胞表型的化合物在相关衰老和癌症模型系统中的药理作用。 发现治疗遗传性罕见致盲性视网膜变性疾病的药物：ADST 已进入公私合作伙伴关系，旨在减轻和治愈致盲性视网膜变性疾病，该伙伴关系利用 ADST 在测定开发、化学生物学和 qHTS 方面的专业知识来开发测定和化学库测试范式，以确定可以挽救和恢复受视网膜疾病（例如色素性视网膜炎和年龄相关性黄斑变性）影响的人的视力的药物和化合物。使用原代啮齿类动物视网膜细胞受益于ADST 采用的低容量测定技术。 通过传染病模型生物评估的化学文库的新来源：为了探究化学方法支持的文库、特权化学型集合、分离的天然产物 (NP) 和预分级可培养 NP 提取物 (NPE) 的生物活性，ADST测试这些化学库在模式生物微生物和寄生虫中的抗感染和抗寄生虫潜力。 这些传染病检测使用 qHTS 来提供文库活性的药理学评估。 此前，我们使用 qHTS 将五个不同地理来源的恶性疟原虫品系暴露于化学库中，以评估红细胞内活力，作为疟疾血源性传播阶段的模型。 ADST 正在开发细菌、真菌和线虫重组生物，以了解寄生物种中独特目标酶的重要性和脆弱性。