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: 10263804
• 负责人: James Inglese
• 金额: $ 47.57万
• 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/10263804
• 关键词: AMP Deaminase; Academia; Adrenal Cortex Hormones; Adult; Affect; Age related macular degeneration; Age-Years; Aging; Alleles; American; Anemia; Animal Model; Anti-Infective Agents; Antimalarials; Antiparasitic Agents; Applications Grants; Autoimmune Process; Autophagocytosis; Basic Science; Biological; Biological Assay; Biological Models; Biology; Blindness; Blood; Blood Platelets; Bone Marrow Transplantation; Cancer Model; Carbon; Cell Aging; Cells; Chemicals; Child; Chromosome abnormality; Chromosomes; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Collaborations; Collection; Communicable Diseases; Communities; Congenital Disorders; Cytometry; DNA Sequence Alteration; Deamination; Defect; Development; Diagnosis; Diamond-Blackfan anemia; Disease; Disease model; Doctor of Medicine; Doctor of Philosophy; Drug Compounding; Drug Targeting; Engineering; Enzymes; Equilibrium; Erythropoiesis; Ethnic group; Evaluation; Extramural Activities; Failure; Fission Yeast; Foundations; Frequencies; Funding; Gametogenesis; Gene Mutation; Gene Silencing; Genes; Genomics; Geography; Glucose; Growth; Half-Life; Human; Individual; Inflammatory; Inherited; Investigation; Iron Chelation; Knowledge; Laboratories; Lasers; Leukocytes; Libraries; Life; Link; Live Birth; Malaria; Malignant Neoplasms; Measures; Mediating; Medical center; Messenger RNA; Metabolic Pathway; Methodology; Microbe; Minority; Modeling; Molecular Biology; Multienzyme Complexes; Muscle Development; Muscle Weakness; Myositis; National Heart, Lung, and Blood Institute; National Human Genome Research Institute; Natural Products; Nematoda; Nonsense Codon; Organism; Palliative Care; Parasites; Parents; Patients; Pattern; Pharmaceutical Preparations; Pharmacology; Phenocopy; Phenotype; Plasmodium falciparum; Production; Progressive Disease; Protein Isoforms; Quality Control; Quantitative Microscopy; RNA Interference; Recombinants; Reporter; Research Personnel; Research Project Grants; Residual state; Reticulocytes; Retina; Retinal Degeneration; Retinal Diseases; Retinitis Pigmentosa; Rodent; Role; Scientist; Siblings; Signal Transduction; Skeletal Muscle; Source; Synthesis Chemistry; System; TP53 gene; Technology; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transfusion; Translational Research; Translations; Uniparental Disomy; United States National Institutes of Health; Vision; Work; Yeasts; assay development; base; cell type; curative treatments; design; drug discovery; experimental study; fighting; gene product; genome editing; glucose metabolism; high throughput screening; human disease; immunoregulation; loss of function; mRNA Decay; method development; muscle metabolism; neglect; novel; peripheral blood; pharmacophore; programs; prototype; public-private partnership; repository; research and development; screening; sensor; small molecule; small molecule libraries; transcription activator-like effector nucleases

This project includes the development of assays to phenocopy inherited genetic mutations leveraging disease knowledge from basic and clinical research programs with recent advances in molecular biology (e.g., application of TALEN and CRISPR-mediated genome editing), and engineering model organism assays of infectious disease. The assay designs are considered in the context of analysis and progression strategies for evaluation of approved drugs and investigational agents using high throughput screening 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: In collaboration with K. Nagaraju (Children's National Medical Center) the ADST laboratory is developing a coincidence reporter assay to aid in the discovery of compounds that increase expression of the enzyme AMP deaminase implicated in the disease idiopathic inflammatory myositis (IIM). IIM is a rare autoimmune progressive disease that afflicts the skeletal muscle of patients. IIM is 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. Diamond Blackfan Anemia (DBA): In collaboration with D. Bodine (NHGRI, NIH) and G. Thomas (U. Cincinnati) the ADST laboratory 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. The diagnosis is usually made in the 1st yr. of life, as a severe anemia that requires transfusion. Unlike other anemias, 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 small minority of patients, bone marrow transplantation from a healthy sibling is a curative therapy. For patients without a suitable donor 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. Discovery of Drugs for inherited rare blinding retinal degenerations: D. J. Zack, M.D., Ph.D., a Johns Hopkins researcher funded by the Foundation Fighting Blindness, is collaborating with Dr. Ingleses ADST laboratory in a public-private partnership aimed at alleviating and curing blinding retinal degenerative diseases. Together they are utilizing ADSTs expertise in assay development, chemical biology and quantitative high-throughput screening (qHTS) to develop assays and chemical library testing paradigms to identify drugs and compounds that have the potential to save and restore vision for people affected by devastating retinal diseases such as retinitis pigmentosa and age-related macular degeneration, the leading cause of blindness in Americans over 50 years of age. The use of primary rodent retinal cells, a limiting cell type for typical HTS experiments, benefits from the low-volume assay technology employed in the Inglese laboratory at NCATS. Glucose-regulating multienzyme compartment high throughput quantitative microscopy-based assay. In collaboration with S. An (UMBC) the ADST laboratory of NCATS is developing a 1536-well plate format high throughput quantitative microscopy-based assay for discovery of compounds that modulate the formation of a dynamically assembled multienzyme complex regulating glucose-derived carbon flux in cells. Given that glucose metabolism is the central metabolic pathway that balances the cellular needs for both energy and building blocks, 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. In turn, extensive high throughput screening is anticipated to discover novel pharmacophores, which promote or disrupt the assembly in human disease models, for therapeutic intervention in the treatment of glucose metabolism-associated human diseases. Controlling cellular senescence with small molecule compounds modulating the 133p53 isoform: In collaboration with C. Harris (NCI) the ADST laboratory of NCATS is developing a 1536-well plate format high throughput laser-cytometry based assay for discovery of compounds that modulate the cellular distribution and half-life of a specific p53 isoform in cells. The demonstrated functional and regulatory role served by 133p53 between cellular senescence and autophagy is relevant to both aging and cancer. Compounds effective in modulating cellular phenotypes based on expression of a GFP- 133p53 sensor will be explored for their pharmacological effect in relevant aging and cancer model systems. 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. In situations where residual function of a truncated gene product could mitigate a pathophysiologic phenotype NMD suppression may provide a therapeutic strategy. Based on our prior NMD assay used in gene-silencing studies (Baird TD, et al. 2018) we have 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. In collaboration with J. R. Hogg (NHLBI, NIH) we plan to develop secondary phenotypic assays to 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. Working with S. Grewal (NCI, NIH) the ADST laboratory is reengineering a medium throughput yeast assay to a higher throughput system capable of qHTS for the evaluation of a large-scale chemical libraries. Novel sources of chemical libraries evaluated by model organisms of infectious disease: To probe the biological activity of academias diversity of synthetic chemistry methodology-enabled libraries, privileged-chemotype collections, isolated natural products (NPs), and pre-fractionated culturable NP extracts (NPEs) the ADST laboratory is testing the anti-infective and anti-parasitic potential of these chemical repositories in model organism microbes and parasites. Based on either well-established or novel designs, these infectious disease assays are complementarily configured to our quantitative high throughput screening (qHTS) technology to provide a comprehensive pharmacological assessment of library activity. In previous efforts to discover new antimalarial drugs and targets, we exposed five Plasmodium 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. Currently the ADST laboratory is developing bacterial, fungal and nematode recombinant organisms to approximate the essentiality and vulnerability of unique target enzymes in highly infectious parasitic species.

该项目包括利用基础和临床研究项目的疾病知识以及分子生物学的最新进展（例如 TALEN 和 CRISPR 介导的基因组编辑的应用）开发表型遗传基因突变测定法，以及传染病的工程模型生物测定法。分析设计是在使用高通量筛选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) 缺乏症：ADST 实验室与 K. Nagaraju（国家儿童医疗中心）合作，正在开发一种巧合报告测定法，以帮助发现可增加与特发性炎症性肌炎相关的 AMP 脱氨酶表达的化合物（IIM）。 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。 IIM 由免疫调节和内在肌肉代谢的错误组成。 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。 Diamond Blackfan 贫血 (DBA)：ADST 实验室与 D. Bodine（NHGRI、NIH）和 G. Thomas（辛辛那提大学）合作，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 DBA。所有族裔群体，发病频率为每百万活产儿 7 例。诊断通常在第一年做出。严重贫血，需要输血。与其他贫血不同，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生成不受影响。 对于少数患者来说，健康兄弟姐妹的骨髓移植是一种治疗方法。 对于没有合适捐赠者的患者，可以采取多种姑息疗法，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。 发现治疗遗传性罕见致盲性视网膜变性的药物：约翰·霍普金斯大学研究员 D. J. Zack 博士在抗击失明基金会的资助下，正在与 Ingleses ADST 博士实验室建立公私合作伙伴关系，旨在缓解和治愈该病致盲的视网膜退行性疾病。他们共同利用 ADST 在检测开发、化学生物学和定量高通量筛选 (qHTS) 方面的专业知识来开发检测和化学库测试范式，以识别有潜力挽救和恢复受毁灭性视网膜疾病影响的人们视力的药物和化合物例如色素性视网膜炎和年龄相关性黄斑变性，这是导致 50 岁以上美国人失明的主要原因。原代啮齿动物视网膜细胞是典型 HTS 实验的限制细胞类型，其使用受益于 NCATS Inglese 实验室采用的低容量测定技术。 葡萄糖调节多酶室高通量定量显微镜检测。 NCATS 的 ADST 实验室与 S. An (UMBC) 合作，正在开发一种 1536 孔板形式的高通量定量显微镜测定法，用于发现调节动态组装多酶复合物形成的化合物，该复合物调节葡萄糖衍生的碳通量细胞。 鉴于葡萄糖代谢是平衡细胞对能量和构建模块的需求的中心代谢途径，对支持或反对组装的药理学活性小分子的初步筛选将有助于了解细胞中组装的生物学意义。反过来，广泛的高通量筛选预计会发现新的药效团，这些药效团可促进或破坏人类疾病模型中的组装，从而用于治疗与葡萄糖代谢相关的人类疾病的治疗干预。 用调节 133p53 同工型的小分子化合物控制细胞衰老：NCATS 的 ADST 实验室与 C. Harris (NCI) 合作，正在开发一种基于 1536 孔板格式高通量激光细胞计数的测定法，用于发现调节细胞分布的化合物以及细胞中特定 p53 亚型的半衰期。 133p53 在细胞衰老和自噬之间所发挥的功能和调节作用已被证明与衰老和癌症相关。 将探索基于 GFP-133p53 传感器的表达有效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。 用于鉴定无义介导的 mRNA 衰减 (NMD) 调节剂的测定：对导致含有过早终止密码子的 mRNA 的基因突变进行无义介导的 mRNA 衰减 (NMD) 质量控制。在截短基因产物的残余功能可以减轻病理生理表型的情况下，NMD 抑制可能提供一种治疗策略。 基于我们之前用于基因沉默研究的 NMD 测定（Baird TD 等人，2018），我们开发了一种 NMD 敏感的功能丧失、信号增益测定，使用我们的巧合报告技术来检查化学库发现新型 NMD 调节化学型。我们计划与 J. R. Hogg（NHLBI、NIH）合作，开发次级表型测定法，以测量在等位基因特异性疾病基因突变背景下调节 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 实验室与 S. Grewal（NCI、NIH）合作，正在将中等通量酵母测定重新设计为能够进行 qHTS 的更高通量系统，用于评估大规模化学库。 通过传染病模型生物评估的化学文库的新来源：探讨学术界合成化学方法支持的文库多样性、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 实验室正在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。 这些传染病检测基于成熟或新颖的设计，与我们的定量高通量筛选 (qHTS) 技术相辅相成，以提供文库活性的全面药理学评估。 在之前发现新的抗疟药物和靶点的努力中，我们使用 qHTS 将 5 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。目前，ADST 实验室正在开发细菌、真菌和线虫重组生物体，以了解高传染性寄生虫物种中独特目标酶的必要性和脆弱性。