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: 10683013
• 负责人: James Inglese
• 金额: $ 50.46万
• 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/10683013
• 关键词: AMP Deaminase; Adrenal Cortex Hormones; Adult; Alleles; Animal Model; Anti-Infective Agents; Antiparasitic Agents; Applications Grants; Autoimmune; Basic Science; Biological; Biological Assay; Biological Models; Blood; Blood Platelets; Bone Marrow Transplantation; CD86 gene; Carbon; Cell model; Cells; Cellular Assay; Chemicals; Chemistry; Chromosome abnormality; Chromosomes; Clinical Research; Clustered Regularly Interspaced Short Palindromic Repeats; Collection; Communicable Diseases; Communities; Congenital Disorders; DNA Sequence Alteration; Deamination; Defect; Development; Diamond-Blackfan anemia; Disease; Disease model; Engineering; Enzymes; Erythropoiesis; Ethnic group; Evaluation; Extramural Activities; Failure; Fission Yeast; Frequencies; Gametogenesis; Gene Mutation; Gene Silencing; Genes; Genetic Screening; Genomics; Geography; Glucose; Growth; Human; Immunity; Individual; Infection; Inflammatory; Inherited; Interferons; Investigation; Iron Chelation; Knowledge; Laboratories; Leukocytes; Libraries; Life; Link; Live Birth; Malaria; Malignant Neoplasms; Measures; Mediating; Membrane; Messenger RNA; Metabolic; Methodology; Microbe; Microscopy; Minority; Missense Mutation; Modality; Modeling; Molecular; Molecular Biology; Multienzyme Complexes; Mus; Muscle Development; Muscle Weakness; Mutation; Myositis; National Center for Advancing Translational Sciences; Natural Products; Nematoda; Nonsense Codon; Organism; Palliative Care; Parasites; Parents; Pathway interactions; Patients; Pattern; Pharmaceutical Preparations; Pharmacology; Phenocopy; Phenotype; Plasmodium falciparum; Production; Progressive Disease; Protein Isoforms; Quality Control; Quantitative Microscopy; RNA Interference; RNA Splicing; RPE65 protein; Recombinants; Reporter; Reporter Genes; Reporting; Research Project Grants; Residual state; Reticulocytes; Retinal Degeneration; Retinal Dystrophy; Retinitis Pigmentosa; Scientist; Siblings; Signal Transduction; Site; Skeletal Muscle; Source; System; T-Cell Activation; Technology; Testing; Therapeutic; Therapeutic Intervention; Tissues; Transfusion; Translational Research; Translations; Ubiquitination; Uniparental Disomy; Work; Yeasts; assay development; base; curative treatments; design; drug discovery; early screening; gene product; genome editing; genome-wide; glucose metabolism; high throughput screening; human disease; immunoregulation; inhibitor; innovation; interest; loss of function; mRNA Decay; malaria infection; method development; muscle metabolism; neglect; novel; peripheral blood; pharmacophore; programs; protein expression; prototype; repository; research and development; response; screening; small molecule; small molecule libraries; 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. 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. Discovery of Drugs for inherited rare blinding retinal degenerations: RPE65 missense mutations result in a spectrum of retinal dystrophies. RPE65 mutations are generally recessively inherited, however the c.1430A>G (p.(D477G)) mutation has been reported to cause autosomal dominant retinitis pigmentosa via splicing of Rpe65 mRNA by generating an ectopic splice site leading to disruption of RPE65 protein expression. Novel assays designed based on ADST innovation in reporter gene technology to identify molecular modalities that can modify aberrant splicing are being developed in the context of the RPE65 mutation. 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.

该项目包括基于细胞和模式生物的检测的发展，以信号传导、代谢和增殖途径为目标，并利用基础和临床研究项目的疾病知识对遗传基因突变进行表型分析。分析设计利用分子生物学的最新进展和新颖的报告系统，使有效的化合物文库筛选。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，分析设计被考虑。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检测和筛选效率。