Phenotypic Assay Design and Development for Rare and Neglected Diseases

National Center For Advancing Translational Sciences

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Abstract

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.