Phenotypic Assay Design and Development for Rare and Neglected Diseases

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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.