Phenotypic Assay Design and Development

National Center For Advancing Translational Sciences

Investigators

Linked publications, trials & patents

Abstract

- Delta133p53alpha (d133p53alpha), an isoform of the tumor suppressor p53, acts as a dominant-negative of full-length p53 activity, for example, causing delay or block of p53-mediated cellular senescence while conversely potentially facilitating DNA repair. Therefore, agents that can increase levels of d133p53alpha may possibly act as therapies to prevent or treat diseases associated with excessive cellular senescence, such as neurodegenerative diseases (e.g., Alzheimer's), and premature aging diseases (e.g., Hutchinson–Gilford progeria syndrome). The levels of cellular d133p53alpha expression are regulated by mechanisms that include protein degradation via chaperone-assisted selective autophagy (CASA). In our collaboration with Dr. C. Harris and his team at NCI, we developed a novel phenotypic cell-based assay to search for compounds that upregulate levels of fluorescently labeled d133p53alpha protein. Quantitative high-throughput screening (qHTS) enabled the concentration-response profiling of over 10,000 small-molecule compounds leading to the identification of two candidates, AZD1981 and celastrol that were subsequently shown to upregulate endogenous d133p53alpha in primary human astrocytes and the normal lung fibroblasts MRC-5. The phenotypic qHTS assay developed and validated in this study has demonstrated that small molecule compounds capable of upregulating d133p53alpha can be identified and potentially translated into new therapies for senescence-associated diseases. - Peroxisome biogenesis disorders (PBDs) are a spectrum of autosomal recessive disorders caused by defects in peroxisome functions that result from mutations in peroxin (PEX) genes required for normal peroxisome assembly, biogenesis, and downstream metabolic functions. The majority of PBD patients fall within the category of Zellweger spectrum disorder (ZSD), which is a disease continuum. While severely affected newborns do not survive past infancy, most ZSD patients have milder forms of disease that typically result in intellectual disabilities and progressive vision and hearing loss, liver dysfunction, osteopenia, kidney stones, and enamel hypoplasia. Through a collaborative effort with our academic colleagues (Prof. J. Hacia, USC; Prof. N. Braverman, McGill University; A. Moser and Dr. P. Watkins, JHU) studying peroxisome biogenesis and long chain fatty oxidation, our laboratory has designed high content imaging qHTS assays assessing and quantifying chemical rescue of peroxisome assembly in a fibroblast model system expressing a common genetic mutation. These assays have been used to interrogate genes and chemical libraries at NCATS including a repository of approved drugs and collections of diverse chemical substances. Analysis of qHTS imaging data has been ongoing. - 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. In a previous genome-wide genetic screen of early P. yoellii infection in mice, the laboratory of Dr. Su (NIAID, NIH), 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. In our collaborative research on this project, a coincidence reporter-based cellular quantitative high throughput screening (qHTS) assay was developed and used to identify inhibitors of the MARCH1 pathway. From small molecule chemical libraries of approved drugs and investigational agents various MARCH1 pathway inhibitors were identified, including cyclin-dependent kinases (CDK) inhibitors. The effect of selected CDK inhibitors is being investigated in models of malaria infection and host-mediated immunity.