A Quantitative High-Throughput Screen Identifies Compounds that Upregulate the p53 Isoform Δ133p53α and Inhibit...

Δ133p53α, a human/primate-specific p53 protein isoform, delays or inhibits the induction of the cellular senescence and associated secretory phenotype (SASP) in various types of human cells, including astrocytes from neurodegenerative diseases, Hutchinson-Gilford progeria syndrome (HGPS) fibroblasts, and exhausted CD8+ T-cells. A major regulatory mechanism of Δ133p53α expression in these human cells is protein degradation via chaperone-assisted selective autophagy (CASA). In this study, we describe a novel cell-based quantitative high-throughput screening (qHTS) assay using fluorescently labeled Δ133p53α to screen large chemical libraries for the identification of compounds that upregulate the Δ133p53α protein level. qHTS enabled the comprehensive and reliable profiling of over 10,000 small-molecule compounds. It also provided an opportunity to repurpose compounds and gain new mechanistic insights into the pathways regulating Δ133p53α expression. We successfully identified two candidate compounds, AZD1981 and celastrol, which were shown to upregulate the fluorescently labeled Δ133p53α protein, as well as the endogenous Δ133p53α protein in primary human astrocytes and the normal lung fibroblasts MRC-5, leading to reduced induction of cellular senescence and SASP factor secretion. The identification of celastrol, an inducer of heat shock protein 70 (HSP70) chaperones, is consistent with the CASA-mediated regulation of Δ133p53α protein turnover. Together with our previous findings of Δ133p53α overexpression-induced restoration of cell proliferation and function in cells otherwise approaching senescence, we propose that these two compounds may have therapeutic potential in senescence- and aging-associated diseases. The qHTS assay developed in this study could be used to identify new therapies for these diseases.