High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes
| Autor: | Richard Johnstone, Zhao Ren, Myung K. Shin, Maarten Hoek, Haihong Zhou, Anthony Kreamer, Mary-Jo Wildey, Jason E. Imbriglio, Stephen F. Previs, Xi Ai, Andrea M. Peier, Steve Cifelli, Lufei Hu, Ashmita Saigal, Josephine Johnson, Richard Visconti, Yanqing Kan, Lin-Lin Shiao, William T. McElroy, Jonathan W. Choy, Adam B. Weinglass, Ying Lei, Robert Ramos, Andy Liaw, Michelle Chen |
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| Rok vydání: | 2021 |
| Předmět: |
Apolipoprotein L2
Phenotypic screening High-throughput screening Cell 030232 urology & nephrology Biochemistry Analytical Chemistry law.invention Small Molecule Libraries 03 medical and health sciences 0302 clinical medicine law Drug Discovery medicine Humans Viability assay Cytotoxicity 030304 developmental biology 0303 health sciences Podocytes Chemistry Drug discovery Apolipoprotein L1 High-Throughput Screening Assays Cell biology medicine.anatomical_structure Molecular Medicine Suppressor Biotechnology |
| Zdroj: | SLAS Discovery. 26:1225-1237 |
| ISSN: | 2472-5552 |
| Popis: | High-throughput phenotypic screening is a key driver for the identification of novel chemical matter in drug discovery for challenging targets, especially for those with an unclear mechanism of pathology. For toxic or gain-of-function proteins, small-molecule suppressors are a targeting/therapeutic strategy that has been successfully applied. As with other high-throughput screens, the screening strategy and proper assays are critical for successfully identifying selective suppressors of the target of interest. We executed a small-molecule suppressor screen to identify compounds that specifically reduce apolipoprotein L1 (APOL1) protein levels, a genetically validated target associated with increased risk of chronic kidney disease. To enable this study, we developed homogeneous time-resolved fluorescence (HTRF) assays to measure intracellular APOL1 and apolipoprotein L2 (APOL2) protein levels and miniaturized them to 1536-well format. The APOL1 HTRF assay served as the primary assay, and the APOL2 and a commercially available p53 HTRF assay were applied as counterscreens. Cell viability was also measured with CellTiter-Glo to assess the cytotoxicity of compounds. From a 310,000-compound screening library, we identified 1490 confirmed primary hits with 12 different profiles. One hundred fifty-three hits selectively reduced APOL1 in 786-O, a renal cell adenocarcinoma cell line. Thirty-one of these selective suppressors also reduced APOL1 levels in conditionally immortalized human podocytes. The activity and specificity of seven resynthesized compounds were validated in both 786-O and podocytes. |
| Databáze: | OpenAIRE |
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