High-Throughput Screening to Identify Small Molecules That Selectively Inhibit APOL1 Protein Level in Podocytes. (October 2021)

• Record Type: Journal Article
• Authors: Choy, Jonathan; Kan, Yanqing; Cifelli, Steve; Johnson, Josephine; Chen, Michelle; Shiao, Lin-Lin; Zhou, Haihong; Previs, Stephen; Lei, Ying; Johnstone, Richard; Liaw, Andy; Saigal, Ashmita; Hu, Lufei; Ramos, Robert; Visconti, Richard; McElroy, William T.; Kreamer, Anthony; Wildey, Mary-Jo; Peier, Andrea; Shin, Myung K.; Imbriglio, Jason; Ren, Zhao; Hoek, Maarten; Weinglass, Adam; Ai, Xi
• Is Part Of: SLAS discovery. Volume 26:Number 9(2021)
• Page Start: 1225
• Page End: 1237
• Publication Date: 2021-10
• Subjects: apolipoprotein L1 -- podocytes -- high-throughput screening -- suppressor screen -- homogeneous time-resolved fluorescence (HTRF) assay; Drugs -- Analysis -- Periodicals; Drugs -- Testing -- Periodicals; Biomolecules -- Analysis -- Periodicals; Biomolecules -- Analysis; Drugs -- Analysis; Drugs -- Testing; Drug Evaluation, Preclinical; Molecular Biology -- methods; Periodicals; Periodicals; 615.1
• DOI: 10.1177/24725552211026245
• Languages: English
• ISSNs: 2472-5552

Abstract

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.