A no-wash, high-throughput, cell-based fluorescence assay using Mirrorball technology to discover small molecule therapeutics for Niemann-Pick disease type A

Authors

• K Gorshkov
• M Pradhan
• M Xu
• S Yang
• M Shen
• W Zheng

Abstract

Niemann-Pick disease type A (NPA) is a rare and fatal lysosomal storage disease caused by a deficiency of acid sphingomyelinase (ASM) that results in lysosomal sphingomyelin (SM) accumulation. There is no treatment available for NPA. In this study, we have developed a washless, 384-well plate, quantitative high-throughput screening (qHTS) fluorescence assay to identify compounds that alleviate the SM accumulation phenotype by restoring ASM function or other mechanisms. The cellular imaging data consisting of cell count and fluorescent intensity measurements were analyzed using the Cellista software. DMSO and delta tocopherol were used as negative and positive controls, respectively. We first screened the LOPAC drug library consisting of 1280 bioactive compounds producing 19 positive hits. The hit selection criteria consisted of greater than 30% reduction green fluorescence (decreased SM accumulation) without a decrease in cell counts. Seven hits clustered into two distinct classes targeting Receptor A or B, suggesting a common mechanism of action. From the follow-up screen, the EC50s for lead compounds A and B were 1.39 nM and 10.1 nM with efficacies of 62.5 percent and 50.0 percent, respectively. All hits were previously FDA-approved for other indications. In summary, our assay advantages include (1) no wash, (2) high throughput, and (3) live or fixed cells. In contrast, traditional imaging assays require multiple plate washes to remove the background signal, long scan and analysis times, and large data files. Therefore, our Mirrorball-based qHTS method advances high-throughput screening by increasing sensitivity, throughput, and efficiency while reducing assay artifacts.

Scientific Focus Area: Cell Biology

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