Development and pilot screen of novel high content assay for down regulators of expression of heterogenous nuclear ribonuclear protein H2

作者：Juan Diez, Sumitha Rajendrarao, Shadi A. Baajour, Praathibha Sripadhan, Timothy Spicer, Louis Scampavia, Dmitriy Minond · 发表于：bioRxiv (Cold Spring Harbor Laboratory) · 年份：2020 · DOI：10.1101/2020.10.05.326116 · 研究领域：Viral Infectious Diseases and Gene Expression in Insects、Computational Drug Discovery Methods、Molecular Biology Techniques and Applications

ABSTRACT Despite recent advances in melanoma drug discovery, the average overall survival of patients with late stage metastatic melanoma is approximately 3 years, suggesting a need for new approaches and melanoma therapeutic targets. Previously we identified heterogeneous nuclear ribonucleoprotein H2 as a potential target of anti-melanoma compound 2155-14 (Palrasu et al , Cell Physiol Biochem 2019;53:656-86). In the present study, we endeavored to develop an assay to enable a high throughput screening campaign to identify drug-like molecules acting via down regulation of heterogeneous nuclear ribonucleoprotein H that can be used for melanoma therapy and research. Results We established a cell-based platform using metastatic melanoma cell line WM266-4 expressing hnRNPH2 conjugated with green fluorescent protein to enable assay development and screening. High Content Screening assay was developed and validated in 384 well plate format, followed by miniaturization to 1,536 well plate format. All plate-based QC parameters were acceptable: %CV = 6.7±0.3, S/B = 21±2.1, Z’ = 0.75±0.04. Pilot screen of FDA-approved drug library (n=1,400 compounds) demonstrated hit rate of 0.5%. Two compounds demonstrated pharmacological response and were authenticated by western blot analysis. Conclusions We developed a highly robust HTS-amenable high content screening assay capable of monitoring down regulation of hnRNPH2. This assay is thus capable of identifying authentic down regulators of hnRNP...