Medicines Discovery Institute, School of Biosciences, Cardiff University, Cardiff CF10 3AT, UK.
Sheffield Institute for Translational Neuroscience, Department of Neuroscience, University of Sheffield, Sheffield S10 2HQ, UK.
Nucleic Acids Res. 2022 Nov 11;50(20):e119. doi: 10.1093/nar/gkac771.
Paraspeckles are ribonucleoprotein granules assembled by NEAT1_2 lncRNA, an isoform of Nuclear Paraspeckle Assembly Transcript 1 (NEAT1). Dysregulation of NEAT1_2/paraspeckles has been linked to multiple human diseases making them an attractive drug target. However currently NEAT1_2/paraspeckle-focused translational research and drug discovery are hindered by a limited toolkit. To fill this gap, we developed and validated a set of tools for the identification of NEAT1_2 binders and modulators comprised of biochemical and cell-based assays. The NEAT1_2 triple helix stability element was utilized as the target in the biochemical assays, and the cellular assay ('ParaQuant') was based on high-content imaging of NEAT1_2 in fixed cells. As a proof of principle, these assays were used to screen a 1,200-compound FDA-approved drug library and a 170-compound kinase inhibitor library and to confirm the screening hits. The assays are simple to establish, use only commercially-available reagents and are scalable for higher throughput. In particular, ParaQuant is a cost-efficient assay suitable for any cells growing in adherent culture and amenable to multiplexing. Using ParaQuant, we identified dual PI3K/mTOR inhibitors as potent negative modulators of paraspeckles. The tools we describe herein should boost paraspeckle studies and help guide the search, validation and optimization of NEAT1_2/paraspeckle-targeted small molecules.
核内无膜结构分隔的多蛋白颗粒（paraspeckles）是由 NEAT1_2 lncRNA 组装而成的核糖核蛋白颗粒，NEAT1_2 是核内无膜结构分隔的多蛋白颗粒组装转录物 1（NEAT1）的一种异构体。NEAT1_2/paraspeckles 的失调与多种人类疾病有关，这使其成为一个有吸引力的药物靶点。然而，目前针对 NEAT1_2/paraspeckles 的转化研究和药物发现受到有限的工具包的限制。为了填补这一空白，我们开发并验证了一组用于鉴定 NEAT1_2 结合物和调节剂的工具，包括生化和基于细胞的测定法。生化测定法中使用 NEAT1_2 三螺旋稳定性元件作为靶标，而基于固定细胞中 NEAT1_2 的高内涵成像的细胞测定法（'ParaQuant'）是作为靶标。作为原理验证，这些测定法用于筛选 1,200 种 FDA 批准的药物化合物库和 170 种激酶抑制剂化合物库，并确认筛选结果。这些测定法易于建立，仅使用市售试剂，并且可扩展至更高的通量。特别是，ParaQuant 是一种经济高效的测定法，适用于任何在贴壁培养中生长的细胞，并且易于进行多重分析。使用 ParaQuant，我们确定了双重 PI3K/mTOR 抑制剂是 paraspeckles 的有效负调节剂。本文中描述的工具应能促进 paraspeckles 研究，并有助于指导针对 NEAT1_2/paraspeckle 的小分子的搜索、验证和优化。
