A 1536-well fluorescence polarization assay to screen for modulators of the MUSASHI family of RNA-binding proteins.
A 1536-well fluorescence polarization assay to screen for modulators of the MUSASHI family of RNA-binding proteins.
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DOI：
10.2174/1386207317666140609122714
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发表时间：
2014
影响因子：
1.8
通讯作者：
Kharas MG
中科院分区：
文献类型：
--
作者：
Minuesa G;Antczak C;Shum D;Radu C;Bhinder B;Li Y;Djaballah H;Kharas MG
RNA 结合蛋白 (RBP) 可以充当干细胞调节剂和致癌驱动因素，但作为癌症的潜在治疗靶点，在很大程度上被制药行业忽视。 MUSASHI (MSI) 家族最近被证明是最具侵袭性癌症的一个有吸引力的临床靶点。因此，小分子抑制剂的发现和开发可以提供一种新的治疗策略。为了寻找具有 MSI RNA 结合抑制活性的新型化合物，我们开发了一种荧光偏振 (FP) 测定法，并对其进行了优化，用于 1536 孔微量滴定板形式的高通量筛选 (HTS)。我们利用包含 6,208 种化合物的化学库，针对 MSI1 和 MSI2 进行了初步筛选，最终鉴定出 7 个针对 MSI1 的分子、15 个针对 MSI2 的分子以及 5 个抑制两者的分子。二次 FP 剂量反应筛选验证了 3 种 MSI 抑制剂，其 IC50 低于 10μM。在二次筛选中重新测试的 25 种化合物中，只有 8 种因高荧光而表现出光学干扰。利用基于 SYBR 的 RNA 电泳迁移率变动分析 (EMSA)，我们进一步验证了前 3 种化合物的 MSI 抑制作用。令人惊讶的是，尽管文库中存在多种氨基糖苷类药物，但它们未能证明 MSI 抑制剂活性，这挑战了这些化合物对 RBP 具有泛活性的概念。总之，我们开发了一种使用 FP HTS 平台识别 MSI 特异性抑制剂的体外策略，这将有助于此类 RBP 的新药发现。
RNA-binding proteins (RBPs) can act as stem cell modulators and oncogenic drivers, but have been largely ignored by the pharmaceutical industry as potential therapeutic targets for cancer. The MUSASHI (MSI) family has recently been demonstrated to be an attractive clinical target in the most aggressive cancers. Therefore, the discovery and development of small molecule inhibitors could provide a novel therapeutic strategy. In order to find novel compounds with MSI RNA binding inhibitory activity, we have developed a fluorescence polarization (FP) assay and optimized it for high throughput screening (HTS) in a 1536-well microtiter plate format. Using a chemical library of 6,208 compounds, we performed pilot screens, against both MSI1 and MSI2, leading to the identification of 7 molecules for MSI1, 15 for MSI2 and 5 that inhibited both. A secondary FP dose-response screen validated 3 MSI inhibitors with IC50 below 10μM. Out of the 25 compounds retested in the secondary screen only 8 demonstrated optical interference due to high fluorescence. Utilizing a SYBR-based RNA electrophoresis mobility shift assay (EMSA), we further verified MSI inhibition of the top 3 compounds. Surprisingly, even though several aminoglycosides were present in the library, they failed to demonstrate MSI inhibitor activity challenging the concept that these compounds are pan-active against RBPs. In summary, we have developed an in vitro strategy to identify MSI specific inhibitors using an FP HTS platform, which will facilitate novel drug discovery for this class of RBPs.
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影响因子：
46.9
作者：
Einav, Shirit;Gerber, Doron;Bryson, Paul D.;Sklan, Ella H.;Elazar, Menashe;Maerkl, Sebastian J.;Glenn, Jeffrey S.;Quake, Stephen R.
通讯作者：
Quake, Stephen R.
DOI：
10.1084/jem.20130736
发表时间：
2014-01-13
期刊：
The Journal of experimental medicine
影响因子：
--
作者：
Park SM;Deering RP;Lu Y;Tivnan P;Lianoglou S;Al-Shahrour F;Ebert BL;Hacohen N;Leslie C;Daley GQ;Lengner CJ;Kharas MG
通讯作者：
Kharas MG
前列腺癌的基因表达谱揭示了多个分子途径在转移过程中的参与。
影响因子：
3.8
作者：
Chandran, Uma R.;Ma, Changqing;Dhir, Rajiv;Bisceglia, Michelle;Lyons-Weiler, Maureen;Liang, Wenjing;Michalopoulos, George;Becich, Michael;Monzon, Federico A.
通讯作者：
Monzon, Federico A.
影响因子：
5.3
作者：
Imai, T;Tokunaga, A;Okano, H
通讯作者：
Okano, H
影响因子：
--
作者：
Visco, Carlo;Perrera, Claudia;Magnaghi, Paola
通讯作者：
Magnaghi, Paola