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Understanding the characteristics of nonspecific binding of drug-like compounds to canonical stem-loop RNAs and their implications for functional cellular assays
RNA ( IF 4.5 ) Pub Date : 2020-10-07 , DOI: 10.1261/rna.076257.120
Megan L Kelly 1 , Chia-Chieh Chu 1 , Honglue Shi 2 , Laura R Ganser 1 , Hal P Bogerd 3 , Kelly Huynh 1 , Yuze Hou 1 , Bryan R Cullen 3 , Hashim M Al-Hashimi 1, 2
Affiliation  

Identifying small molecules that selectively bind an RNA target while discriminating against all other cellular RNAs is an important challenge in RNA-targeted drug discovery. Much effort has been directed toward identifying drug-like small molecules that minimize electrostatic and stacking interactions that lead to non-specific binding of aminoglycosides and intercalators to many stem-loop RNAs. Many such compounds have been reported to bind RNAs and inhibit their cellular activities. However, target engagement and cellular selectivity assays are not routinely performed, and it is often unclear whether functional activity directly results from specific binding to the target RNA. Here, we examined the propensities of three drug-like compounds, previously shown to bind and inhibit the cellular activities of distinct stem-loop RNAs, to bind and inhibit the cellular activities of two unrelated HIV-1 stem-loop RNAs: the transactivation response element (TAR) and the rev response element stem IIB (RREIIB). All compounds bound TAR and RREIIB in vitro, and two inhibited TAR-dependent transactivation and RRE-dependent viral export in cell-based assays while also exhibiting off-target interactions consistent with non-specific activity. A survey of X-ray and NMR structures of RNA-small molecule complexes revealed that aminoglycosides and drug-like molecules form hydrogen bonds with functional groups commonly accessible in canonical stem-loop RNA motifs, in contrast to ligands that specifically bind riboswitches. Our results demonstrate that drug-like molecules can non-specifically bind stem-loop RNAs most likely through hydrogen bonding and electrostatic interactions and reinforce the importance of assaying for off-target interactions and RNA selectivity in vitro and in cells when assessing novel RNA-binders.

中文翻译:

了解类药物化合物与经典茎环 RNA 的非特异性结合的特征及其对功能性细胞测定的影响

识别选择性结合 RNA 靶点同时区分所有其他细胞 RNA 的小分子是 RNA 靶向药物发现的一个重要挑战。很多努力都致力于识别药物样小分子,这些小分子可最大限度地减少导致氨基糖苷类和嵌入剂与许多茎环 RNA 的非特异性结合的静电和堆积相互作用。据报道,许多此类化合物可结合 RNA 并抑制其细胞活性。然而,靶标参与和细胞选择性测定并未常规进行,并且通常不清楚功能活性是否直接来自与靶标 RNA 的特异性结合。在这里,我们检查了三种药物样化合物的倾向,这些化合物以前显示可以结合和抑制不同茎环 RNA 的细胞活性,结合并抑制两种不相关的 HIV-1 茎环 RNA 的细胞活性:反式激活反应元件 (TAR) 和 rev 反应元件茎 IIB (RREIIB)。所有化合物在体外与 TAR 和 RREIIB 结合,两种化合物在基于细胞的试验中抑制 TAR 依赖性反式激活和 RRE 依赖性病毒输出,同时还表现出与非特异性活性一致的脱靶相互作用。一项对 RNA 小分子复合物的 X 射线和 NMR 结构的调查显示,与特异性结合核糖开关的配体相比,氨基糖苷类和药物样分子与典型茎环 RNA 基序中通常可接近的官能团形成氢键。
更新日期:2020-10-07
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