Department of Chemistry, Duke University, 124 Science Drive, Durham, North Carolina 27705, United States.
Department of Biochemistry, Duke University School of Medicine, Nanaline H. Duke, Durham, North Carolina, 27710, United States.
ACS Chem Biol. 2022 Sep 16;17(9):2437-2447. doi: 10.1021/acschembio.2c00124. Epub 2022 Aug 19.
The rapidly accelerating characterization of RNA tertiary structures has revealed their pervasiveness and active roles in human diseases. Small molecule-mediated modulation of RNA tertiary structures constitutes an attractive avenue for the development of tools for therapeutically targeting and/or uncovering the pathways associated with these RNA motifs. This potential has been highlighted by targeting of the triple helix present at the 3'-end of the noncoding RNA MALAT1, a transcript implicated in several human diseases. This triplex has been reported to decrease the susceptibility of the transcript to degradation and promote its cellular accumulation. While small molecules have been shown to bind to and impact the stability of the MALAT1 triple helix, the small molecule properties that lead to these structural modulations are not well understood. We designed a library utilizing the diminazene scaffold, which is underexplored but precedented for nucleic acid binding, to target the MALAT1 triple helix. We employed multiple assays to holistically assess what parameters, if any, could predict the small molecule affinity and effect on triplex stability. We designed and/or optimized competition, calorimetry, and thermal shift assays as well as an enzymatic degradation assay, the latter of which led to the discovery of bidirectional modulators of triple helix stability within the scaffold-centric library. Determination of quantitative structure-activity relationships afforded predictive models for both affinity- and stability-based assays. This work establishes a suite of powerful orthogonal biophysical tools for the evaluation of small molecule:RNA triplex interactions that generate predictive models and will allow small molecule interrogation of the growing body of disease-associated RNA triple helices.
RNA 三级结构的快速特征分析揭示了它们在人类疾病中的普遍性和积极作用。小分子介导的 RNA 三级结构调节为开发靶向治疗工具和/或揭示与这些 RNA 基序相关的途径提供了诱人的途径。这一潜力已通过针对非编码 RNA MALAT1 3'末端存在的三聚体的靶向得到了强调，MALAT1 转录本与几种人类疾病有关。据报道，该三聚体降低了转录本的降解敏感性并促进其细胞积累。虽然已经表明小分子可以结合并影响 MALAT1 三聚体的稳定性，但导致这些结构修饰的小分子特性尚不清楚。我们设计了一个利用二甲苯胺骨架的文库，该骨架的核酸结合作用尚未得到充分探索，但已有先例，以靶向 MALAT1 三聚体。我们采用了多种测定方法来全面评估哪些参数（如果有的话）可以预测小分子的亲和力和对三聚体稳定性的影响。我们设计和/或优化了竞争、量热法和热位移测定法以及酶促降解测定法，后者导致在以骨架为中心的文库中发现了三聚体稳定性的双向调节剂。定量构效关系的确定为基于亲和力和稳定性的测定法提供了预测模型。这项工作建立了一套强大的正交生物物理工具，用于评估小分子：RNA 三聚体相互作用，这些工具可生成预测模型，并允许小分子对不断增长的疾病相关 RNA 三聚体进行研究。
