Basic Research Division, Veritas In Silico Inc., Shinagawa, Tokyo 141-0031, Japan.
Graduate School of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565-0871, Japan.
ACS Chem Biol. 2023 Nov 17;18(11):2368-2376. doi: 10.1021/acschembio.3c00287. Epub 2023 Oct 19.
Interest in small molecules that target RNA is flourishing, and the expectation set on them to treat diseases with unmet medical needs is high. However, several challenges remain, including difficulties in selecting suitable tools and establishing workflows for their discovery. In this context, we optimized experimental and computational approaches that were previously employed for the protein targets. Here, we demonstrate that a fluorescence-based assay can be effectively used to screen small molecule libraries for their ability to bind and stabilize an RNA stem-loop. Our screen identified several fluoroquinolones that bind to the target stem-loop. We further probed their interactions with the target using biolayer interferometry, isothermal titration calorimetry (ITC), and nuclear magnetic resonance spectroscopy. The results of these biophysical assays suggest that the fluoroquinolones bind the target in a similar manner. Armed with this knowledge, we built models for the complexes of the fluoroquinolones and the RNA target. Then, we performed fragment molecular orbital (FMO) calculations to dissect the interactions between the fluoroquinolones and the RNA. We found that the binding free energies obtained from the ITC experiments correlated strongly with the interaction energies calculated by FMO. Finally, we designed fluoroquinolone analogues and performed FMO calculations to predict their binding free energies. Taken together, the results of this study support the importance of conducting orthogonal assays in binding confirmation and compound selection and demonstrate the usefulness of FMO calculations in the rational design of RNA-targeted small molecules.
对靶向 RNA 的小分子的兴趣正在蓬勃发展，人们对它们治疗具有未满足医疗需求的疾病的期望很高。然而，仍存在一些挑战，包括在选择合适的工具和建立发现工作流程方面的困难。在这种情况下，我们优化了以前用于蛋白质靶标的实验和计算方法。在这里，我们证明了一种基于荧光的测定法可以有效地用于筛选小分子文库，以确定它们结合和稳定 RNA 发夹的能力。我们的筛选鉴定了几种能够与靶标发夹结合的氟喹诺酮类药物。我们进一步使用生物层干涉法、等温滴定量热法 (ITC) 和核磁共振波谱法探测它们与靶标的相互作用。这些生物物理测定法的结果表明，氟喹诺酮类药物以相似的方式与靶标结合。有了这些知识，我们为氟喹诺酮类药物和 RNA 靶标的复合物构建了模型。然后，我们进行了片段分子轨道 (FMO) 计算，以剖析氟喹诺酮类药物与 RNA 之间的相互作用。我们发现，从 ITC 实验获得的结合自由能与 FMO 计算的相互作用能密切相关。最后，我们设计了氟喹诺酮类药物类似物并进行了 FMO 计算以预测它们的结合自由能。总之，这项研究的结果支持在结合确认和化合物选择中进行正交测定的重要性，并证明了 FMO 计算在合理设计靶向 RNA 的小分子中的有用性。
