PO.ET02.09 · 实验与分子治疗
用于发现RNA解旋酶抑制剂的通用型高通量解旋实验
A generic, high throughput unwinding assay for discovery of RNA helicase inhibitors
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
DEAD-box（DDX）解旋酶催化ATP依赖性的RNA及核酸-蛋白复合物重塑。由于它们在RNA代谢和先天免疫信号传导中的重叠作用，正成为肿瘤学、自身免疫和抗病毒药物研发中重要的治疗靶点。为实现选择性解旋酶抑制剂的发现和机制表征，我们开发了一组高通量生化实验，以均相（混合即读）形式并采用远红荧光读出来测量解旋酶的酶学功能。我们此前已展示使用Transcreener® ADP 2实验检测一组DDX酶的RNA依赖性ATP酶活性。在此，我们描述了Heliscreener™解旋实验的开发与验证，该实验采用荧光去猝灭策略监测标记的RNA双链的分离。我们评估了多种报告RNA与捕获RNA配对，并鉴定出一种单一组合，可稳健、通用地检测多种解旋酶（包括DDX3、DDX5、DHX9和DDX17）的解旋活性。对ATP和RNA浓度的系统优化产生了在1.7至66 nM酶浓度下提供强信号背景比的条件。在这些条件下，Heliscreener能够以连续或终点形式检测解旋活性，Z'因子>0.8，支持其适用于高通量筛选。对1,280种生物活性化合物的初步筛选鉴定出20个抑制剂命中物和6个表观激活物，其中约50%因荧光干扰而被排除。抑制剂选择性谱在ATP酶和解旋实验之间总体一致，但效力常有差异，大多数化合物在解旋实验中表现出更高的表观效力，这与ATP水解与双链分离的部分解偶联相符。这些结果凸显了联合ATP酶和解旋读出以获得机制见解的价值，这些见解可能影响RNA解旋酶抑制剂的疗效和选择性。
查看英文原文 English abstract
DEAD-box (DDX) helicases catalyze ATP-dependent remodeling of RNA and nucleic acid-protein complexes. Because of their overlapping roles in RNA metabolism and innate immune signaling, they are emerging as important therapeutic targets in oncology, autoimmunity, and antiviral drug discovery. To enable discovery and mechanistic characterization of selective helicase inhibitors, we have developed a panel of high-throughput biochemical assays for measuring helicase enzymatic functions in homogenous (mix-and-read) format with far red fluorescent readouts. We previously demonstrated detection of RNA-dependent ATPase activity for a panel of DDX enzymes using the Transcreener® ADP 2 assay. Here, we describe the development and validation of the Heliscreener™ unwinding assay, which uses a fluorescence dequenching strategy to monitor separation of a labeled RNA duplex. We evaluated multiple reporter and capture RNA pairs and identified a single combination that supports robust, generic detection of unwinding by several helicases, including DDX3, DDX5, DHX9, and DDX17. Systematic optimization of ATP and RNA concentrations yielded conditions that provide strong signal-to-background with enzyme concentrations from 1.7 to 66 nM. Under these conditions, Heliscreener enables detection of unwinding activity in continuous or endpoint formats with Z′ factors >0.8, supporting its suitability for high-throughput screening. A pilot screen of 1,280 bioactive compounds identified 20 inhibitor hits and 6 apparent activators, ~50% of which were eliminated as fluorescent interference. Inhibitor selectivity profiles were generally concordant between ATPase and unwinding assays, but potencies often differed, with most compounds showing higher apparent potency in the unwinding assay, consistent with partial uncoupling of ATP hydrolysis from duplex separation. These results highlight the value of combining ATPase and unwinding readouts to obtain mechanistic insights that may impact the efficacy and selectivity of RNA helicase inhibitors.
利益披露 Disclosure
H. Pham, None..
R. Lowery, None.