Poster abstracts
Poster number 84 submitted by Kaba Tandjigora
Optimization of a fluorescence-based assay for high throughput screening of small molecule inhibitors of the DEAD-box RNA helicase DDX3X
Kaba Tandjigora (The Center for RNA Science and Therapeutics), Eckhard Jankowsky (The Center for RNA Science and Therapeutics)
Abstract:
DEAD-box RNA helicases utilize ATP to bind and remodel RNA and RNA protein complexes. They are involved in virtually all aspects of RNA metabolism. Deregulation of many DEAD-box helicases have been associated with various diseases, particularly to cancer and infectious diseases. Although several DEAD-box helicases are attractive targets for anti-cancer and antiviral therapeutics, few efforts have been made to develop inhibitors for specific DEAD-box helicases. Here, we developed a fluorescence-based, endpoint assay suitable for High Throughput Screening (HTS) of small molecule inhibitors of the unwinding activity of DDX3X, a human DEAD-box RNA helicase. DDX3X has been linked to various cancers and is targeted by multiple viruses. We expressed and purified recombinant DDX3X and characterized enzymatic activities. We then developed a fluorescence-based assay for duplex unwinding that relies on fluorescence quenching of a cy3-labeled RNA strand by a label on the complementary RNA strand. The fluorescence signal increases upon unwinding. The assay was optimized for end-point determination and proved to be DMSO tolerant. Stability of the fluorescence signals and Z’ factor indicate high robustness, sensitivity, and reproducibility. The data set the stage for HTS screening of small molecule inhibitors of the helicase activity of DDX3X. The developed HTS assay is potentially applicable for other RNA helicases.
Keywords: DDX3X, HTS, Small molecules