Identification of a selective DDX3X inhibitor with newly developed quantitative high-throughput RNA helicase assays

Area of Science:

• Biochemistry
• Molecular Biology
• Cancer Research

Background:

• DEAD-box RNA helicases, including eIF4A3 and DDX3X, are vital for mRNA regulation and implicated in cancer progression.
• Existing small-molecule inhibitors targeting these helicases show promise but require more potent compounds and efficient screening methods.
• There is a significant need for high-throughput assays to identify novel RNA helicase inhibitors for therapeutic development.

Purpose of the Study:

• To develop novel fluorescence resonance energy transfer (FRET)-based high-throughput assays for eIF4A3 and DDX3X.
• To utilize these assays to identify and characterize small-molecule inhibitors of eIF4A3 and DDX3X.
• To assess the correlation between ATPase inhibition and helicase activity inhibition for identified compounds.

Main Methods:

• Development of FRET-based high-throughput assays for quantifying eIF4A3 and DDX3X RNA helicase activity.
• Screening of small molecules using the developed assays to identify inhibitors.
• Characterization of identified inhibitors, including selectivity assessment (e.g., C1 for DDX3X over eIF4A3).

Main Results:

• Successful development of FRET-based high-throughput RNA helicase assays for eIF4A3 and DDX3X.
• Identification of several eIF4A3 allosteric inhibitors with a strong correlation between ATPase and helicase activity inhibition.
• Discovery of a selective DDX3X inhibitor (C1) from a pool of eIF4A3 ATPase inhibitors.

Conclusions:

• The developed high-throughput assays are effective tools for evaluating RNA helicase inhibitory activity.
• Small-molecule inhibitors of DEAD-box RNA helicases can aid in understanding their molecular mechanisms.
• These assays will facilitate the discovery of potent therapeutic agents targeting cancer-driving RNA helicases.