Abstract 460: A generic, high throughput unwinding assay for discovery of RNA helicase inhibitors.

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® ADP2 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. Citation Format: Ha Pham, Robert Lowery. A generic, high throughput unwinding assay for discovery of RNA helicase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 460.