(1403-D) Interrogating RIG-I and MDA5 with the Transcreener® ADP2 Assay
Tuesday, February 6, 2024
2:00 PM – 3:00 PM EST
Location: Exhibit Halls AB
Abstract: RIG-1 (retinoic acid-inducible gene 1) and MDA5 (melanoma differentiation-associated gene 5) are members of the RLR (RIG-I-like receptors) family of immune sensors that bind double-stranded RNA (dsRNA) and trigger an IFN1 response in response to microbial infection and tumorigenesis. RLR agonists are being investigated for enhancing tumor immunity, and related RNA helicases in the DEAD box (DDX) helicase family are being targeted for cancer; therefore robust HTS assays for RIG-1 and MDA5 are needed. RNA helicases use cycles of ATP binding and hydrolysis to drive local unwinding of dsRNA, so activity can be measured by detecting ADP formation. We used the Transcreener ADP2 Assay, which relies on direct, homogenous immunodetection of ADP to develop high throughput assays for MDA5 and RIG-I with FP, FI, and TR-FRET readouts. We produced recombinant RIG-1 and MDA5 in baculovirus-infected insect cells, determined their RNA and ATP requirements, and optimized assays for initial velocity detection of RNA-dependent ATPase activity with Z’ values greater than 0.7. We validated the assays for HTS by screening a collection of bioactives (Tocris 2.0) and confirming the hits in dose response assays. These assays will facilitate screening and hit-to-lead/SAR for RIG-I and MDA5 agonists and for assessing off target activity of DDX helicase inhibitors.