Abstract 480: An HTS assay for detection of XRN1 exoribonuclease activity with unmodified RNA.

Abstract XRN1 is a 5’ to 3’ exoribonuclease that degrades both single- and double-stranded RNA to nucleotide 5’ monophosphate products. It plays an important role in mRNA turnover and in innate immunity, by damping activation of cytosolic RNA sensors, e.g., PKR and MDA5. Loss of XRN1 has been linked to high interferon signaling in cancer cell culture models, making it an attractive target for cancer immunotherapy. Here we describe development of HTS-compatible assays for screening and profiling inhibitors using the Transcreener AMP2/GMP2 Assay. Existing assays for RNAses rely on fluorescently labeled RNAs, which impose limitations on the substrates that can be used and may affect enzyme binding or catalysis. WIth the Transcreener AMP2/GMP2 Assay, purine monophosphates produced by XRN1 are directly detected by a competitive immunoassay with a far-red, fluorescence polarization (FP) or time-resolved Förster-resonance-energy-transfer (TR-FRET) readout. This format is homogenous (mix-and-read), compatible with diverse RNA substrates, and resistant to interference from screening compounds. Using full length human XRN1 produced in BaV-infected insect cells, we tested a number of RNA substrates, including synthetic double and single strand oligos with different ends (blunt, overhang, etc.) as well as total yeast RNA. Rates of AMP/GMP formation ranging from 0.054 sec-1 to 0.52 sec-1 were observed, with single strand RNAs yielding higher activity than double strand substrates; polyA was used for more detailed studies. Though the Km for poly-A was not length-dependent, the rate and extent of degradation was higher for a 100-mer than a 300-mer. A probe inhibitor, pAp, was tested in dose response experiments yielding an IC50 of 114.8 nM, which aligns well with the value measured using a fluorescently labeled DNA/RNA hybrid. The assays were validated for HTS by screening a collection of 1280 bio-actives, yielding a Z’ 0.9 and identification of hits that were confirmed in dose response experiments. Citation Format: Mahbbat Ali, Robert Lowery. An HTS assay for detection of XRN1 exoribonuclease activity with unmodified RNA 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 480.