Targeting OAS Enzymes with the Transcreener® 2-5A OAS Assay

Oligoadenylate synthetases, including OAS1, OAS2, OAS3, are pattern recognition receptors that comprise an important component of the innate immune response against invading pathogens.  Binding of dsRNA activates OAS-proteins to produce linear 2’-5’ oligoadenylates, which in turn activate endoribonuclease L (RNase L), resulting in degradation of viral RNA. 

OAS enzymes and the closely related dsDNA-sensing PRR, cyclic GAMP synthase are emerging as potential therapeutic targets for autoimmune diseases and cancer.  To enable screening for small molecule modulators of OAS enzymes and for selectivity profiling of cGAS modulators, we developed a robust, HTS-compatible assay method for measuring OAS activity by using a coupling enzyme, with the Transcreener AMP²/GMP² Assay, a competitive fluorescence polarization (FP) immunoassay for purine monophosphates. 

Because the 2’-5’ oligoadenylates produced by OAS enzymes are not available commercially, we used an iterative empirical approach to optimize OAS-1 and coupling enzyme concentrations to ensure that all the product formed was converted to AMP.   As expected, the reaction was dependent on the presence of dsRNA and ATP.  We were able to obtain robust assay signals ( >100mP) with less than 10 nM OAS1 and approximately 100 nM OAS2; OAS1 yielded a Z’ value of 0.87. The assay was validated in a pilot screen of 1,280 pharmacologically active molecules, which identified several inhibitors, at least one of which demonstrated dose-dependency; suramin was also found to inhibit OAS1 with an IC₅₀ of 162 nM. The Transcreener 2-5A OAS Assay will provide a robust tool for discovery of OAS inhibitors or selectivity profiling of compounds targeting related enzymes.