Assay development and HTS of PAPD5 inhibitors using an innovative mass spectrometry platform

The Poly Adenosine Polymerase Associated domain 5 and 7 proteins regulate RNA stability by adding adenosine nucleotides to the 3’ end of various RNAs, including telomeric RNA, oncogenic microRNAs (miRNA) and viral RNA such as Hepatitis B Virus (HBV). Inhibitors of PAPD5 and PAPD7 offer promising therapeutic avenues to combat certain cancers, telomere-biology disorders, and HBV. Traditional biochemical assays to measure polyadenylation often rely on indirect measurements such as the generation of a pyrophosphate biproduct, FRET assays that utilize a third component to bind to the newly synthesized poly A tail, or luminescence that inform on the ATP to ADP reaction. These optical assays display poor sensitivity and are prone to false positives due to inhibition of secondary enzymes and optical interference of small molecule inhibitors. Here we develop a biochemical assay that combines surface chemistry and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS) for a label-free and high-throughput readout and direct analysis of the poly A products. The assay was developed to inform on the addition of distinct adenylated products and measure enzyme linearity. Next, we measured the kinetic parameters of the RNA substrate and ATP cofactor and measured the robustness and sensitivity of the assay compared to a traditional ADP-Glo format. In addition to the direct readout, the MS assay offered a 100-fold increase in sensitivity and signal to background. The MS assay was used to screen 30,000 compounds from a pharmacophore-like library to identify candidate inhibitors. Initial hits were validated in concentration response curves and demonstrated activity on PAPD5 and PAPD7. Importantly, this assay is flexible and could be applied to other RNA and DNA polymerases, opening new avenues for drug discovery.