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. 

Assay development and high-throughput screen for Sortase A inhibitors using an innovative mass spectrometry platform to combat antibiotic resistant bacteria.

Abstract:

Bacterial virulence is a driving mechanism of infection and disease. In Methicillin-sensitive and -resistant Staphylococcus aureus (MRSSA/MRSA), Sortase A plays a crucial role in bacterial virulence, where its transpeptidase activity anchors specific proteins like Staphylococcal protein A (SpA) to the cell wall enabling colonization and infection as well as facilitating resistance to antibiotics. Sortase A therefore represents a promising therapeutic target to combat infection and reduce the spread of antibiotic-resistant bacteria. Assays to measure Sortase A activity rely on optical readouts that measure changes in fluorescence or FRET signals. When screening for inhibitors, these optical formats are not optimal and are prone to false positive and negative results due to interference of library compounds and other assay artifacts. Here, we describe the development of a label-free and high-throughput assay that combines surface chemistry using polymeric enrichment arrays and matrix assisted laser desorption ionization (MALDI) mass spectrometry (MS). After optimizing the assay to reveal kinetically balanced conditions, we completed a screen of over 200,000 compounds in two days to identify several hits against Sortase A (0.1% hit rate). When retested from fresh powder, >80% of compounds validated with IC50s < 50 uM, supporting the quality and reproducibility of the assay. To determine selectivity, the compounds were tested in a panel of protease assays including a mutant Sortase A, SARS-CoV-2 3CLpro, and human Cathepsin L. Finally, the compounds were tested in bacterial cultures to monitor the in vivo efficacy of the inhibitors to reduce SpA anchoring. Our novel MS platform offers several solutions for measuring Sortase A activity and identifying inhibitors. Firstly, many top hits from the MS screen would have been missed in a canonical optical assay due to optical interference. Secondly, compounds that contain primary amines can act as non-specific competitive inhibitors, resulting in false positive hits. Our MS platform resolves this behavior, saving time and resources. Thirdly, the use of polymeric enrichment arrays enabled rapid purification of the substrates and products from the complex reaction, overcoming challenges with ion suppression common to MS readouts. Taken together, the results reveal novel compounds that open therapeutic avenues for dangerous bacterial infections and demonstrate the power of innovative MS approaches for label-free and high-throughput screening assays for Sortase and many other biochemical activities.