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.