Assessing HTS Performance Using BioAssay Ontology: Screening and Analysis of a Bacterial Phospho-N-Acetylmuramoyl-Pentapeptide Translocase Campaign

Abstract

With the public availability of biochemical assays and screening data constantly increasing, new applications for data mining and method analysis are evolving in parallel. One example is BioAssay Ontology (BAO) for systematic classification of assays based on screening setup and metadata annotations. In this article we report a high-throughput screening (HTS) against phospho-N-acetylmuramoyl-pentapeptide translocase (MraY), an attractive antibacterial drug target involved in peptidoglycan synthesis. The screen resulted in novel chemistry identification using a fluorescence resonance energy transfer assay. To address a subset of the false positive hits, a frequent hitter analysis was performed using an approach in which MraY hits were compared with hits from similar assays, previously used for HTS. The MraY assay was annotated according to BAO and three internal reference assays, using a similar assay design and detection technology, were identified. Analyzing the assays retrospectively, it was clear that both MraY and the three reference assays all showed a high false positive rate in the primary HTS assays. In the case of MraY, false positives were efficiently identified by applying a method to correct for compound interference at the hit-confirmation stage. Frequent hitter analysis based on the three reference assays with similar assay method identified additional false actives in the primary MraY assay as frequent hitters. This article demonstrates how assays annotated using BAO terms can be used to identify closely related reference assays, and that analysis based on these assays clearly can provide useful data to influence assay design, technology, and screening strategy.

A Modular Approach to Phosphoglycosyltransferase Inhibitors Inspired by Nucleoside Antibiotics

“…Together, these studies provide the proof‐of‐concept for our approach, and pave the way for increasing the potency and specificity for diverse monotpic PGTs and potentially the more complex polytopic PGTs by performing structure optimization at the diverse point of contact with the enzyme. Of paramount importance in these studies was the establishment of a reproducible enzyme purification protocol, and the development of a reliable activity assay, which in contrast to existing fluorescence‐based assays for MraY, represents a significant challenge. We found that both assays described herein report accurately on PglC activity.…”

A Rapid and Efficient Luminescence-based Method for Assaying Phosphoglycosyltransferase Enzymes

“…PGTs represent important targets for inhibitor design due to their centrality in bacterial glycan synthesis. Except for MraY, for which a high-throughput fluorescent assay has been developed 28 , advances in inhibitor development for the PGTs that transfer simple sugar phosphate moieties have been hampered by the traditional challenging and cumbersome kinetic assays discussed above. The 96-well plate-based format makes the UMP-Glo assay highly attractive for inhibitor screening.…”

Good Research Practice in Non-Clinical Pharmacology and Biomedicine

“…They extracted metrics on the utilization of assay design and detection approaches and considered over-vs. underutilization of potential technologies. BAO terms were also used to identify similar assays from which hits were acknowledged as frequent false-positive results in high-throughput screening (Moberg et al 2014;Schürer et al 2011).…”