Bart Blanchaert

An overview of analytical methods for monitoring bacterial transglycosylation

Transpeptidation as the last step in peptidoglycan synthesis has been a successful antibacterial target for many decades, whereas inhibitors of the preceding transglycosylation did not lead to any commercialized drugs although both reactions are essential for cell survival. Since the substrate, Lipid II, has become more easily available, a lot of research towards synthesis of new inhibitors of transglycosylation has been done. Several assay types have been elaborated to evaluate them. The main problem in the development is the lack of UV-chromophores in the substrate leading to the need for radioactive and fluorescence labeling. Also, high-throughput screens that allow screening of large libraries of compounds have been conceived. This review gives an overview of the different assay types and highlights some cleverly engineered screens.

Development of a liquid chromatography/mass spectrometry assay for the bacterial transglycosylation reaction through measurement of Lipid II

Transglycosylation is the second to last step in the production of bacterial peptidoglycan. It is catalyzed by a transglycosylation site in class A penicillin‐binding proteins (PBPs) or monofunctional glycosyl transferases. Several potential inhibitors have been suggested and need to be tested for activity. In this article, we describe the development and validation of an LC/MS assay for Lipid II, the substrate for transglycosylation. The developed assay can be used to monitor the transglycosylation activity of Staphylococcus aureus PBP2. There was no need for modification of Lipid II with a fluorescent tag that could alter affinity of inhibitors toward Lipid II. Recombinant PBP2 was produced in Escherichia coli and has been tested for activity. This LC/MS method is suitable for a transglycosylation assay for PBP2 and since it is relatively fast, it can be used to test inhibitors.

Bioanalytical LC/MS study of potential bacterial transglycosylation inhibitors

Bacterial transglycosylation is an interesting target in antibiotic drug development. An in vitro transglycosylation assay was developed and used to search for possible inhibitors of Staphylococcus aureus Penicillin Binding Protein 2-mediated transglycosylation. Since the substrate, Lipid II, has no UV-chromophore, the assay relies on LC coupled to MS for analysis of the incubation mixtures. Extracts from Thymus sipyleus, Salvia verticillata, Salvia virgata and Oolong tea were tested, as well as epigallocatechin gallate and ursolic acid, which are chemical compounds derived from plants. Matrix effects hampered Lipid II quantification in samples treated with very high concentrations of extracts. None of these extracts or isolated compounds appeared to have inhibitory activities towards the transglycosylation function of Penicillin Binding Protein 2.

Abl1 inhibitory contaminants leach from plastic tubes

Abstract Plastic materials are widely used in research laboratories. Disposable plasticware facilitates life science research in the storage, transportation and manipulation of biological samples. However, recent findings have shown that some disposable plasticwares release bioactive contaminants. The bioactive leachates from plastic tubes, used as Abl1 catalytic incubator in this report, were noticed to interfere with the activity of Abl1. Extraction of these bioactive leachates was performed, and their inhibitory activity against Abl1 and cytotoxicity were tested. Results indicated that the tube extracts had no significant cytotoxicity but could inhibit the activity of Abl1. Therefore, these bioactive leachates from plastic tubes might be a specific inhibitor of tyrosine kinase.