The Journal of Antibiotics https://doi.org/10.1038/s41429-018-0120-5 BRIEF COMMUNICATION Fluorescence assay to predict activity of the glycopeptide antibiotics Vladimir Vimberg 1 ● Radek Gazak 1 ● Zsolt Szűcs 2 ● Aniko Borbás 2 ● Pal Herczegh 2 ● Jorunn Pauline Cavanagh 3,4 ● Leona Zieglerova 1 ● Jan Závora 5 ● Václava Adámková 5 ● Gabriela Balikova Novotna 1 Received: 25 June 2018 / Revised: 13 August 2018 / Accepted: 4 October 2018 © The Author(s) under exclusive licence to the Japan Antibiotics Research Association 2018 Abstract Here, we describe a fluorescent assay developed to study competitive binding of the glycopeptide antibiotics to live bacteria cells. This assay demonstrated that the mechanism of action of the lipoglycopeptide antibiotics strongly depends on the hydrophobicity of the substitutes, with the best antibacterial activity of the glycopeptide antibiotics equally sharing properties of binding to D-Ala–D-Ala residues of the nascent peptidoglycan and to the membrane. For many years, vancomycin and teicoplanin were the only glycopeptide antibiotics used clinically in treatment of severe infections caused by gram-positive pathogens. However, the spread of glycopeptide-resistant enterococci and staphylococci has led to a renewed interest in the development of novel derivatives of the glycopeptide anti- biotics [1–3]. Semisynthetic lipoglycopeptide antibiotics telavancin, dalbavancin and oritavancin, with improved antibacterial activity and pharmacokinetics in comparison to vancomycin and teicoplanin, were recently approved for clinical usage. The improved activity of these lipoglyco- peptide antibiotics was associated with lipophilic substitutes introduced to glycopeptides, which enhanced interaction of the antibiotics with cell-wall precursors and with the membrane, leading to inhibition of cell wall synthesis, as well as disruption of cell membrane integrity [4–6]. Because of the complexity of the binding of glycopeptide antibiotics to the cell wall, involving binding to the terminal D-Ala–D-Ala residues of the nascent peptidoglycan, pepti- doglycan bridges, interaction with the membrane and anti- biotic dimerization [7], direct comparison of the efficiency of binding of different glycopeptide antibiotics to live bacterial cells was lacking. Fluorescently labelled antibiotics have been used success- fully to characterize changes in the cell wall of the bacteria [8, 9]. We decided to use fluorescently labelled glycopeptide antibiotics to compare the binding of the glycopeptide anti- biotics to the live bacterial cells. We saturated exponentially growing Staphylococcus aureus ATCC29213 cells with fluorescently labelled vancomycin (FL-VAN), available from Sigma-Aldrich, or fluorescently labelled teicoplanin (FL-TEI), labelled with rhodamine B isothiocyanate (Fig. 1a). Protocol of synthesis of FL-TEI is described in Fig. S1. Detailed protocol of the fluorescent assay is described in Supplemen- tary Materials. Then we tracked the release of the fluorescent glycopeptides bound to the cells by treating the cells for 10 min at room temperature with increasing amounts of non- fluorescent glycopeptides: vancomycin—VAN (V2002, Sigma-Aldrich), teicoplanin—TEI (T0578, Sigma-Aldrich), dalbavancin—DALB (HY-17586, MedChemExpress), orita- vancin—ORI (SML1586, Sigma-Aldrich) and recently pub- lished novel lipoglycopeptide antibiotic derivatives with substitutes to the primary amino function of teicoplanin pseudoaglycon: MA79, MA72, ERJ390, LTS3, SZZS-12 [10–12] (Fig. 1b, Fig S1). As a proof of concept of the experiment, we showed nearly 100% release of the FL-VAN by N-acetyl-D-Ala–D-Ala dipeptide (D-Ala–D-Ala) (Sigma- * Vladimir Vimberg vladimir.vimberg@biomed.cas.cz 1 Institute of Microbiology v. v. i., Czech Academy of Sciences, Vestec, Czech Republic 2 Department of Pharmaceutical Chemistry, University of Debrecen, Debrecen, Hungary 3 Department of Pediatrics, University Hospital of North Norway, Tromsø, Norway 4 Department of Clinical Medicine, UiT The Arctic University of Norway, Tromsø, Norway 5 Clinical Microbiology and ATB Centre, Institute of Medical Biochemistry and Laboratory Diagnostics, General University Hospital, Prague, Czech Republic Electronic supplementary material The online version of this article (https://doi.org/10.1038/s41429-018-0120-5) contains supplementary material, which is available to authorized users. 1234567890();,: 1234567890();,: