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. 2022 Feb 27;23(5):2637.
doi: 10.3390/ijms23052637.

An Efficient Way to Screen Inhibitors of Energy-Coupling Factor (ECF) Transporters in a Bacterial Uptake Assay

Spyridon Bousis  1   2   3 , Steffen Winkler  1 , Jörg Haupenthal  1 , Francesco Fulco  1 , Eleonora Diamanti  1 , Anna K H Hirsch  1   2   3
Affiliations

An Efficient Way to Screen Inhibitors of Energy-Coupling Factor (ECF) Transporters in a Bacterial Uptake Assay

Spyridon Bousis et al. Int J Mol Sci. .

Abstract

Herein, we report a novel whole-cell screening assay using Lactobacillus casei as a model microorganism to identify inhibitors of energy-coupling factor (ECF) transporters. This promising and underexplored target may have important pharmacological potential through modulation of vitamin homeostasis in bacteria and, importantly, it is absent in humans. The assay represents an alternative, cost-effective and fast solution to demonstrate the direct involvement of these membrane transporters in a native biological environment rather than using a low-throughput in vitro assay employing reconstituted proteins in a membrane bilayer system. Based on this new whole-cell screening approach, we demonstrated the optimization of a weak hit compound (2) into a small molecule (3) with improved in vitro and whole-cell activities. This study opens the possibility to quickly identify novel inhibitors of ECF transporters and optimize them based on structure-activity relationships.

Keywords: B-type vitamins; antimicrobials; bacterial uptake assay; energy-coupling factor transporters; screening.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Schematic representation of group I and II ECF transporters. The heterodimeric ATPases (ECFA and ECFA’) are represented in light and dark red color, while the T-component is shown in blue. The S-components belonging to the ECF group I and II are colored in light green and yellow, respectively.
Figure 2
Figure 2
Chemical structures of the two identified inhibitors of the group II ECF-FolT2 transporter [9].
Figure 3
Figure 3
Illustration of the key steps of the new plated whole-cell uptake assay of ECF FolT transporter. (A) Depicting the cell-preparation steps of L. casei. Wash: PBS (50 mM, pH 7.4). MRS: De Man, Rogosa and Sharpe agar. (B) Depicting the crucial steps during the assay. Wash: PBS (10 mM, pH 7.2) citrate buffer (20 mM, pH 6, 2% D-Glucose, 8 g/L NaCl, 0.2 g/L KCl) and 5% DMSO.
Figure 4
Figure 4
Substrate-dependent folate uptake in L. casei in PBS (50 mM, pH 7.4, 2% Glucose). With KM = 49 (±4) nM and Vmax = 83 (±2) counts per minute (cpm).
Figure 5
Figure 5
Chemical evolution of 2 to 3 presented on this work.
See this image and copyright information in PMC

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