Synthesis of vanoxerine analogues

Vanoxerine and GBR12935 were purchased from Adooq Bioscience and Carbosynth and were used without further purification. All other reagents were purchased from either Sigma-Aldrich (Merck), Alfa Aesar, Acros Organics, Fisher Scientific, VWR or Fluorochem, and were used as sold within the synthetic routes summarised in Fig. 1.

Fig. 1

Synthesis of vanoxerine analogues 3–11

Vanoxerine analogues 3–11 were synthesised from commercially available alcohol 1 according to the Fig. 1, using methods adapted from the literature [16]. The full synthetic protocols and characterisation are provided in the supplementary material. For compounds 5–8, the HCl salts were stable. Compounds 3–4 and 9–11 proved unstable to HCl; however, the maleic and bis-maleic acid salts were found to be suitable and stable salt forms for use in biological testing.

Bacterial strains and growth conditions

The bacterial strains used were M. smegmatis mc2155, M. bovis BCG Pasteur and E. faecium E745. These were grown in either BHI broth (E. faecium), 7H9 broth + Tween-80 0.05% (M. smegmatis) or 7H9 broth + Tween-80 0.05% + oleic acid, dextrose, albumin, and catalase (OADC, M. bovis BCG). All strains were grown by inoculating a single colony into broth and growing at 37 °C, either shaking at 180 rpm (E. faecium, M. smegmatis) or statically (M. bovis BCG).

MIC determination

Cultures of bacterial strains were grown to mid-log (OD600 = 0.5–1.0), before being diluted to a final OD600 of 0.05. An intermediate plate was prepared, performing a two-fold serial dilution of vanoxerine or its analogues into DMSO. Each concentration (1 µl) was transferred into a 96-well plate, followed by addition of the diluted cells (99 µl). The plate was then incubated (37 °C) for either 21 h (M. smegmatis), 24 h (E. faecium) or 144 h (M. bovis BCG). For E. faecium, the OD600 was measured immediately. For the mycobacteria strains, resazurin (0.02%, 30 µl per well) was added and the plate was incubated for a further 3 or 24 h (M. smegmatis or M. bovis BCG). The resazurin was converted to resorufin in metabolically active cells, hence the resorufin fluorescence (excitation at 544 nm, emission at 590 nm) was measured. Both sets of data were normalised relative to DMSO and antibiotic controls, and the percentage survival calculated.

Ethidium bromide accumulation and efflux assay

This method has been described previously [22], but is briefly outlined here: to a 96-well assay plate, DMSO, test compounds and verapamil were added, to achieve final concentrations of 1%, 50–100 µM and 102 µM, respectively. Bacterial cultures were grown to mid-log (OD600 = 0.5–1.0), and then washed and resuspended in PBS + Tween-80 0.05% + 0.625 µg ml−1 ethidium bromide (OD600 = 0.8). For accumulation, glucose 0.4% was added, and the cells immediately added to the assay plate. For efflux, 50 µg ml−1 verapamil was added, and the culture incubated (1 h, 37 °C). The cells were then centrifuged (3000 g, 8 min, 4 °C) and resuspended in PBS + Tween-80 0.05% (OD600 = 0.8). The ethidium bromide-loaded cells were either immediately added to the assay plate, or supplemented with glucose 0.4% prior to their addition. For both assays, the fluorescence (emission at 544 nm, emission at 590 nm) was measured every 60 s for 1 h at 37 °C in a BMG Labtech POLARstar omega plate reader.

Membrane electric potential assay

This method has been described previously [22], but is briefly outlined here: mid-log bacterial cultures were washed and resuspended in PBS + Tween-80 0.05% + 30 µM 3,3’-diethyloxacarbocyanine iodine (DiOC2(3)). The culture was incubated (37 °C, 2 h), before addition (99 µl) into a 96-well assay plate. Baseline fluorescence readings (excitation at 485 nm, emission at 520 and 620 nm) were taken every 90 s for 9 min, in a BMG Labtech POLARstar omega plate reader at 37 °C. DMSO, CCCP and test compounds were added, to achieve final concentrations of 1%, 25 µM and 100 µM, respectively. The fluorescence was read for a further 80 min.

Dopamine transport inhibition assay

This assay was performed by Eurofins Discovery and is based on a reported method [24]. Transfected Chinese hamster ovary (CHO) cells (2.5 × 103 cells/well) were incubated (90 minutes) at room temperature with [3H] dopamine (0.3 µM) in the absence or presence of test compounds in assay buffer (5 mM Tris-HCl pH 7.4, 7.5 mM HEPES/Tris, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 5.0 mM glucose and 1.0 mM ascorbic acid). Following incubation, the amount of [3H] dopamine was quantified using a scintillation counter (Topcount, Packard). The results were expressed as a percentage inhibition relative to the control uptake of [3H] dopamine. If the tested concentrations resulted in inhibition values of less than 50%, then an inhibition curve was estimated to calculate an IC50.