Figure 6: Target identification of dosabulin.

(a) Confocal microscopy in U2OS cells of both enantiomers of dosabulin show that the (S)-enantiomer disrupts the tubulin network in interphase cells, similarly to nocodazole, whilst the (R)-enantiomer does not. U2OS cells were stained with DAPI (blue) and an α-tubulin specific antibody (green). Scale bar: 15 μm; (b) in vitro tubulin polymerization data shows that (S)-dosabulin causes a decrease in tubulin polymerization, confirming this as a mechanism of action. Porcine brain tubulin (3 mg ml⁻¹) kept at 4 °C was incubated with test compounds for 60 min at 37 °C. Compound concentrations: (S)-dosabulin and (R)-dosabulin used at 20 μM, nocodazole at 5 μM. OD=optical density; (c) fluorescence polarization (FP) data using FL-BODIPY-Vinblastine (2 μM) as a tracer shows that (S)-dosabulin does not bind tubulin in the vinblastine site. FP values were normalized to the DMSO control and data points are mean±s.e.m. of an experiment conducted in triplicate (ex/em wavelengths 485/520 nm). CPD=compound; (d) fluorescence intensity (F) measurements using colchicine show that (S)-dosabulin (red) causes a dose-dependent decrease in fluorescence without completely inhibiting the interaction, and thus is likely to bind in the vicinity or allosterically to colchicine. F-values were normalized to the DMSO control and data points are mean±s.e.m. of an experiment conducted in triplicate (ex/em wavelengths 365/435 nm).