ELR510444, a novel microtubule disruptor with multiple mechanisms of action

Abstract

Although several microtubule-targeting drugs are in clinical use, there remains a need to identify novel agents that can overcome the limitations of current therapies, including acquired and innate drug resistance and undesired side effects. In this study, we show that ELR510444 has potent microtubule-disrupting activity, causing a loss of cellular microtubules and the formation of aberrant mitotic spindles and leading to mitotic arrest and apoptosis of cancer cells. ELR510444 potently inhibited cell proliferation with an IC(50) value of 30.9 nM in MDA-MB-231 cells, inhibited the rate and extent of purified tubulin assembly, and displaced colchicine from tubulin, indicating that the drug directly interacts with tubulin at the colchicine-binding site. ELR510444 is not a substrate for the P-glycoprotein drug transporter and retains activity in βIII-tubulin-overexpressing cell lines, suggesting that it circumvents both clinically relevant mechanisms of drug resistance to this class of agents. Our data show a close correlation between the concentration of ELR510444 required for inhibition of cellular proliferation and that required to cause significant loss of cellular microtubule density, consistent with its activity as a microtubule depolymerizer. ELR510444 also shows potent antitumor activity in the MDA-MB-231 xenograft model with at least a 2-fold therapeutic window. Studies in tumor endothelial cells show that a low concentration of ELR510444 (30 nM) rapidly alters endothelial cell shape, similar to the effect of the vascular disrupting agent combretastatin A4. These results suggest that ELR510444 is a novel microtubule-disrupting agent with potential antivascular effects and in vivo antitumor efficacy.

Fig. 1.

A, chemical structure of ELR510444. B and C, dose-dependent effects of ELR510444 (B) and CA-4 (C) on the proliferation of the MDA-MB-231 breast cancer cell line. D, the SRB assay was used to determine the IC₅₀ values for inhibition of proliferation of MDA-MB-231, MDA-MB-435, 2H-11, HeLa, WTβIII, SK-OV-3, and SK-OV-3/MDR-1-6/6 cells by ELR510444 and CA-4. Rr values were calculated for both sets of drug-resistant cell lines, and paclitaxel was used as a positive control. E, ELR510444 is not a substrate for the Pgp drug efflux pump, as indicated by an efflux ratio less than 2 in the MDR1-MDCK permeability assay. Cyclosporin A (CSA) was used as a competitive inhibitor for Pgp transport.

Fig. 2.

ELR510444 causes loss of cellular microtubules. A, A-10 cells were treated with vehicle (DMSO), 12.5 nM CA-4, or 50 nM ELR510444 for 18 h. Microtubules were visualized by indirect immunofluorescence for β-tubulin. The loss of cellular microtubules can be seen in CA-4- and ELR510444-treated cells. B, the percent microtubule depolymerization observed at each concentration of ELR510444 was estimated visually, the effects of a range of concentrations were plotted, and the EC₅₀ value of 21 nM was calculated from linear regression analysis.

Fig. 3.

ELR510444 causes mitotic arrest and aberrant mitotic spindles. HeLa cells were treated with vehicle alone (DMSO), 10 nM CA-4, or 25 nM ELR510444 for 18 h. A, cells were analyzed for cell cycle distribution by propidium iodide staining followed by flow cytometry. B, microtubules were visualized by indirect immunofluorescence.

Fig. 4.

ELR510444 directly inhibits the polymerization of tubulin. A, purified porcine brain tubulin (Cytoskeleton) was incubated in the presence of vehicle (DMSO), 5 μM CA-4, or 5 or 10 μM ELR510444 in general tubulin buffer with 10% glycerol and 1 mM GTP at 37°C. Tubulin polymerization was monitored by absorbance at 340 nm. B, ELR510444 displaces colchicine from tubulin. The fluorescence of tubulin alone (1); 2 μM colchicine and 2 μM tubulin (2); 2 μM colchicine, 2 μM tubulin, and 50 μM ELR510444 (3); 2 μM colchicine, 2 μM tubulin, and 100 μM ELR510444 (4); and 2 μM colchicine, 2 μM tubulin, and 200 μM ELR510444 (5) was determined. The fluorescence measured with 2 μM colchicine and 2 μM tubulin was set at 100%.

Fig. 5.

In vivo antitumor activity of ELR510444. A, dose-dependent antitumor activity was evaluated in the MDA-MB-231 xenograft model with oral dosing of ELR510444 at 3, 6, or 12.5 mg/kg once daily or ABT-751 at 75 mg/kg once daily. B, the plasma compound concentration over time was determined by liquid chromatography-tandem mass spectrometry after a single oral dose of 25 mg/kg.

Fig. 6.

Effects of ELR510444 on endothelial cell morphology. 2H-11 endothelial cells were treated with vehicle, 3 nM CA-4, or 30 nM ELR510444 for 1 h. The actin cytosketelon (red) and DNA (blue) were visualized by tetramethylrhodamine B isothiocyanate-phalloidin and DAPI staining, respectively.