EAPB0203, EAPB0503 and imiquimod bind tubulin on the colchicine binding site.

<p><b>(A)</b> Interaction modes of EAPB0203, EAPB0503 and imiquimod on the colchicine site as predicted by AutoDock Vina. Molecules interact with two hydrophobic pockets (blue and orange). (<b>B)</b> Competition of EAPB0203, EAPB0503, imiquimod, nocodazole (positive control) and vinorelbine (negative control) with colchicine for tubulin binding. Fluorescence of the colchicine-tubulin complex was measured after excitation at 365 nm. F/F0: relative fluorescence intensity, F0: fluorescence intensity of the colchicine-tubulin complex alone.</p

EAPB0203, EAPB0503 and imiquimod display an antagonistic effect with colchicine, but synergistic with vinorelbine.

<p>A375 cells were treated with EAPB0203, EAPB0503, colchicine and vinorelbine–alone and with constant ratio combination—at concentrations surrounding their previously determined IC₅₀. Cell viability was assessed 48h after treatment, using the MTT test. Chou and Talalay method was used to calculate a combination index: CI<1, CI = 1 and CI>1 respectively mean a synergy, an additive effect or an antagonism. The grey frame embodies the 90% confidence interval as determined by Calcusyn software. Fa = fraction affected by the dose.</p

EAPB0203 and EAPB0503, and imiquimod at high concentrations, inhibit polymerization of tubulin <i>in vitro</i>.

<p>Purified tubulin polymerization was quantitated by turbidimetry measurement at 350 nm with or without (blank) various concentrations of EAPB0203, EAPB0503, imiquimod, and colchicine as positive control, or warfarin (1,600 μM) as negative control. Various parameters representative of the polymerization process were calculated: A_max (maximum absorbance plateau value), t_1/10 (abscissa value of the A_max/10 absorbance), p (slope of the plot log(A(t)/A_max) versus log(t) during the elongation process, i.e. from 1 min to the time of 80% A_max), and k_obs (pseudo-first order rate constant of elongation, determined by plotting ln(1 –A(t)/ A_max) as a function of time). The ratio to blank values are reported here. The red line embodies the ratio of 1, meaning identity to blank. Mean ± SD, n = 1 to 6. (<b>A)</b> EAPB0203, EAPB0503, imiquimod and colchicine at 0.1 to 10 μM concentrations. (<b>B)</b> Imiquimod at 160 to 1,600 μM concentrations. Mean ± SD, n = 1 or 2.</p

EAPB0203, EAPB0503 and imiquimod bind tubulin.

<p>Binding levels of EAPB0203, EAPB0503 and imiquimod were determined by surface plasmon resonance on immobilized tubulin at different concentrations (n = 4, except for colchicine n = 1).</p

EAPB0203, EAPB0503 and imiquimod disturb microtubule network in A375 cancer cell line.

<p>A375 cells were treated by EAPB0203, EAPB0503 and imiquimod at the indicated concentrations for 24h. Beta-tubulin was stained using a mouse monoclonal anti-β-tubulin antibody and a secondary Rhodamine-labeled anti-mouse antibody. Nuclei were stained with Hoechst. Microtubule network (green) and nuclear DNA (red) were visualized using a Leica DMRM fluorescence microscope with a 63x magnification. Representative images are displayed here.</p

EAPB0203 and EAPB0503 block cell cycle in G2 and M phases and induce apoptosis.

<p>A375 cells were treated with EAPB0203 or EAPB0503 at the indicated concentrations. (<b>A</b>) After 24 hours, cells were stained with propidium iodide alone (G2/M) or with propidium iodide and anti-phospho histone H3 antibody (M). Flow cytometry was used to determine the percentage of cells in G2/M and M phases of the cell cycle. (<b>B)</b> After the indicated time, cells were stained with 7-AAD and Annexin V. We used flow cytometry to determine the percentage of necrotic and secondary necrotic cells (Annexin V and 7-AAD positive) and early stage apoptotic cells (Annexin V positive and 7-AAD negative).</p

Chemical structures of studied compounds.

<p>The imidazo[1,2-<i>a</i>]quinoxalines family, nick-named <i>imiqualines</i>, was developed from structural analogy with imiquimod. The compound EAPB0203 (<i>N</i>-methyl-1-(2-phenylethyl)imidazo[1,2-a]quinoxalin-4-amine) was identified as the initial leader of the series. Further pharmacomodulation of imiqualines lead to the new compound EAPB0503 (1-(3-methoxyphenyl)-<i>N</i>-methylimidazo[1,2-<i>a</i>]quinoxalin-4-amine).</p

EAPB0203 and EAPB0503 inhibit cell proliferation and display cytotoxicity.

<p>A375 cells were treated with EAPB0203 or EAPB0503 at the indicated concentrations, harvested at various times post-treatment, and counted using Casy cell counter. Percentage of dead cells was determined on Malassez hemocytometer after trypan blue staining, and dead cells removed from the total cell count. The thick grey line corresponds to the number of living cells as counted at time zero.</p