Imidazoquinoxaline anticancer derivatives and imiquimod interact with tubulin: Characterization of molecular microtubule inhibiting mechanisms in correlation with cytotoxicity

• Published in: PloS one 2017-08, Vol.12 (8), p.e0182022-e0182022
• Main Authors: Courbet, Alexis, Bec, Nicole, Constant, Caroline, Larroque, Christian, Pugniere, Martine, El Messaoudi, Safia, Zghaib, Zahraa, Khier, Sonia, Deleuze-Masquefa, Carine, Gattacceca, Florence
• Format: Article
• Language: English
• Subjects: Antineoplastic Agents - pharmacology; Antineoplastics Agents/pharmacology; Antiviral drugs; Apoptosis; Apoptosis - drug effects; Binding Sites; Biology and Life Sciences; Cancer; Cell cycle; Cell Cycle - drug effects; Cell death; Cell Death - drug effects; Cell Line, Tumor; Cell Proliferation - drug effects; Clinical trials; Colchicine; Colchicine - pharmacology; Competition; Complications and side effects; Computer applications; Computer Simulation; Computor simulation; Cytotoxicity; Dosage and administration; Drug development; Drug dosages; Drug therapy; Genetic aspects; Humans; Imiquimod; In vitro methods and tests; Leukemia; Life Sciences; Medicine and Health Sciences; Melanoma; Metabolism; Models, Molecular; Molecular docking; Molecular Docking Simulation; Pharmaceutical sciences; Pharmacology; Physical Sciences; Physiological aspects; Polymerization; Protein Binding; Quinoxalines; Quinoxalines - pharmacology; Quinoxalynes/pharmacology; Research and Analysis Methods; Signal transduction; Skin cancer; Toxicity; Tubulin; Tubulin - metabolism; Tubulin Modulators - pharmacology
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0182022

Displaying a strong antiproliferative activity on a wide variety of cancer cells, EAPB0203 and EAPB0503 belong to the imidazo[1,2-a]quinoxalines family of imiquimod structural analogues. EAPB0503 has been shown to inhibit tubulin polymerization. The aim of the present study is to characterize the interaction of EAPB0203 and EAPB0503 with tubulin. We combine experimental approaches at the cellular and the molecular level both in vitro and in silico in order to evaluate the interaction of EAPB0203 and EAPB0503 with tubulin. We examine the influence of EAPB0203 and EAPB0503 on the cell cycle and fate, explore the binding interaction with purified tubulin, and use a computational molecular docking model to determine the binding modes to the microtubule. We then use a drug combination study with other anti-microtubule agents to compare the binding site of EAPB0203 and EAPB0503 to known potent tubulin inhibitors. We demonstrate that EAPB0203 and EAPB0503 are capable of blocking human melanoma cells in G2 and M phases and inducing cell death and apoptosis. Second, we show that EAPB0203 and EAPB0503, but also unexpectedly imiquimod, bind directly to purified tubulin and inhibit tubulin polymerization. As suggested by molecular docking and binding competition studies, we identify the colchicine binding site on β-tubulin as the interaction pocket. Furthermore, we find that EAPB0203, EAPB0503 and imiquimod display antagonistic cytotoxic effect when combined with colchicine, and disrupt tubulin network in human melanoma cells. We conclude that EAPB0203, EAPB0503, as well as imiquimod, interact with tubulin through the colchicine binding site, and that the cytotoxic activity of EAPB0203, EAPB0503 and imiquimod is correlated to their tubulin inhibiting effect. These compounds appear as interesting anticancer drug candidates as suggested by their activity and mechanism of action, and deserve further investigation for their use in the clinic.