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.
Les imidazoquinoxalines (imiqualines), molecules bioactives originales analogues chimiques de l’imiquimod et presentant un important potentiel anticancereux, ont ete explorees afin d’elucider leurs mecanismes d'action. Les molecules EAPB0203 et EAPB0503, identifiees lors d’etudes anterieures comme etant les « tetes de series », ont montre un effet cytotoxique puissant in vitro sur des lignees cellulaires cancereuses humaines de melanome et de lymphomes T. Nous avons etudie l’effet cytotoxique de 13 imiqualines nouvellement synthetisees sur une lignee cellulaire de melanome humain (A375). Tous les composes ont montre un effet cytotoxique important. L’effet cytotoxique a ete demontre sur d’autres lignees cellulaires cancereuses humaines (colon, sein et lymphome T de l’adulte).Un blocage du cycle cellulaire en phase G2/M a ete mis en evidence par cytometrie en flux sur des cellules A375 traitees par EAPB0203 et EAPB0503. Cet arret du cycle cellulaire semble etre en relation avec un effet inhibiteur de la polymerisation de la tubuline. En effet, nos resultats ont montre que l’EAPB0503 et 3 autres imiqualines inhibent la polymerisation de la tubuline. L’etude de modelisation moleculaire de la liaison a de la tubuline a montre que ces composes se fixent sur le site de la colchicine.Une analyse transcriptomique sur EAPB0503 a ete effectuee pour elucider le mecanisme d'action des imiqualines. Cette etude a ete faite sur la lignee A375 en comparaison avec 13 anticancereux de reference. L’etude transcriptomique a montre que EAPB0503 a une composante antitubuline tout en revelant un mecanisme d’action original. Deux hypotheses mecanistiques pour EAPB0503 ont ainsi ete identifiees : 1/ alteration de la voie de signalisation liee aux integrines, 2/ alteration de la voie de signalisation du recepteur TNFR et de FasL.Des analyses fonctionnelles in vitro nous ont permis d’explorer ces hypotheses. Ainsi, une alteration uniquement des voies de signalisation PI3K/AKT et RAS/MAPK, toutes deux liees aux integrines, a ete observee. La premiere hypothese semble donc validee. De plus, ce resultat a ete confirme par l’etude de l’expression et de la phosphorylation de ERK.Finalement, nous avons developpe une formulation injectable par voie intraveineuse de EAPB0503 a base de nanocapsules. Cette formulation a d’abord ete testee in vitro et in vivo sur un modele de lymphome. Ces etudes ont pu mettre en evidence l’absence de toxicite des nanoparticules vides et le maintien de l’activite cytotoxique de EAPB0503 encapsule in vitro, avec un effet immunomodulateur in vivo qui demande a etre explore.
Objective: EAPB0503, lead compound of imiqualines, presented high antitumor activities but also a very low water solubility which was critical for further preclinical studies. To apply to EAPB0503, a robust and safe lipid formulation already used for poor soluble anticancer agents for injectable administration at a concentration higher than 1 mg/mL. Materials and Methods: Physicochemical properties of EAPB0503 were determined to consider an adapted formulation. In a second time, lipid nanocapsules (LNC) formulations based on the phase-inversion process were developed for EAPB0503 encapsulation. Then, EAPB0503 loaded-LNC were tested in vitro on different cell lines and compared to standard EAPB0503 solutions. Results: Optimized EAPB0503 LNC displayed an average size of 111.7 ± 0.9 nm and a low polydispersity index of 0.059 ± 0.002. The obtained loading efficiency was higher than 96% with a drug loading of 1.7 mg/mL. A stability study showed stability during 4 weeks stored at 25°C. In vitro results highlighted similar efficiencies between LNC and standard EAPB0503 solutions prepared in dimethyl sulfoxide. Conclusion: In view of results obtained for loading efficiency and drug loading, the use of a LNC formulation is very interesting to permit the solubilization of a lipophilic drug and to improve its bioavailability. Preliminary tested pharmaceutical formulation applied to EAPB0503 significantly improved its water solubility and will be soon considered for future preclinical in vivo studies.
