Interaction of 4-Arylcoumarin Analogues of Combretastatins with Microtubule Network of HBL100 Cells and Binding to Tubulin †

ABSTRACT : The synthesis of different 4-arylcoumarin analogues of combretastatin A-4 led to theidentification of two new compounds (1 and 2) with potent cytotoxic activity on a CEM leukemia cellline and a third one completely inactive (compound 3). It was suggested that the cytotoxicity of compounds1 and 2 may be related to their interaction with microtubules and tubulin, since these compounds inhibitmicrotubule formation from purified tubulin in vitro [Bailly et al. (2003) J. Med. Chem. 46 (25), 5437-5444]. In the present study, tubulin was identified as the main target of these molecules. We studiedstructure-activity relationships of these compounds using biological experiments specific for tubulinbinding. The modification of cell cycle progression induced by compounds 1 and 2 was characterized byan apoptotic induction on human breast cells (HBL100). In addition, these two molecules disturbed cellsurvival by depolymerizing the microtubule network, leading to a mitotic block. We then determined thethermodynamic parameters of their interaction with purified tubulin by fluorescence spectroscopy andisothermal microcalorimetry. These results, together with a superimposition of the molecule on colchicinein the X-ray-determined three-dimensional structure model of tubulin-colchicine complex, allowed us toidentify the pharmacophore of the combretastatin A-4 analogues responsible for their biological activity.The microtubule network is an essential component of thecytoskeleton in eukaryotic cells. The cytostatic drug colchi-cine was shown to bind tubulin, the main constitutive proteinof microtubules. This fundamental step in the developmentof new antimitotic agents then led to new cancer chemo-therapy approaches and to a better knowledge of microtubulepharmacology and biochemistry. Antimitotic drugs that bindtubulin, also called antitubulin agents, are classified asmicrotubule-stabilizing agents or microtubule-destabilizingagents. Taxoids (paclitaxel and docetaxel) enhance thetubulin polymerization and interact directly with microtu-bules, stabilizing them against depolymerization. The Vincaalkaloids and the colchicinoids inhibit tubulin polymerizationbut differ in their mechanism of action and in their bindingsite on tubulin (1). Until now, only molecules from Vincaalkaloids and taxoid groups were extensively used in cancerchemotherapies. Considering the success of the antitubulinpharmacological class, numerous researches are now focusedon the discovery and clinical trial development of newderivatives. Combretastatin A-4, already known as a powerfulinhibitor of tubulin polymerization and cell growth (2), alsohas an antivascular activity at low concentration (3). Thisnew low-concentration activity of combretastatin A-4 led tothe renewal of interest for the possible clinical use ofcolchicine analogues often discarded for their large neuro-toxicity. Moreover, tumor cells can acquire resistanceproperties to various anticancer agents. Overexpression ofthe drug efflux transporter P-glycoprotein causes this mul-tidrug-resistance phenotype and contributes to the failure ofcancer chemotherapy. The combretastatin A-4 is not recog-nized by this glycoprotein. Because of this latter characteristicand of its antiangiogenic property, combretastatin A-4became the most interesting antitubulin agent which interactswith the colchicine binding site. However, natural combre-tastatin A-4 suffers from serious drawbacks: it isomerizesto an inactive form (4) and has a low aqueous solubility.Therefore, efforts have focused on making soluble combre-tastatin A-4 derivatives and blocking isomerization (5). Aset of 4-arylcoumarin analogues of combretastatin A-4 havethus been synthesized, and their cytotoxicity on humanleukemia CEM cell line have been assessed (6). Among thisset of molecules, compounds 1