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Journal of Molecular and Clinical Medicine  2018, Vol. 1 Issue (2): 67-76    DOI: 10.31083/j.jmcm.2018.02.003
Research article | Next articles
A Small Molecule Tubulin Depolymerizing Agent Identified by a Phenotypic Drug Discovery Approach
Carla Ríos-Luci1, Elena Díaz-Rodríguez2, Rubén M. Buey3, Inês J. Sousa4, Miguel X. Fernandes1, 4, Atanasio Pandiella2, José M. Padrón1, *()
1 Instituto Universitario de Bio-Orgánica "Antonio González" (IUBO-AG), Centro de Investigaciones Biomédicas de Canarias (CIBICAN), Universidad de La Laguna, C/Astrofísico Francisco Sánchez 2, 38206 La Laguna, Spain
2 Instituto de Biología Molecular y Celular del Cáncer, Campus Miguel de Unamuno, 37007 Salamanca, Spain
3 Metabolic Engineering Group, Dept. Microbiology and Genetics, Universidad de Salamanca, Campus Miguel de Unamuno, 37007 Salamanca, Spain
4 Centro de Química da Madeira, Universidade da Madeira, Campus da Penteada, 9000-390 Funchal, Portugal
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In the scenario of drug discovery, numerous in vitro testing initiatives had been established. Thus far, no general methodology is reputable and literature on this hot topic is scarce. In this respect, we propose a strategy based on a Phenotypic Drug Discovery approach. Within our program directed at the discovery of new antitumor agents, we have focused our attention on compounds that disturb the cell cycle. Our strategy relies on the use of a set of biological assays organized in a modular fashion. Herein, we exemplified this strategy with a family of propargylic enol ether derivatives. Using different assays in sequential stages and in a stepwise manner, our studies allowed us to understand the bioactivity of this family of compounds and led us to identify tubulin as the main molecular target.

Key words:  Anti-cancer      Colchicine binding site      Drug-target interaction      Microtubule-targeting agents     
Submitted:  03 November 2017      Accepted:  08 December 2017      Published:  20 April 2018     
*Corresponding Author(s):  José M. Padrón     E-mail:

Cite this article: 

Carla Ríos-Luci, Elena Díaz-Rodríguez, Rubén M. Buey, Inês J. Sousa, Miguel X. Fernandes, Atanasio Pandiella, José M. Padrón. A Small Molecule Tubulin Depolymerizing Agent Identified by a Phenotypic Drug Discovery Approach. Journal of Molecular and Clinical Medicine, 2018, 1(2): 67-76.

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Fig.1.  (A) Chemical structure of DTA0100. (B) DTA0100 did not inhibit TOPO II $\alpha$ activity of SW1573 nuclear extracts. Nuclear preparations were exposed to increasing concentrations of DTA0100 for 1 h, after which the supercoiled relaxation assay was performed.

Fig.2.  (A) DTA0100 induces a G$_2$/M arrest in a time-dependent manner. Cell cycle histograms of untreated cells (control) and cells treated with DTA0100 for the indicated time. Cell cycle stage was determined by flow cytometric analysis of DNA content in individual cells. (B) DTA0100 inhibited cell growth and induced mitotic cell accumulation. Cells were treated with the indicated concentrations of DTA0100 for 24 h. Then, cell cycle stage was determined by flow cytometric analysis of DNA content and phosphohistone 3 (pH3) levels in individual cells. (C) DTA0100 specifically blocked cell cycle at mitosis. Cells were treated with 4 $\mu$M DTA0100 for the indicated time. Protein extracts were obtained for each condition and protein levels of pH3, cyclin B1, BubR1 and GAPDH were determined by western blotting.

Fig.3.  (A) DTA0100 induces apoptotic cell death. HBL-100 and HeLa cells were treated at the indicated concentrations with DTA0100. After 24 h and 48 h of DTA0100 treatment, samples were taken and stained for annexin V-FITC and propidium iodide. (B) DTA0100 provoked MMP. HBL-100 and HeLa cells were treated with DTA0100 for the indicated times and $\Delta\Psi$m was evaluated using TMRE as a dye. Live single cells were gated and fluorescence intensity decrease of TMRE was quantified by flow cytometry. (C) DTA0100 caused cell death through a caspase-dependent mechanism. HBL-100 and HeLa cells were treated for the indicated times with DTA0100 and the expression of PARP, caspase 3, caspase 7, caspase 9 and caspase 8 were analyzed by western blotting. GADPH was used as protein loading control.

Fig.4.  DTA0100 disrupts the mitotic spindle. (A) SW1573 and (B) HBL-100 cells were treated with DTA0100 for 24 h and 48 h, fixed, stained with anti $\beta$-tubulin antibody (green) and counterstained with DAPI (blue). Representative confocal images of cellular preparations are shown. Scale bar: 10 $\mu$m. (C) Induction of mitotic slippage by prolonged DTA0100 exposure in SW1573 cells. SW1573 cells were treated with DTA0100 for 48 h. Nuclear pore complex re-formation in cells upon mitotic slippage with the appearance of micronucleated cells in a tetraploid G$_0$-state. Scale bar: 10 $\mu$m. (D) Tetraploid G$_0$ arrest induced by DTA0100. Western blot analysis of cyclin D1, pRbSer780/811, p53 and p21. Cytoplasmic GAPDH was used as loading control.

Fig.5.  DTA0100 inhibits microtubule polymerization in a dose-dependent manner. (A) Tubulin was polymerized for 30 min at 37$^{\circ}$C in the presence of 20 $\mu$M of the indicated compounds. Colchicine (Col) and paclitaxel (PXT) were used as controls. After ultracentrifugation, the supernatant (S) and pellet (P) were analyzed by SDS-PAGE and stained with Coomassie. (B) Tubulin was polymerized for 30 min at 37 $^{\circ}$C in the presence of 10 $\mu$M of the indicated molecules, including nocodazole (NOC) and the absorbance at 340 nm was measured. (C) Tubulin was polymerized for 30 min at 37$^{\circ}$C in the presence of different concentrations of DTA0100. (D) DTA0100 impeded NOC-arrested cells recovery. After nocodazole treatment, cells were released in fresh medium with and without DTA0100 for the indicated period of times. Cells were fixed, stained with $\beta$-tubulin antibody (green) and counterstained with DAPI (blue). Representative confocal images for cellular preparations are shown. Scale bar 10 $\mu$m.

Fig.6.  Displacement of MTC from the colchicine binding domain by DTA0100. Fluorescence emission spectra of MTC-tubulin complex co-incubated with different concentrations of (A) DTA0100 and (B) colchicine in 10 mM sodium phosphate buffer pH 7.0 containing 0.1 mM GTP at 25$^{\circ}$C.

Fig.7.  Comparison of R-DTA0100 and S-DTA0100 conformations at the colchicine binding domain of tubulin. Localization of docked configurations of ligands are primarily in $\beta$-tubulin. (A) R-DTA0100 and (B) S-DTA0100. Residues thought to interact with the compounds are represented as thin dashed lines. Compounds are shown in stick representation.

Table 1  Anti-proliferative activity (GI$_{50}$) of DTA100 and tubulin-interacting drugs in SW1573 and SW1573/P-gp cell lines$^{\rm a}$
-Verapamil +Verapamil
SW1573 SW1573/P-gp Rfb SW1573 SW1573/P-gp Rf
DTA0100 1029 士 221 2902 士 476 3 656 士 252 2637 士 713 4
Paclitaxel 1.5 士 0.5 196 士 53 128 1.6 士 0.2 4.2 士 0.9 3
Colchicine 71 士 15 531 士 95 7 46 士 11 131 士 7 3
Vincristine 9.7 士 3.6 180 士 68 18 1.5 士 0.3 3.8 士 0.5 3
Vinblastine 0.9 士 0.3 2051 士 682 2388 0.8 士 0.2 1.0 士 0.5 1
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