Abstract

Uma estratégia importante para a terapia do câncer é o planejamento de modulares que interferem na dinâmica dos microtúbulos através de sua ligação específica à subunidade β da tubulina. No presente trabalho, estudos de análise comparativa dos campos moleculares (CoMFA) foram realizados com uma série de análogos do discodermolídeo com ação antimitótica. Resultados significativos foram obtidos (CoMFA (i) , q 2 = 0,68, r 2 = 0,94; CoMFA (ii) , q 2 = 0,63, r 2 = 0,91), indicando a elevada consistência interna e externa dos modelos gerados empregando duas estratégias independentes de alinhamento estrutural. Os modelos foram validados externamente com um conjunto teste e os valores preditos apresentaram boa concordância com os resultados experimentais. Os modelos de QSAR e os mapas de contorno 3D forneceram importantes informações sobre as bases químicas e estruturais envolvidas no processo de reconhecimento molecular dessa família de análogos do discodermolídeo, sendo uma valiosa ferramenta no planejamento de novos moduladores específicos da β-tubulina com potente atividade antitumoral. An important approach to cancer therapy is the design of small molecule modulators that interfere with microtubule dynamics through their specific binding to the β-subunit of tubulin. In the present work, comparative molecular field analysis (CoMFA) studies were conducted on a series of discodermolide analogs with antimitotic properties. Significant correlation coefficients were obtained (CoMFA (i) , q 2 = 0.68, r 2 = 0.94; CoMFA (ii) , q 2 = 0.63, r 2 = 0.91), indicating the good internal and external consistency of the models generated using two independent structural alignment strategies. The models were externally validated employing a test set, and the predicted values were in good agreement with the experimental results. The final QSAR models and the 3D contour maps provided important insights into the chemical and structural basis involved in the molecular recognition process of this family of discodermolide analogs, and should be useful for the design of new specific β-tubulin modulators with potent anticancer activity. Keywords: cancer, drug design, discodermolide, microtubule, β-tubulin Introduction A rational approach to cancer therapy involves the design of small molecule ligands that interfere with microtubule dynamics through their specific binding to the β-subunit of tubulin, leading to mitotic arrest and cell death. 1-6 Taxol (paclitaxel, 6-10 These include non-taxane microtubule-stabilizing natural products, such as discodermolide, epothilone and dictyostatin 11-15 The marine polyketide discodermolide is one of the most potent MSAAs known. In addition to its potent antiproliferative and apoptosis-inducing activities, discodermolide is more water soluble than paclitaxel and retains substantial activity against taxane-resistant cell lines. These physicochemical and biological characteristics have qualified discodermolide as a lead compound for the development of new, more effective and safer anticancer agents. As part of our ongoing research program aimed at designing new β-tubulin modulators, a rational structurebased design strategy was employed to investigate the molecular recognition patterns required for specific β-tubulin binding, as schematically shown in Experimental Data set The data set used for the QSAR analyses contains 42 discodermolide analogs. Computational approach The QSAR modeling analyses, calculations, and visualizations for CoMFA were performed using the SYBYL 8.0 package (Tripos Inc., St. Louis, MO, USA) running on Red Hat Enterprise Linux workstations. The 3D structures of the small molecule discodermolide modulators were constructed using standard geometric parameters of the SYBYL 8.0 package. Each single optimized conformation of each molecule in the data set was energetically minimized employing the Tripos force field and Gasteiger-Huckel charges. 38,39 A hierarchical cluster analysis of the data set was carried out with Tsar 3D (Accelrys, San Diego, CA). Molecular alignment Alignment is a crucial component in 3D QSAR studies, and a variety of useful approaches have been described in the literature. [18] (ii) Receptor-based. Predicted binding modes of discodermolide and its analogs into the β-tubulin cavity have been previously proposed by using an integration of experimental evidences and computer-aided studies. 25 Docking and scoring protocols as implemented in GOLD 3.1 (Cambridge Crystallographic Data Centre, Cambridge, UK) were used to investigate the possible binding conformations of the ligands within the discodermolide binding domain of β-tubulin. 25,40 The X-ray crystallographic data for αβ-tubulin complexed with paclitaxel and epothilone (PDB IDs, 1JFF and 1TVK, respectively) used in the docking studies were retrieved from the Protein Data Bank (PDB). 3D QSAR studies To better understand and explore the electrostatic and steric contributions to the binding of the discodermolide analogs, and to build predictive 3D QSAR models, CoMFA studies were performed based on the two molecular alignments described 43,44 Results and Discussion Chemical and biological data 3D QSAR CoMFA models were derived for a series of 42 discodermolide analogs for which IC 50 values for the inhibition of A549 cell growth were collected 32-37 The IC 50 values vary from 3.7 to 10000 nM, a factor of about 2700. The discodermolide analogs of the data set present structural variations at the C 21 -side chain, C 19 -carbamate group, C 14 -and C 7 -substituents, and in the lactone ring, as depicted in The generation of consistent statistical models depends on the quality of both training and test sets in terms of Vol. 20, No. 4, 2009 structural diversity and property value distributions. From the original data set, 33 compounds (1-33, Molecular alignment strategies The determination of the spatial structural alignments of the discodermolide analogs in the binding pocket of β-tubulin can critically affect the outcome of the CoMFA studies, since the analyses are highly dependent on the quality of the alignments. 3D structural information on the interaction of small molecule modulators with the β-tubulin cavity is limited to crystal structures of the complexes of αβ-tubulin dimers with paclitaxel, docetaxel and epothilone A (PBD IDs JFF, 1TUB and 1TVK, respectively). Taxanes and epothilones have been found to overlap in their occupation of a rather expansive common binding cavity of β-tubulin, exploiting the binding pocket in a unique and different manner. Considering the experimental evidence that indicates that the β-tubulin cavity can accommodate discodermolide and its synthetic analogs, as well as a variety of structurally diverse tubulin modulators, the crystal structures of β-tubulin complexed with paclitaxel (PDB ID 1JFF) and epothilone A (PDB ID 1TVK) were used in the receptor-based molecular docking studies, in such a way to explore the significant conformational flexibility of both receptor cavity and ligands. 3D QSAR models The CoMFA method is based on the assumption that changes in ligand-binding affinities are related to changes in molecular properties represented by 3D molecular fields. Therefore, in order to investigate the structural and chemical features related to the biological activity of this series of discodermolide analogs, the two molecular alignments developed in this work were analyzed using the CoMFA steric and electrostatic fields. Although the q 2 LOO and q 2 LMO procedures may give a suitable representation of the predictive power of the models for untested tubulin modulators, the external validation process can be considered the most valuable validation method. As can be seen, the receptor-based CoMFA model possesses higher predictive ability than the corresponding rigid-body fit model, as indicated by its predictive r 2 value and lower residual values The specialized molecular fields can be analyzed considering two aspects in the CoMFA contour maps. Unfavorable steric regions are represented in yellow and favorable steric regions in green, while red contours represent regions where electronegative substituents may increase the biological activity, and blue contours indicate regions where electropositive groups would contribute to enhance the antimitotic potency. For comparison purposes, the CoMFA electrostatic contour maps for the rigid-body fit and receptor-based models are shown in 25,48 The 3D receptor-based model generated in this study is compatible with the chemical environment of the β-tubulin binding cavity, as shown in Conclusions The understanding of protein-ligand intermolecular interactions is essential for the design of compounds with improved affinity and biological potency.