FULL PAPER DOI: 10.1002/chem.200800039 The Bound Conformation of Microtubule-Stabilizing Agents: NMR Insights into the Bioactive 3D Structure of Discodermolide and Dictyostatin** Angeles Canales,[a] Ruth Matesanz,[a] Nicola M. Gardner,[b] JosØ Manuel Andreu,[a] Ian Paterson,[b] J. Fernando Díaz,*[a] and Jesffls JimØnez-Barbero*[a] Abstract: A protocol based on a com- skeleton between the major conformer spect to the major conformer in solu- bination of NMR experimental data in water solution and that bound to as- tion, and still displays mobility even with molecular mechanics calculations sembled microtubules. For DCT, the when bound. The bound conformer of and docking procedures has been em- deduced bound geometry presents DCT resembles that of DDM and pro- ployed to determine the microtubule- some key conformation differences vides very similar contacts with the re- bound conformation of two microtu- around certain torsion angles, with re- ceptor. Competition experiments indi- bule-stabilizing agents, discodermolide cate that both molecules compete with (DDM) and dictyostatin (DCT). The the taxane-binding site. A model of the Keywords: anticancer agents · con- data indicate that tubulin in assembled binding mode of DDM and DCT to tu- formation analysis · microtubules · microtubules recognizes DDM through bulin is proposed. molecular modeling · a conformational selection process, molecular recognition with minor changes in the molecular Introduction essential components of the cytoskeleton, are involved in cell motility, intracellular transport, and maintenance of cell A major challenge of contemporary medicine is in cancer shape, and form part of the mitotic spindle that allows chro- chemotherapy. A cancerous cell is exquisitely well adapted mosome separation during cell division.[3,4] for survival and, although a wide range of drugs targeting In this context, the development of the multidrug-resist- the different cell functions necessary for cell division are ance (MDR) phenotype in paclitaxel-treated patients[5,6] has now available, resistance to chemotherapy is frequently en- countered in the clinic. One of the most commonly used chemotherapeutic agents is paclitaxel and its derivative docetaxel (a semisynthetic an- alogue).[1,2] These compounds target microtubules, which are [a] Dr. A. Canales, R. Matesanz, Prof. Dr. J. M. Andreu, Dr. J. F. Díaz, Prof. Dr. J. JimØnez-Barbero Centro de Investigaciones Biológicas, CSIC Ramiro de Maeztu 9, 28040 Madrid (Spain) Fax : (+34)91-564-4853 E-mail: [email protected] [email protected] [b] N. M. Gardner, Prof. Dr. I. Paterson Department of Chemistry, University of Cambridge Lensfield Road, Cambridge, CB2 1EW (UK) [**] The Bound Conformation of Microtubule-Stabilizing Agents, Part 2; for Part 1 see: J. JimØnez-Barbero, A. Canales, P. T. Northcote, R. M. Buey, J. M. Andreu, J. F. Díaz, J. Am. Chem. Soc. 2006, 128, 8757– 8765. Supporting information for this article is available on the WWW under http://www.chemeurj.org/ or from the author. Chem. Eur. J. 2008, 00, 0 – 0 2008 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim &1& These are not the final page numbers! ÞÞ triggered the search for novel compounds that display the analogues with improved activity. Thus, in this context, we same mechanism of action but are less prone to resistance herein report the NMR investigation of the bioactive con- effects. Indeed, in the last 10 years, many new, structurally formations of both discodermolide and dictyostatin when unrelated compounds that stabilize microtubules and mimic bound to microtubules. We compare the bound conforma- the activity of paclitaxel/docetaxel have been found from tions with their geometries in the free state in different sol- various sources (including corals, marine sponges, bacteria, vents,[33,34] as well as with the conformation of discodermo- and plants).[7–15] In a previous study,[16] these microtubule- lide in the presence of unassembled tubulin reported by Car- stabilizing agents (MSA) were classified according to their lomagno and co-workers.[31] binding site, either the paclitaxel-binding site or the alterna- tive laulimalide-binding site.[14,15] From a structural perspective, the paclitaxel-binding site, Experimental Section located in the lumen of the microtubules,[17] is not easily ac- cessible to the paclitaxel molecules in solution. However, it Protein and chemicals: Purified calf-brain tubulin and chemicals were [35] was also shown that paclitaxel binds very quickly to micro- used as described previously. Discodermolide and dictyostatin were synthesized as reported by Paterson et al.[13,36,37] The compounds were di- tubules[18] and that a fluorescent tag attached to paclitaxel luted in dimethylsulfoxide (DMSO) solution to give a final concentration [19] bound to microtubules can be recognized by an antibody, of 10 mm and stored at À208C. which indicates an additional, and external, accessible bind- Preparation of samples for the NMR experiments: The preparation of ing site. This apparent paradox of a hidden but easily acces- the samples has been described already.[35] However, we repeat the basic sible luminal binding site was elucidated by the discovery of features of the protocol for the sake of clarity. A slowly hydrolyzable nu- [20] cleotide analogue, guanosine 5’-(a,b-methylenetriphosphate) a new external binding site, to which a covalently binding [38] [12,21] (GMPCPP), was employed to assemble tubulin. Guanosine 5’-triphos- MSA, cyclostreptin, binds before being internalized to phate (GTP)-bound tubulin in 10 mm sodium phosphate buffer with m m the luminal site. 6m MgCl2·H2O and 1 m GTP at pH 6.7 was unable to assemble into Although paclitaxel and epothilone are known to bind to microtubules at concentrations up to 200 mm.[32] However, when the GTP the luminal site,[17,22] detailed knowledge of where the other was substituted by GMPCPP and potassium in the buffer, the critical concentration is very low (4.6 mm at 378C). Thus, the protein was equili- paclitaxel mimics bind remains elusive. Since blocking of brated in a buffer comprising 10 mm potassium phosphate, 6 mm magnesi- the external site stops paclitaxel binding,[20] all the known m um chloride, and 0.1 m GMPCPP in D2O (99.9 %, Merck) at pD 7.0 by paclitaxel-binding-site ligands should be in competition. a two-step procedure. Sucrose and GTP were removed by a drained cen- Among the taxoid mimetics, discodermolide (DDM),[10] trifuge column of Sephadex G-25 (6 1 cm) equilibrated in a buffer con- m m originally isolated from the deep-sea sponge Discodermia sisting of 10 m potassium phosphate and 10 m GTP in D2O (99.9%, Merck) at pD 7.3. This was followed by a second chromatography process dissoluta, displays the highest binding constant to the pacli- in a cold Sephadex G-25 column equilibrated in 10 mm potassium phos- [16] m taxel site. It shows potent antiproliferative activity against phate in D2O (99.9%, Merck) at pD 7.3, and then 6 m magnesium chlo- a wide range of human cancer cell lines, including P-glyco- ride and 0.1 mm GMPCPP were added to the solution to give a pD value protein-overexpressing cell lines.[10,23] of 7.0. Immediately before the experiments were performed, the protein concentration was adjusted to 20 mm, the desired amount of target ligand Another potent taxoid mimetic is dictyostatin (DCT), was added to give a final 100–400 mm concentration, and the sample was originally isolated from a marine sponge of the Spongia incubated for 30 minutes at 298 or 310 K (depending on the temperature genus.[24] Its binding constant for the paclitaxel site is 20 of the experiment). The sample was found to be completely stable for times larger than that of paclitaxel itself.[16] From a structur- overnight experiments and only started to slowly degrade after 20 h. Under these conditions, the critical concentration of tubulin is 1.5 mm at al perspective, there are strong similarities between disco- m [25] 298 K and 0.9 m at 310 K. This means that either 92.5 or 95.5% of the dermolide and dictyostatin, which suggests that their bio- sample (at 298 or 310 K, respectively) is assembled into microtubules. A active conformations may be similar. Indeed, a hybrid mole- portion of the formed polymers was adsorbed onto formvar/carbon- cule has recently been designed on these grounds.[26] coated 300 mesh copper grids, negatively stained with 1 % uranyl acetate, Despite apparently binding to the same site, the existence observed with a Jeol 1230 transmission electron microscope (JEOL, Tokyo, Japan), and found to consist of microtubules. of synergy in the effects of paclitaxel and discodermolide on Computational methods cells and microtubules has been reported.[27–30] These obser- Conformational searching and dynamics: Molecular mechanics calcula- vations could indicate binding to an additional site, such as tions on DDM and DCT were performed by using the Maestro 7.5 pack- the recently discovered pore site present in unassembled tu- age[39] and the MM3* force field.[40] Bulk-water solvation was simulated bulin, although this is merely speculative. by using the generalized Born/surface area (GB/SA) continuum solvent In elegant recent work by Carlomagno and co-workers,[31] model.[41] The conformational searches were carried out with 20000 steps the unassembled-tubulin-bound conformation of discoder- of the usage-directed Monte Carlo/energy minimization (MC/EM) proce- dure. Extended nonbonded cutoff distances (a van der Waals cutoff of molide was reported. Because high-affinity taxane binding 8.0 and an electrostatic cutoff of 20.0 ) were used. does not take place in unassembled tubulin but in assembled For the Monte Carlo/stochastic dynamics (MC/SD) simulations, van der [20,32] microtubules, it is uncertain whether the tubulin-bound Waals and electrostatic cutoffs of 25 , together with a hydrogen-bond conformation described therein[31] represents the conforma- cutoff of 15 , were used.