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53 p.-7 fig.+ 7 p.-1 tab.supl.-6 fig.supl. Saez Calvo, Gonzalo et al., Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells., This work was supported in part by grants BFU2016‐75319‐R (AEI/FEDER, UE) (JFD), BFU2014‐51823‐R (to JMA) from Ministerio de Economia y Competitividad and 31003A_166608 from the Swiss National Science Foundation (M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by nature ‐ from natural products chemistry to drug discovery”. The SAXS experiments were performed at BL11‐NCD beamline at ALBA Synchrotron with the collaboration of ALBA staff. X‐ray data were collected at beamline X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland).

13 páginas, 13 figuras, 3 tablas -- PAGS nros. 7557-7569, A protocol based on a combination of NMR experimental data with molecular mechanics calculations and docking procedures has been employed to determine the microtubule-bound conformation of two microtubule-stabilizing agents, discodermolide (DDM) and dictyostatin (DCT). The data indicate that tubulin in assembled microtubules recognizes DDM through a conformational selection process, with minor changes in the molecular skeleton between the major conformer in water solution and that bound to assembled microtubules. For DCT, the deduced bound geometry presents some key conformation differences around certain torsion angles, with respect to the major conformer in solution, and still displays mobility even when bound. The bound conformer of DCT resembles that of DDM and provides very similar contacts with the receptor. Competition experiments indicate that both molecules compete with the taxane-binding site. A model of the binding mode of DDM and DCT to tubulin is proposed, We thank the Ministery of Education and Science of Spain and Comunidad Autonoma de Madrid for funding (grant nos. CTQ2006-10874-C02-01 to J.J.-B., BIO2007-61336 to J.F.D., and BIPPED S-BIO-0214-2006 to J.M.A., J.F.D., and J.J.-B.).I.P. thanks the Engineering and Physical Sciences Research Council and AstraZeneca (CASE studentship to N.M.G.) for funding

The new arylthioindole (ATI) derivatives 10, 14-18, and 21-24, which bear a halogen atom or a small size ether group at position 5 of the indole moiety, were compared with the reference compounds colchicine and combretastatin A-4 for biological activity. Derivatives 10, 11, 16, and 21-24 inhibited MCF-7 cell growth with IC50 values, This work was supported by Grants BFU2004-00358 from the Dirección General de Investigación Científica y Tecnológica (DGICYT) and CAM200520M061 from Comunidad Autonoma de Madrid. This research was also partially supported by a grant of the Fondazione Banca del Monte di Lombardia (Pavia, Italy) to A.I.S. For S.K. we would like to acknowledge the Embassy of the Arab Republic of Egypt for the award of a PhD scholarship

49 p.-7 tab.- 7 fig., We have found that four taxanes with chemical modifications at positions C10 and C13 were active against all types of taxane resistant cell lines, resistant by p-Gp overexpression, by mutations in the β-tubulin binding site or by overexpression of the highly dynamic βIII-tubulin isotype. We have characterized the interaction of taxanes with high activity on chemotherapy resistant tumoural cells with microtubules, and also studied their cellular effects. The biochemical property enhanced in comparison with other taxanes is their potency at inducing tubulin assembly, despite the fact that their interactions with the microtubule binding sites (pore and luminal) are similar as studied by NMR and SAXS. A differential interaction with the S7-S9 loop (M-loop) is responsible for their enhanced assembly induction properties. The chemical changes in the structure also induce changes in the thermodynamic properties of the interaction, indicating a higher hydrophilicity and also explaining their properties on p-Gp and βIII overexpressing cells and on mutant cells. The effect of the compounds on the microtubular network is different from those observed with the classical (docetaxel and paclitaxel) taxanes, inducing different bundling in cells with microtubules being very short, indicating a very fast nucleation effect and reflecting their high assembly induction power., This research was supported by BIPPED2 (S2010/BMD‐2457) (JFD), and MHit (CAM S2010/BMD-2353 to JJB) projects of the Comunidad de Madrid, the BIO2010‐16351 (to JFD) and CTQ2012-32025 (to JJB) projects from the Ministry of Economy and Competitiveness of Spain, a NSFC grant (No. 30930108) to (WF), the NIH/NCI CA103314 project (to IO) and a Ramon y Cajal grant to Dr. A. Canales.

Ten novel taxanes bearing modifications at the C2 and C13 positions of the baccatin core have been synthesized and their binding affinities for mammalian tubulin have been experimentally measured. The design strategy was guided by (i) calculation of interaction energy maps with carbon, nitrogen and oxygen probes within the taxane-binding site of β-tubulin, and (ii) the prospective use of a structure-based QSAR (COMBINE) model derived from an earlier series comprising 47 congeneric taxanes. The tubulin-binding affinity displayed by one of the new compounds (CTX63) proved to be higher than that of docetaxel, and an updated COMBINE model provided a good correlation between the experimental binding free energies and a set of weighted residue-based ligand-receptor interaction energies for 54 out of the 57 compounds studied. The remaining three outliers from the original training series have in common a large unfavourable entropic contribution to the binding free energy that we attribute to taxane preorganization in aqueous solution in a conformation different from that compatible with tubulin binding. Support for this proposal was obtained from solution NMR experiments and molecular dynamics simulations in explicit water. Our results shed additional light on the determinants of tubulin-binding affinity for this important class of antitumour agents and pave the way for further rational structural modifications. © The Royal Society of Chemistry 2013., National Natural Science Foundation of China (NSFC grant 30930108 to W.S.F.); Spanish Ministerio de Ciencia e Innovación (BIO2008-04384 and BIO-2010-16351); Comisión Interministerial de Ciencia y Tecnología (SAF2009-13914-C02-02), and Comunidad de Madrid (S-BIO/0214/2006 and S2010-BMD-2457)

We have investigated the target and mechanism of action of a new family of cytotoxic small molecules of marine origin. PM050489 and its dechlorinated analogue PM060184 inhibit the growth of relevant cancer cell lines at subnanomolar concentrations. We found that they are highly potent microtubule inhibitors that impair mitosis with a distinct molecular mechanism. They bind with nanomolar affinity to unassembled αβ-tubulin dimers, and PM050489 binding is inhibited by known Vinca domain ligands. NMR TR-NOESY data indicated that a hydroxyl-containing analogue, PM060327, binds in an extended conformation, and STD results define its binding epitopes. Distinctly from vinblastine, these ligands only weakly induce tubulin self-association, in a manner more reminiscent of isohomohalichondrin B than of eribulin. PM050489, possibly acting like a hinge at the association interface between tubulin heterodimers, reshapes Mg2+-induced 42 S tubulin double rings into smaller 19 S single rings made of 7 ± 1 αβ-tubulin dimers. PM060184-resistant mutants of Aspergillus nidulans map to β-tubulin Asn100, suggesting a new binding site different from that of vinblastine at the associating β-tubulin end. Inhibition of assembly dynamics by a few ligand molecules at the microtubule plus end would explain the antitumor activity of these compounds, of which PM060184 is undergoing clinical trials., We wish to thank J. M. Fernandez Sousa (PharmaMar) for useful discussions and support, E. Hamel (NCI) for providing eribulin, C. Scazzocchio and G. Diallinas for useful advice on mutant screening, H. N. Arst for advice on mutant screening and mapping and for kindly providing strains MAD3688 and MAD4655, T. J. Fitzgerald (A&M University) for MTC and C. Alfonso (CIB) for AUC analysis. We also thank Rhône Poulenc Rorer Aventis for supplying docetaxel and Matadero Municipal Vicente de Lucas de Segovia for providing the calf brains for tubulin purification. B.P. had a contract from Comunidad de Madrid, and A.C. had a Ramon y Cajal contract, J.R.-S. had a fellowship from “Programa de Cooperación Científica entre el Ministerio de Ciencia, Tecnologías y Medio Ambiente de la República de Cuba (CITMA) y el CSIC”. This work was supported by grants BIO2010-16351 (J.F.D.), BQU2009-08536 (J.J.-B.), CAM S2010/BMD-2457 (J.F.D.), CAM S2010/BMD-2353 (J.J.-B., J.M.A.), IPT-2011-0752-900000 and BIO2012-30965 (M.A.P.), BFU2011-23416 (J.M.A.) and PharmaMar-CSIC contracts.

13 páginas, 7 figuras, 1 tabla -- PAGS nros. 329-341, Cyclostreptin is the first microtubule-stabilizing agent whose mechanism of action was discovered to involve formation of a covalent bond with tubulin. Treatment of cells with cyclostreptin irreversibly stabilizes their microtubules because cyclostreptin forms a covalent bond to β-tubulin at either the T220 or the N228 residue, located at the microtubule pore or luminal taxoid binding site, respectively. Because of its unique mechanism of action, cyclostreptin overcomes P-glycoprotein-mediated multidrug resistance in tumor cells. We used a series of reactive cyclostreptin analogues, 6-chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and [14C-acetyl]-8-acetyl-cyclostreptin, to characterize the cellular target of the compound and to map the binding site. The three analogues were cytotoxic and stabilized microtubules in both sensitive and multidrug resistant tumor cells. In both types of cells, we identified β-tubulin as the only or the predominantly labeled cellular protein, indicating that covalent binding to microtubules is sufficient to prevent drug efflux mediated by P-glycoprotein. 6-Chloroacetyl-cyclostreptin, 8-chloroacetyl-cyclostreptin, and 8-acetyl-cyclostreptin labeled both microtubules and unassembled tubulin at a single residue of the same tryptic peptide of β-tubulin as was labeled by cyclostreptin (219-LTTPTYGDLNHLVSATMSGVTTCLR-243), but labeling with the analogues occurred at different positions of the peptide. 8-Acetyl-cyclostreptin reacted with either T220 or N228, as did the natural product, while 8-chloroacetyl-cyclostreptin formed a cross-link to C241. Finally, 6-chloroacetyl-cyclostreptin reacted with any of the three residues, thus labeling the pathway for cyclostreptin-like compounds, leading from the pore where these compounds enter the microtubule to the luminal binding pocket, This work was supported in part by Grant BIO2010-16351 from Ministerio de Ciencia e Innovacioń (to J.F.D.) and grant BIPPED2 from Comunidad Autónoma de Madrid. The CNIC is supported by the Ministerio de Ciencia e Innovación and the Fundación Pro CNIC

Three fluorescent probes 3a, 3b, and 4 have been synthesized through conjugation of fluorescein and difluorescein groups to the 7-OH of C-2 modified paclitaxel and cephalomannine derivatives with very high affinity to microtubules. All these probes exhibited potent tubulin assembly promotion and tumor cell killing activities, thus may be useful as tools for the determination of thermodynamic parameters and exploration of ligand-microtubule interactions., This work was supported by NSFC (20572135 and 30630069), MOST grants (2006DFA31490) to W.S.F., and BIO2007-61336 from MEC and BIPPED-CM (S2006/BIO-0214) grants to J.F.D.

14 páginas, 1 figura, 2 tablas, 6 esquemas -- PAGS nros. 10144-10157, A series of epothilone B and D analogues bearing isomeric quinoline or functionalized benzimidazole side chains has been prepared by chemical synthesis in a highly convergent manner. All analogues have been found to interact with the tubulin/microtubule system and to inhibit human cancer cell proliferation in vitro, albeit with different potencies (IC(50) values between 1 and 150 nM). The affinity of quinoline-based epothilone B and D analogues for stabilized microtubules clearly depends on the position of the N-atom in the quinoline system, while the induction of tubulin polymerization in vitro appears to be less sensitive to N-positioning. The potent inhibition of human cancer cell growth by epothilone analogues bearing functionalized benzimidazole side chains suggests that these systems might be conjugated with tumor-targeting moieties to form tumor-targeted prodrugs, We are indebted to the Swiss National Science Foundation (S.A.D.; project numbers 200021-107876 and 205320-117594) and the ETH Zürich for generous financial support. This work was supported in part by grant BIO2007–61336 from MEC and BIPPED-CM from the Comunidad de Madrid to J.F.D.

15 páginas, 1 figura, 1 tabla, 10 esquemas -- PAGS nros. 5060-5074, A total, stereoselective synthesis of the naturally occurring, cytotoxic macrolide FD-891 and of its non-natural (Z)-C12 isomer is described. Three fragments of the main carbon chain were stereoselectively prepared by using asymmetric aldol and allylation reactions as the key steps. The molecule was then assembled by using two Julia–Kocienski olefinations to connect the three fragments and a Yamaguchi reaction to close the macrolactone ring. Some specific biological properties (cytotoxicity, binding to tubulin) have been determined for both macrolides. The E configuration of the C12–C13 olefinic bond seems to be an important feature in determining the cytotoxicity but the precise biological mechanism of the latter still remains to be cleared, Financial support has been granted (to J.A.M. and M.C.) by the Spanish Ministry of Education and Science (projects BQU2002–00468 and CTQ2005–06688-C02–01/02, including financial support by FEDER funds), and by the Consellería d′Empresa, Universitat i Ciencia de la Generalitat Valenciana (projects GV05/52, ACOMP06/006 and ACOMP06/008). J.G.-F. and J. M. thank the Spanish Ministry of Education and Science for a FPU pre-doctoral fellowship and a Ramón y Cajal contract, respectively. Partial support (to J.F.D) by the Spanish Ministry of Education and Science (project BFU2004–00358) and by CAM (BIPPED project) is also acknowledged

In combination with chemical modifications, bioassays, and computational simulation techniques, C-2 benzoylthio, and benzylthio taxoids were synthesized, biologically evaluated, and their binding conformations rationalized, in order to probe the interaction of taxane molecule with β-tubulin., The authors thank FANEDD (Grant No. 199949), NSFC (Grant No. 20572135), and Fok Ying Tong Education Foundation (Grant No. 91037) (WSF), Comunidad Autonoma de Madrid (BIPPED-CM), Ministerio de Educación y Ciencia of Spain (Grant BFU-2004-0038) (JFD) for financial supports. JPS and AAA are grateful to Professor Dennis Liotta (Emory University) for encouragement and support, JFD thanks Prof. J.M. Andreu for useful discussions and support and Matadero Madrid Norte S.A. and Carnicas Madrid-Canarias S.L. for providing the brains for tubulin purification.

Cyclostreptin (1), a natural product from Streptomyces sp. 9885, irreversibly stabilizes cellular microtubules, causes cell cycle arrest, evades drug resistance mediated by P-glycoprotein in a tumor cell line and potently inhibits paclitaxel binding to microtubules, yet it only weakly induces tubulin assembly. In trying to understand this paradox, we observed irreversible binding of synthetic cyclostreptin to tubulin. This results from formation of covalent crosslinks to β-tubulin in cellular microtubules and microtubules formed from purified tubulin in a 1:1 total stoichiometry distributed between Thr220 (at the outer surface of a pore in the microtubule wall) and Asn228 (at the lumenal paclitaxel site). Unpolymerized tubulin was only labeled at Thr220. Thus, the pore region of β-tubulin is an undescribed binding site that (i) elucidates the mechanism by which taxoid-site compounds reach the kinetically unfavorable lumenal site and (ii) explains how taxoid-site drugs induce microtubule formation from dimeric and oligomeric tubulin. © 2007 Nature Publishing Group.