Structural Determinants of the Dictyostatin Chemotype for Tubulin Binding Affinity and Antitumor Activity Against Taxane- and Epothilone-Resistant Cancer Cells

A combined biochemical, structural, and cell biology characterization of dictyostatin is described, which enables an improved understanding of the structural determinants responsible for the high-affinity binding of this anticancer agent to the taxane site in microtubules (MTs). The study reveals that this macrolide is highly optimized for MT binding and that only a few of the structural modifications featured in a library of synthetic analogues resulted in small gains in binding affinity. The high efficiency of the dictyostatin chemotype in overcoming various kinds of clinically relevant resistance mechanisms highlights its potential for therapeutic development for the treatment of drug-resistant tumors. A structural explanation is advanced to account for the synergy observed between dictyostatin and taxanes on the basis of their differential effects on the MT lattice. The X-ray crystal structure of a tubulin−dictyostatin complex and additional molecular modeling have allowed the rationalization of the structure−activity relationships for a set of synthetic dictyostatin analogues, including the highly active hybrid 12 with discodermolide. Altogether, the work reported here is anticipated to facilitate the improved design and synthesis of more efficacious dictyostatin analogues and hybrids with other MT-stabilizing agents.We thank Peter T. Northcote for peloruside A, W.-S. Fang for Flutax-2, K. H. Altmann for epothilone D, Dr. Paraskevi Giannakakou (Weill Cornell Medical Center, New York) for the 1A9, PTX10, PTX22, and A8 cell lines, and Prof. Richard Ludueñ a (University of Texas) for the HeLa βIII-transfected cells. We thank Matadero INCOVA (Segovia) for the calf brains for tubulin purification. This work was supported in part by grants BIO2013-42984-R (J.F.D.) and SAF2012-39760-C02-02 (F.G.) from Ministerio de Economia y Competitividad, grant S2010/ ́ BMD-2457 BIPEDD2 from Comunidad Autonoma de Madrid ́ (F.G. and J.F.D.), and the Swiss National Science Foundation grants 310030B_138659 and 31003A_166608 (M.O.S.). The authors acknowledge networking contribution by the COST Action CM1407 “Challenging organic syntheses inspired by naturefrom natural products chemistry to drug discovery” and the COST action CM1470. I.P. thanks the EPSRC and AstraZeneca for funding, Dr. John Leonard (AstraZeneca) for useful discussions, Dr. Stuart Mickel (Novartis) for the provision of chemicals, and the EPSRC UK National Mass Spectrometry Facility at Swansea University for mass spectra

Structural Determinants of the Dictyostatin Chemotype for Tubulin Binding Affinity and Antitumor Activity Against Taxane- and Epothilone-Resistant Cancer Cells

A combined biochemical, structural, and cell biology characterization of dictyostatin is described, which enables an improved understanding of the structural determinants responsible for the high-affinity binding of this anticancer agent to the taxane site in microtubules (MTs). The study reveals that this macrolide is highly optimized for MT binding and that only a few of the structural modifications featured in a library of synthetic analogues resulted in small gains in binding affinity. The high efficiency of the dictyostatin chemotype in overcoming various kinds of clinically relevant resistance mechanisms highlights its potential for therapeutic development for the treatment of drug-resistant tumors. A structural explanation is advanced to account for the synergy observed between dictyostatin and taxanes on the basis of their differential effects on the MT lattice. The X-ray crystal structure of a tubulin−dictyostatin complex and additional molecular modeling have allowed the rationalization of the structure−activity relationships for a set of synthetic dictyostatin analogues, including the highly active hybrid 12 with discodermolide. Altogether, the work reported here is anticipated to facilitate the improved design and synthesis of more efficacious dictyostatin analogues and hybrids with other MT-stabilizing agents.We thank Peter T. Northcote for peloruside A, W.-S. Fang for Flutax-2, K. H. Altmann for epothilone D, Dr. Paraskevi Giannakakou (Weill Cornell Medical Center, New York) for the 1A9, PTX10, PTX22, and A8 cell lines, and Prof. Richard Ludueñ a (University of Texas) for the HeLa βIII-transfected cells. We thank Matadero INCOVA (Segovia) for the calf brains for tubulin purification. This work was supported in part by grants BIO2013-42984-R (J.F.D.) and SAF2012-39760-C02-02 (F.G.) from Ministerio de Economia y Competitividad, grant S2010/ ́ BMD-2457 BIPEDD2 from Comunidad Autonoma de Madrid ́ (F.G. and J.F.D.), and the Swiss National Science Foundation grants 310030B_138659 and 31003A_166608 (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” and the COST action CM1470. I.P. thanks the EPSRC and AstraZeneca for funding, Dr. John Leonard (AstraZeneca) for useful discussions, Dr. Stuart Mickel (Novartis) for the provision of chemicals, and the EPSRC UK National Mass Spectrometry Facility at Swansea University for mass spectra

Probing the Pore Drug Binding Site ofMicrotubules with Fluorescent Taxanes:Evidence of Two Binding Poses

The pore site in microtubules has been studied with the use of Hexaflutax, a fluorescent probe derived from paclitaxel. The compound is active in cells with similar effects to paclitaxel, indicating that the pore may be a target to microtubule stabilizing agents. While other taxanes bind microtubules in a monophasic way, thus indicating a single type of sites, Hexaflutax association is biphasic. Analysis of the phases indicates that two different binding sites are detected, reflecting two different modes of binding, which could arise from different arrangements of the taxane or fluorescein moieties in the pore. Association of the 4-4-20 antifluorescein monoclonal antibody-Hexaflutax complex to microtubules remains biphasic, thus indicating that the two phases observed arise from two different poses of the taxane moiety

Structure-activity relationships of novel substituted naphthalene diimides as anticancer agents

Novel 1,4,5,8-naphthalenetetracarboxylic diimide (NDI) derivatives were synthesized and evaluated for their antiproliferative activity on a wide number of different tumor cell lines. The prototypes of the present series were derivatives 1 and 2 characterized by interesting biological profiles as anticancer agents. The present investigation expands on the study of structureeactivity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3e22, characterized by a different substituent on the aromatic rings and/or a different chain length varying from two to three carbon units, were synthesized and evaluated for their cytostatic and cytotoxic activities. The most interesting compound was 20, characterized by a linker of three methylene units and a 2,3,4-trimethoxy substituent on the two aromatic rings. It displayed antiproliferative activity in the submicromolar range, especially against some different cell lines, the ability to inhibit Taq polymerase and telomerase, to trigger caspase activation by a possible oxidative mechanism, to downregulate ERK 2 protein and to inhibit ERKs phosphorylation, without acting directly on microtubules and tubuline. Its theoretical recognition against duplex and quadruplex DNA structures have been compared to experimental thermodynamic measurements and by molecular modeling investigation leading to putative binding modes. Taken together these findings contribute to define this compound as potential Multitarget-Directed Ligands interacting simultaneously with different biological targets

Insights into the interaction of discodermolide and docetaxel with tubulin. Mapping the binding sites of microtubule-stabilizing agents by using an integrated NMR and computational approach

41 p.-7 fig.-3 fig.-supl.The binding interactions of two antitumor agents that target the paclitaxel site, docetaxel and discodermolide, to unassembled α/β-tubulin heterodimers and microtubules have been studied using biochemical and NMR techniques. The use of discodermolide as a water-soluble paclitaxel biomimetic and extensive NMR experiments allowed the detection of binding of microtubule-stabilizing agents to unassembled tubulin α/β-heterodimers. The bioactive 3D structures of docetaxel and discodermolide bound to α/β-heterodimers were elucidated and compared to those bound to microtubules, where subtle changes in the conformations of docetaxel in its different bound states were evident. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that docetaxel and discodermolide target an additional binding site at the pore of the microtubules, which is different from the internal binding site at the lumen previously determined by electron crystallography. Binding to this pore site can then be considered as the first ligand-protein recognition event that takes place in advance of the drug internalization process and interaction with the lumen of the microtubulesThis work was supported in part by grant BIO2010-16351 and BQU2009-08536 from MICINN (to J.F.D. and J.J.B., respectively), BIPPED-CM from Comunidad de Madrid (J.F.D., J.J.B., and J.M.A.), and EPSRC (I.P.). We also thank CESGA and the CAI NMR of Universidad Complutense for providing access to their facilitiesPeer reviewe