Mecanismo de accion de los macrolidos citotoxicos amphinolidas X y J

Trabajo presentado en el XI Congreso de la Sociedad de Biofísica de España: "XXV años y más allá", celebrado en Murcia (España) , del 1 al 4 de junio de 201

PM060184, a new tubulin binding agent with potent antitumor activity including P-glycoprotein over-expressing tumors

PM060184 belongs to a new family of tubulin-binding agents originally isolated from the marine sponge Lithoplocamia lithistoides. This compound is currently produced by total synthesis and is under evaluation in clinical studies in patients with advanced cancer diseases. It was recently published that PM060184 presents the highest known affinities among tubulin-binding agents, and that it targets tubulin dimers at a new binding site. Here, we show that PM060184 has a potent antitumor activity in a panel of different tumor xenograft models. Moreover, PM060184 is able to overcome P-gp mediated resistance in vivo, an effect that could be related to its high binding affinity for tubulin. To gain insight into the mechanism responsible of the observed antitumor activity, we have characterized its molecular and cellular effects. We have observed that PM060184 is an inhibitor of tubulin polymerization that reduces microtubule dynamicity in cells by 59%. Interestingly, PM060184 suppresses microtubule shortening and growing at a similar extent. This action affects cells in interphase and mitosis. In the first case, the compound induces a disorganization and fragmentation of the microtubule network and the inhibition of cell migration. In the second case, it induces the appearance of multipolar mitosis and lagging chromosomes at the metaphase plate. These effects correlate with prometaphase arrest and induction of caspase-dependent apoptosis or appearance of cells in a multinucleated interphase-like state unrelated to classical apoptosis pathways. Taken together, these results indicate that PM060184 represents a new tubulin binding agent with promising potential as an anticancer agent.This work was supported by grants BIO2010-16351 (JFD), CAM S2010/BMD-2457 (JFD), CAM S2010/BMD-2353 (JMA), BFU2011-23416 (JMA) and PharmaMar-CSIC contracts. BP had a contract from Comunidad de Madrid

New interfacial microtubule inhibitors of marine origin, PM050489/PM060184, with potent antitumor activity and a distinct mechanism

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

Zampanolida, un novedoso agente estabilizador de microtubulos quew se une covalentemente al sitio de paclitaxel

Trabajo presentado en el XI Congreso de la Sociedad de Biofísica de España: "XXV años y más allá", celebrado en Murcia (España) , del 1 al 4 de junio de 201

Corrigendum: Mechanism of Action of the Cytotoxic Macrolides Amphidinolide X and J

Peer reviewe

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 structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, 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. \ua9 2012 Elsevier Masson SAS. All rights reserved

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 structure-activity relationships of prototypes 1 and 2, namely, the influence of the different substituents of the phenyl rings on the biological activity. Derivatives 3-22, 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.This research was supported by a grant from MIUR, Rome (PRIN), University of Bologna (RFO) and Polo Scientifico-Didattico di Rimini (to V.T.). We thank the National Cancer Institute (Bethesda, MD) for the anticancer assays. MICINN (Spanish Government) is acknowledged for grant BIO2010-16351 (to J.F.D.). Lizzia Raffaghello is a recipient of MFAG Grant. Giovanna Bianchi is a recipient of a FIRC fellowship

Thermodynamics as a Tool for the Optimization of Drug Binding, Thermodynamics - Interaction Studies - Solids, Liquids and Gases

20 páginas, 5 figuras, 2 tablas -- PAGS nros. 777-796A non-covalent interaction is a kind of chemical bond, typically between macromolecules, that involves dispersed variations of electromagnetic interactions (Alberts et al. 1994; Connors & Mecozzi 2010). Non-covalent interactions are individually weak as compared with covalent bonds, but their net strength is higher than the sum of that of the individual interactions. There are few drugs that bind irreversibly to their targets, in pharmacology, most drugs establish non-covalent interactions with their target molecules (usually proteins). From a chemical point of view, the affinity constant (Ka) is a very useful measurement for the study of binding reactions as it provides much information about the mechanism. In many cases some chemical or physical properties of ligand or target change with the interaction between them, these changes might help to measure binding constants. It is important to establish the stoichiometry of the complex to be sure that the constants are accurately calculated. From the affinity constants measured it is possible to calculate the standard thermodynamic quantities for the binding reaction: free-energy (ΔG), enthalpy (ΔH) and entropy (ΔS). Our group has already demonstrated that, in some cases, binding affinity measurements are very helpful for the optimization of ligand binding as it can be determined the contribution of every single chemical modification of the ligand to the binding affinity (Buey et al. 2004; Matesanz et al. 2008) One of the objectives of drug development is the search of new or modified compounds with improved properties such as better potency, higher selectivity, better pharmacokinetics or superior drug resistance profiles. An important goal in this objective is the optimization of drugs binding affinity towards their targets, as binding affinity is directly related to potency (Ruben et al. 2006). Moreover, it has been shown that extremely high affinity drugs reflect as well changes in other properties like selectivity (Ohtaka et al. 2004; Ohtaka & Freire 2005) or resistance overcoming ability (Matesanz et al. 2008). Examples of the importance of ligand affinity in drug optimization can be observed in the development of HIV-1 protease inhibitors and statins (cholesterol lowering drugs) over the years as remarked in (Freire 2008). In this chapter we will study the nature of non-covalent interations and the concept of binding constant for these interactions. Examples of methodologies to measure binding constants of small ligands to macromolecules will be introduced and we will emphasize theneed to determine the stoichiometry of the studied system to calculate accurately the constants. Once the thermodynamic concepts were introduced, we will show the use of these kind of studies for the optimization of drug binding to its target. We will detail the role of single chemical modifications in the molecule of study to modulate its binding affinity, and the way to quantify these changes. We will finally further discuss how the selection of the best sustituents can result in the optimization of bindinPeer reviewe

Taxanes with high potency inducing tubulin assembly overcome tumoural cell resistances

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.Peer reviewe