Estudio y caracterización del mecanismo de acción bioquímico y celular de compuestos antitumorales dirigidos contra el citoesqueleto

Abstract

Introduction Microtubules and actin filaments play important biological functions in connection with mitosis, cytokinesis, cell signaling, intracellular transport, and cell motility of eukaryotic cells (1, 2). Any external element that disrupts the correct functioning of tubulin and actin causes harmful effects to the body. Thus, it is reasonable that a majority of pharmacological research has focused on reactive products against proteins, many of which are natural compounds or analogues. There are several clinical drugs based on the stabilization (paclitaxel-like behavior) or destabilization (either vinca-like or colchicine-like behavior) of microtubules with regard to their heterodimeric component, α,β- tubulin (3). On the other hand, none actin-targeting drug has entered yet in clinical studies. Research content and conclusions This work takes a comprehensive approach to structure/function/ mechanistic interaction of different molecules which targets the cytoskeleton. Different compounds received were classified into three groups of different families according to their mechanism of action: microtubule stabilizing agents (dictyostatin and discodermolide family; peloruside A family), microtubule destabilizing compounds (pironetin family; podophyllotoxin family) and ligands acting at the level of actin filaments (amphidinolides family). Given the interest, we have investigated the influence of chemical modifications in the binding affinity of the compounds for tubulin and actin. We employed biochemical and biophysical techniques to determine binding constants and stoichiometries as well as NMR techniques to determine the binding epitopes and bioactive conformations of the compounds. Also we used cell biology techniques to evaluate their effects on a panel of cells both sensitive to chemotherapy and resistant to it through three different mechanisms: overexpression of P-glycoprotein, overexpression of βIII-tubulin isotypes and mutations in the β-tubulin gene.

Dictyostatin and discodermolide family. Since gaining FDA approval in 1992, paclitaxel and subsequently its semisynthetic analogue taxotere (docetaxel) have been widely used in clinical studies in regards to oncology treatments, including breast, ovarian, and lung cancers (4-7). Although the cytotoxic drugs of taxanes have great utility as chemotherapeutic agents, they suffer from low aqueous solubility and tend to induce drug resistance in patients (8), further underlining the continued need for the identification of new microtubule stabilizing agents. This need has led to the search for structurally novel natural product scaffolds that share the same mode of action as the taxanes but are more effective to overcome drug resistance. The naturally occurring compounds discodermolide (9) and dictyostatin (10) bind to microtubules, cause cell cycle arrest in G2/M at nanomolar concentrations, and retain antiproliferative activity in paclitaxel-resistant cell lines (11, 12), making these compounds attractive candidates for development as antineoplastic agents. In this study, we examined a series of dictyostatin analogs and discodermolide/dictyostatina hybrids to probe biological and biochemical structure-activity relationships. A set of ten derivatives of dictyostatin and five hybrids of discodermolide and dictyostatin have been evaluated to determine the structural features required to improve the interaction of these compounds with microtubules. The activity of the agents was evaluated against drug-sensitive and drugresistant cancer cell lines. The compounds studied are cytotoxic and less susceptible than paclitaxel to multidrug resistance arising from overexpression of the P-glycoprotein efflux pump, exhibit remarkable potency against cell lines which have the isotype of βIII-tubulin and are not affected by mutations that affect the taxoid binding site of β-tubulin. In cell proliferation assay, statistically significant synergy was found between some compounds investigated and paclitaxel, and between some of these compounds and the peloruside A. These results confirm that the discodermolide/dictiostatina family's compounds, when added in combination with other microtubule stabilizing agents, act synergistically to enhance the antimitotic action of the drugs, but also highlight the complexity of drug interactions in intact cells. In vitro studies with purified tubulin indicate that these agents bind to the paclitaxel site (the binding is exothermic) and directly induce tubulin polymerization. Removal of the methyl group of dictyostatin at position 6, hydrogenation of the (Z)-alkene between positions 2 and 3, substitution of the hydroxyl at position 9 with a methoxy group and addiction of a double bound between positions 4 and 5 resulted in significant enhancements of dictyostatin and discodermolide’s binding affinity for the site and citotoxicity.

The binding interaction of discodermolide to unassembled α/β-tubulin heterodimers and microtubules has 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 structure of discodermolide bound to α/β-heterodimers was elucidated and compared to those bound to microtubules. Moreover, the combination of experimental TR-NOE and STD NMR data with CORCEMA-ST calculations indicate that discodermolide targets 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 microtubules (13). Peloruside A family. Peloruside A (14) is a microtubule-stabilizing agent that targets the same site as laulimalide. It binds to microtubules with a 1:1 stoichiometry and with a binding affinity in the low-nM range (15, 16). It reduces the number of microtubular protofilaments in the same way as paclitaxel. Although the binding affinity of the compound is comparable to that of the low-affinity stabilizing agent sarcodictyin (17), peloruside is more active in inducing microtubule assembly and is more cytotoxic to tumor cells; this suggests that the peloruside site is a more effective site for stabilizing microtubules. Acetylation of the C24 hydroxyl group results in inactive compounds. Using data-directed molecular docking simulations, we confirm that peloruside A binds within a pocket on the exterior of β-tubulin at a previously unknown ligand site (18), rather than on α-tubulin as suggested in earlier studies (19).

Pironetin family. The action mechanism of a series of pironetin analogues with simplified structure is described in this work. Their cytotoxic activity and their interactions with tubulin have been investigated. It has been found that, while less active than the parent molecule (20-22), the pironetin analogues still share the mechanism of action of the latter and compete for the same binding site to α- tubulin. Variations in the configurations of their stereocenters do not translate into relevant differences between biological activities (23). Podophyllotoxins family. Several pinacol derivatives of podophyllotoxins bearing different side chains and functions at C7 were synthesized through reductive cross-coupling of podophyllotoxone and several aldehydes and ketones. While possessing a hydroxylated chain at C7, the compounds retained their respective hydroxyl group with either the 7α (podo) or 7β (epipodo) configuration. Cytotoxicities against neoplastic cells followed by cell cycle arrest and cellular microtubule disruption were evaluated and mechanistically characterized through tubulin polymerization inhibition and assays of binding to the colchicine site. Compounds of the epipodopinacol (7β-OH) series behaved similarly to podophyllotoxin in all the assays and proved to be the most potent inhibitors. Significantly, 7α-isopropyl-7-deoxypodophyllotoxin (41), without any hydroxyl function, appeared as a promising lead compound for a novel type of tubulin polymerization inhibitors. Experimental results were in overall agreement with modeling and docking studies performed on representative compounds of each series (24).

Amphidinolides family. Biological studies of four small amphidinolides of the amphidinolide family have been reported. Their effect on the proliferation of A2780 (human ovarian carcinoma) and of LoVo (human colon carcinoma) cell lines, as well as on the cytoskeleton proteins tubulin, actin, and intermediate filaments of A549 lung carcinoma cells and PtK2 cells have been investigated. Their effect on actin polymerization was then studied in vitro. The findings indicate that the actin filament cytoskeleton rather than the microtubule cytoskeleton is the biological target of the compounds. We believe that this may also be the case for other small macrolides of the amphidinolide family. The influence of the ligand on the actin assembly in vitro is consistent with the actin disassembly effect observed in treated cells. As the ligands interacted with the disassembled actin (G-actin) in vitro, but they did not show any effect on the polymerized actin (F-actin), we suggest that their mechanism of action involves the inhibition of actin monomer additions to pre-existing filaments. Such an inhibition is similar to, albeit weaker than, that induced by cytochalasin B. Docking calculations of compounds indicate that they may emulate cytochalasin D and B in the inhibition of actin assembly (25). References: (1) Chen, H., Bernstein, B.W., and Bamburg, J.R. (2000). Regulating actin-filament dynamics in vivo. Trends Biochem. Sci 25(1), 19–23. (2) Desai, A., and Mitchison, T.J. (1997). Microtubule polymerization dynamics. Annu Rev Cell Dev Biol 13, 83-117. (3) Perez, E.A. (2009). Microtubule inhibitors: Differentiating tubulininhibiting agents based on mechanisms of action, clinical activity, and resistance. Mol. Cancer Ther 8, 2086–2095. (4) Jordan, M.A., and Wilson, L. (2004). Microtubules as a target for anticancer drugs. Nat Rev Cancer 4, 253-265. (5) Montero, A., Fossella, F., Hortobagyi, G., and Valero, V. (2005). Docetaxel for treatment of solid tumours: a systematic review of clinical data. Lancet Oncol 6, 229-239.

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