RESUMEN
Paclitaxel (Taxol) is a taxane and a chemotherapeutic drug that stabilizes microtubules. While the interaction of paclitaxel with microtubules is well described, the lack of high-resolution structural information on a tubulin-taxane complex precludes a comprehensive description of the binding determinants that affect its mechanism of action. Here, we solved the crystal structure of baccatin III the core moiety of paclitaxel-tubulin complex at 1.9 Å resolution. Based on this information, we engineered taxanes with modified C13 side chains, solved their crystal structures in complex with tubulin, and analyzed their effects on microtubules (X-ray fiber diffraction), along with those of paclitaxel, docetaxel, and baccatin III. Further comparison of high-resolution structures and microtubules' diffractions with the apo forms and molecular dynamics approaches allowed us to understand the consequences of taxane binding to tubulin in solution and under assembled conditions. The results sheds light on three main mechanistic questions: (1) taxanes bind better to microtubules than to tubulin because tubulin assembly is linked to a ßM-loopconformational reorganization (otherwise occludes the access to the taxane site) and, bulky C13 side chains preferentially recognize the assembled conformational state; (2) the occupancy of the taxane site has no influence on the straightness of tubulin protofilaments and; (3) longitudinal expansion of the microtubule lattices arises from the accommodation of the taxane core within the site, a process that is no related to the microtubule stabilization (baccatin III is biochemically inactive). In conclusion, our combined experimental and computational approach allowed us to describe the tubulin-taxane interaction in atomic detail and assess the structural determinants for binding.
Asunto(s)
Taxoides , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Taxoides/farmacología , Taxoides/química , Taxoides/metabolismo , Microtúbulos/metabolismo , Paclitaxel/farmacología , Paclitaxel/químicaRESUMEN
Taxanes are microtubule-stabilizing agents used in the treatment of many solid tumors, but they often involve side effects affecting the peripheral nervous system. It has been proposed that this could be related to structural modifications on the filament upon drug binding. Alternatively, laulimalide and peloruside bind to a different site also inducing stabilization, but they have not been exploited in clinics. Here, we use a combination of the parental natural compounds and derived analogs to unravel the stabilization mechanism through this site. These drugs settle lateral interactions without engaging the M loop, which is part of the key and lock involved in the inter-protofilament contacts. Importantly, these drugs can modulate the angle between protofilaments, producing microtubules of different diameters. Among the compounds studied, we have found some showing low cytotoxicity and able to induce stabilization without compromising microtubule native structure. This opens the window of new applications for microtubule-stabilizing agents beyond cancer treatment.
Asunto(s)
Lactonas , Tubulina (Proteína) , Lactonas/farmacología , Tubulina (Proteína)/metabolismo , Excipientes/análisis , Excipientes/metabolismo , Sitios de Unión , Microtúbulos/metabolismoRESUMEN
Microtubules are dynamic cytoskeletal polymers that spontaneously switch between phases of growth and shrinkage. The probability of transitioning from growth to shrinkage, termed catastrophe, increases with microtubule age, but the underlying mechanisms are poorly understood. Here, we set out to test whether microtubule lattice defects formed during polymerization can affect growth at the plus end. To generate microtubules with lattice defects, we used microtubule-stabilizing agents that promote formation of polymers with different protofilament numbers. By employing different agents during nucleation of stable microtubule seeds and the subsequent polymerization phase, we could reproducibly induce switches in protofilament number and induce stable lattice defects. Such drug-induced defects led to frequent catastrophes, which were not observed when microtubules were grown in the same conditions but without a protofilament number mismatch. Microtubule severing at the site of the defect was sufficient to suppress catastrophes. We conclude that structural defects within the microtubule lattice can exert effects that can propagate over long distances and affect the dynamic state of the microtubule end.
Asunto(s)
Microtúbulos/metabolismo , Moduladores de Tubulina/metabolismo , Fenómenos Biológicos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/ultraestructura , Paclitaxel/metabolismo , Polimerizacion , Unión Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/químicaRESUMEN
Microtubules (MTs) are hollow cylinders made of tubulin, a GTPase responsible for essential functions during cell growth and division, and thus, key target for anti-tumor drugs. In MTs, GTP hydrolysis triggers structural changes in the lattice, which are responsible for interaction with regulatory factors. The stabilizing GTP-cap is a hallmark of MTs and the mechanism of the chemical-structural link between the GTP hydrolysis site and the MT lattice is a matter of debate. We have analyzed the structure of tubulin and MTs assembled in the presence of fluoride salts that mimic the GTP-bound and GDPâ¢Pi transition states. Our results challenge current models because tubulin does not change axial length upon GTP hydrolysis. Moreover, analysis of the structure of MTs assembled in the presence of several nucleotide analogues and of taxol allows us to propose that previously described lattice expansion could be a post-hydrolysis stage involved in Pi release.
Asunto(s)
Microtúbulos/química , Modelos Moleculares , Conformación Molecular , Microscopía por Crioelectrón , Guanosina Difosfato/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/química , Enlace de Hidrógeno , Microtúbulos/metabolismo , Relación Estructura-Actividad , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismoRESUMEN
Microtubules are polymers of tubulin dimers, and conformational transitions in the microtubule lattice drive microtubule dynamic instability and affect various aspects of microtubule function. The exact nature of these transitions and their modulation by anticancer drugs such as Taxol and epothilone, which can stabilize microtubules but also perturb their growth, are poorly understood. Here, we directly visualize the action of fluorescent Taxol and epothilone derivatives and show that microtubules can transition to a state that triggers cooperative drug binding to form regions with altered lattice conformation. Such regions emerge at growing microtubule ends that are in a pre-catastrophe state, and inhibit microtubule growth and shortening. Electron microscopy and in vitro dynamics data indicate that taxane accumulation zones represent incomplete tubes that can persist, incorporate tubulin dimers and repeatedly induce microtubule rescues. Thus, taxanes modulate the material properties of microtubules by converting destabilized growing microtubule ends into regions resistant to depolymerization.
Asunto(s)
Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Taxoides/farmacología , Células HeLa , Humanos , Cinética , Tubulina (Proteína)/metabolismoRESUMEN
Microtubule targeting agents represent a very active arena in the development of anticancer agents. In particular, compounds binding at the colchicine site in tubulin are being deeply studied, and the structural information recently available on this binding site allows structure-directed design of new ligands. Structural comparison of our recently reported high resolution X-Ray structure of the cyclohexanedione derivative TUB075 bound to tubulin and the tubulin-DAMA-colchicine complex has revealed a conformational change in the αT5 loop. By a grid-based computational analysis of the tubulin-DAMA-colchicine binding site, we have identified a new favourable binding area in the colchicine-site that was unexplored by our lead TUB075. Thus, based on a structure-guided design, new cyclohexanedione derivatives have been synthesized and tested for tubulin binding and in cellular assays. As a result, we have identified diphenyl ether derivatives with IC50 values around 10-40â¯nM against three different tumor cell lines and affinity constants for tubulin similar to that of colchicine around 107â¯M-1. As expected, they halted the cell cycle progression at G2/M phase at concentrations as low as 0.08⯵M.
Asunto(s)
Antineoplásicos/farmacología , Colchicina/farmacología , Ciclohexanonas/farmacología , Éteres Fenílicos/farmacología , Tubulina (Proteína)/metabolismo , Antineoplásicos/síntesis química , Antineoplásicos/química , Sitios de Unión/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Colchicina/química , Cristalografía por Rayos X , Ciclohexanonas/química , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Modelos Moleculares , Estructura Molecular , Éteres Fenílicos/síntesis química , Éteres Fenílicos/química , Relación Estructura-ActividadRESUMEN
It has been proposed that one of the mechanisms of taxane-site ligand-mediated tubulin activation is modulation of the structure of a switch element (the M-loop) from a disordered form in dimeric tubulin to a folded helical structure in microtubules. Here, we used covalent taxane-site ligands, including cyclostreptin, to gain further insight into this mechanism. The crystal structure of cyclostreptin-bound tubulin reveals covalent binding to ßHis229, but no stabilization of the M-loop. The capacity of cyclostreptin to induce microtubule assembly compared to other covalent taxane-site agents demonstrates that the induction of tubulin assembly is not strictly dependent on M-loop stabilization. We further demonstrate that most covalent taxane-site ligands are able to partially overcome drug resistance mediated by ßIII-tubulin (ßIII) overexpression in HeLa cells, and compare their activities to pironetin, an interfacial covalent inhibitor of tubulin assembly that displays invariant growth inhibition in these cells. Our findings suggest a relationship between a diminished interaction of taxane-site ligands with ßIII-tubulin and ßIII tubulin-mediated drug resistance. This supports the idea that overexpression of ßIII increases microtubule dynamicity by counteracting the enhanced microtubule stability promoted by covalent taxane-site binding ligands.
Asunto(s)
Microtúbulos/química , Compuestos Policíclicos/química , Tubulina (Proteína)/química , Resistencia a Antineoplásicos , Ácido Edético/química , Células HeLa , Humanos , Espectrometría de Masas , Taxoides/químicaRESUMEN
Microtubule-targeting agents that bind at the colchicine-site of tubulin are of particular interest in antitumoral therapy due to their dual mechanism of action as antimitotics and vascular disrupting agents. Cyclohexanediones derivatives have been described as a new family of colchicine-domain binders with an association constant to tubulin similar to that of colchicine. Here, the high-resolution structures of tubulin in complex with cyclohexanediones TUB015 and TUB075 were solved by X-ray crystallography. A detailed analysis of the tubulin-TUB075 interaction by means of computational affinity maps allowed the identification of two additional regions at the binding site that were addressed with the design and synthesis of a new series of cyclohexanediones with a distal 2-substituted benzofurane. These new compounds showed potent antiproliferative activity with IC50 values in the nM range, arrested cell cycle progression at the G2/M phase and induced apoptosis at sub µM concentrations. Moreover, they caused the destruction of a preformed vascular network in vitro and inhibited the migration of endothelial cells at non-toxic concentrations. Finally, these compounds displayed high affinity for tubulin as substantiated by a K b value of 2.87 × 108 M-1 which, to the best of our knowledge, represents the highest binding constant measured to date for a colchicine-domain ligand.
Asunto(s)
Ciclohexanonas/síntesis química , Moduladores de Tubulina/síntesis química , Tubulina (Proteína)/química , Sitios de Unión , Línea Celular , Línea Celular Tumoral , Movimiento Celular , Colchicina/química , Colchicina/farmacología , Ciclohexanonas/farmacología , Células Endoteliales/efectos de los fármacos , Células Endoteliales/fisiología , Humanos , Ligandos , Simulación del Acoplamiento Molecular , Unión Proteica , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
Based on the conformation of the α-methyl chalcone TUB091 in its complex with tubulin, a series of conformational mimetics have been designed and synthesized where the methyl group of the chalcone has been fused to phenyl ring B resulting in 1,2,3,4-tetrahydronaphthalen-2-yl aryl ketones. Among the synthesized compounds, the 5-amino-6-methoxy derivative, with a similar substitution pattern to that of TUB091, showed antiproliferative activity around 20â¯nM against tumor and endothelial cells. Tubulin binding experiments confirmed its binding to tubulin at the colchicine site with a Kb of 2.4â¯×â¯106â¯M-1 resulting in the inhibition of the in vitro assembly of purified tubulin. Moreover, based on the recently reported complex of combretastatin A4 (CA4) with tubulin, a comparative analysis of the binding mode of CA4 and the α-methyl chalcone to tubulin has been performed.
Asunto(s)
Antineoplásicos/síntesis química , Chalconas/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Benzodioxoles/metabolismo , Sitios de Unión , Biomimética , Línea Celular , Proliferación Celular/efectos de los fármacos , Diseño de Fármacos , Células Endoteliales/efectos de los fármacos , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/patología , Conformación Proteica , Moduladores de TubulinaRESUMEN
In our efforts to improve the efficacy of taxane-based microtubule (MT) stabilizing agents against tumor drug resistance mediated by multiple mechanisms, two clinically relevant factors were focused: i.e., P-glycoprotein and ßIII-tubulin overexpression. Based on the structure of C-seco taxoid 1 m (IDN5390) which was believed to more selectively interact with ßIII-tubulin than paclitaxel, we prepared a series of C-seco taxoids bearing various 7,9-O-linkages and/or different substituents at C2 and C3' positions. Some of them exhibited much more potent binding affinity to MTs and cytotoxicity than their C-seco parent compounds in drug resistant cells with both mechanisms. SAR analysis indicated that C2 modifications significantly enhanced MT binding but brought ambiguous influence to cytotoxicity whereas 7,9-linkage and C3' modifications enhance cytotoxicity more efficiently than improve MT binding. These observations illustrate a better translation of molecular binding effect to cellular activity by C ring closure and C3' modification than C2 modification in C-seco taxoids.