RESUMEN
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
Tubulin-targeted chemotherapy has proven to be a successful and wide spectrum strategy against solid and liquid malignancies. Therefore, new ways to modulate this essential protein could lead to new antitumoral pharmacological approaches. Currently known tubulin agents bind to six distinct sites at α/ß-tubulin either promoting microtubule stabilization or depolymerization. We have discovered a seventh binding site at the tubulin intradimer interface where a novel microtubule-destabilizing cyclodepsipeptide, termed gatorbulin-1 (GB1), binds. GB1 has a unique chemotype produced by a marine cyanobacterium. We have elucidated this dual, chemical and mechanistic, novelty through multidimensional characterization, starting with bioactivity-guided natural product isolation and multinuclei NMR-based structure determination, revealing the modified pentapeptide with a functionally critical hydroxamate group; and validation by total synthesis. We have investigated the pharmacology using isogenic cancer cell screening, cellular profiling, and complementary phenotypic assays, and unveiled the underlying molecular mechanism by in vitro biochemical studies and high-resolution structural determination of the α/ß-tubulin-GB1 complex.
Asunto(s)
Antineoplásicos/síntesis química , Proteínas Bacterianas/síntesis química , Productos Biológicos/síntesis química , Depsipéptidos/síntesis química , Microtúbulos/efectos de los fármacos , Moduladores de Tubulina/síntesis química , Tubulina (Proteína)/química , Antineoplásicos/aislamiento & purificación , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Proteínas Bacterianas/aislamiento & purificación , Proteínas Bacterianas/farmacología , Sitios de Unión , Productos Biológicos/aislamiento & purificación , Productos Biológicos/farmacología , Línea Celular Tumoral , Colchicina/química , Colchicina/farmacología , Cristalografía por Rayos X , Cianobacterias/química , Depsipéptidos/aislamiento & purificación , Depsipéptidos/farmacología , Descubrimiento de Drogas , Células HCT116 , Humanos , Maitansina/química , Maitansina/farmacología , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Modelos Moleculares , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Pironas/química , Pironas/farmacología , Taxoides/química , Taxoides/farmacología , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/aislamiento & purificación , Moduladores de Tubulina/farmacología , Alcaloides de la Vinca/química , Alcaloides de la Vinca/farmacologíaRESUMEN
Invited for the cover of this issue are the groups of Professors Passarella and Pieraccini at the University of Milan, in collaboration with some of the members of TubInTrain consortium. The image depicts work with the elements of nature, in particular the destabilising effect of maytansinol (the constellation) on microtubules (the trees). Read the full text of the article at 10.1002/chem.202203431.
Asunto(s)
Maitansina , Microtúbulos , Investigación , Grupo SocialRESUMEN
Maytansinoids are a successful class of natural and semisynthetic tubulin binders, known for their potent cytotoxic activity. Their wider application as cytotoxins and chemical probes to study tubulin dynamics has been held back by the complexity of natural product chemistry. Here we report the synthesis of long-chain derivatives and maytansinoid conjugates. We confirmed that bulky substituents do not impact their high activity or the scaffold's binding mode. These encouraging results open new avenues for the design of new maytansine-based probes.
Asunto(s)
Antineoplásicos , Maitansina , Tubulina (Proteína)/metabolismo , Antineoplásicos/metabolismo , MicrotúbulosRESUMEN
The aim of the present study is to assess the immediate effects of applying lateral wedge insoles of different heights (0.00, 0.3, 0.4, and 0.6 cm) in patients with chronic ankle instability (CAI) in normal and supinated feet during a Star Excursion Balance Test (SEBT) and in the reflex response of Peroneus Longus (PL), Peroneus Brevis (PB), and Tibialis Anterior (TA) over a 30° inversion of the feet. The effects of the height of the wedges were assessed using a double-blind, crossover design. In total, 25 participants were allocated into two groups, depending on the foot posture (Normal = 12, Supinated = 13) and performed the tests in a random fashion. Reaction time (RT) of stabilizing muscles of the ankle was measured using superficial electromyography (EMG) and postural balance with the SEBT. Foot posture did not show any significant effects on the analyzed variables. Nonetheless, the use of a 0.3 cm external rearfoot wedge (PB p = 0.002; PL p = 0.066 and TA p = 0.006) and 0.6 cm (PB p = 0.043; PL p = 0.058 and TA p = 0.071) reduces RT in stabilizing muscles of the ankle and improves results in SEBT, except for the anterolateral direction, in subjects with CAI. Therefore, our results suggest that the use of lateral wedge insoles could reduce RT and improve dynamic balance in chronic ankle instability.
Asunto(s)
Tobillo , Inestabilidad de la Articulación , Humanos , Articulación del Tobillo/fisiología , Enfermedad Crónica , Extremidad Inferior , Músculo Esquelético/fisiología , Equilibrio Postural/fisiología , Rango del Movimiento Articular/fisiología , Estudios Cruzados , Método Doble CiegoRESUMEN
Maytansinol is a valuable precursor for the preparation of maytansine derivatives (known as maytansinoids). Inspired by the intriguing structure of the macrocycle and the success in targeted cancer therapy of the derivatives, we explored the maytansinol acylation reaction. As a result, we were able to obtain a series of derivatives with novel modifications of the maytansine scaffold. We characterized these molecules by docking studies, by a comprehensive biochemical evaluation, and by determination of their crystal structures in complex with tubulin. The results shed further light on the intriguing chemical behavior of maytansinoids and confirm the relevance of this peculiar scaffold in the scenario of tubulin binders.
Asunto(s)
Maitansina , Neoplasias , Humanos , Maitansina/análogos & derivados , Microtúbulos , Tubulina (Proteína) , Moduladores de TubulinaRESUMEN
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
A new simplified, epoxide-free epothilone analog was prepared incorporating an N-(2-hydroxyethyl)-benzimidazole side chain, which binds to microtubules with high affinity and inhibits cancer cell growth in vitro with nM potency. Building on this scaffold, a disulfide-linked conjugate with the purported EGFR-binding (EGFR, epidermal growth factor receptor) peptide GE11 was then prepared. The conjugate retained significant microtubule-binding affinity, in spite of the size of the peptide attached to the benzimidazole side chain. The antiproliferative activity of the conjugate was significantly lower than for the parent scaffold and, surprisingly, was independent of the EGFR expression status of cells. Our data indicate that the disulfide-based conjugation with the GE11 peptide is not a viable approach for effective tumor-targeting of highly potent epothilones and probably not for other cytotoxics.
Asunto(s)
Citostáticos/síntesis química , Epotilonas/farmacología , Microtúbulos/metabolismo , Péptidos/farmacología , Moduladores de Tubulina/síntesis química , Animales , Bovinos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Citostáticos/farmacología , Epotilonas/química , Receptores ErbB/metabolismo , Células HEK293 , Humanos , Péptidos/química , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/farmacologíaRESUMEN
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
The marine natural product zampanolide and analogues thereof constitute a new chemotype of taxoid site microtubule-stabilizing agents with a covalent mechanism of action. Zampanolide-ligated tubulin has the switch-activation loop (M-loop) in the assembly prone form and, thus, represents an assembly activated state of the protein. In this study, we have characterized the biochemical properties of the covalently modified, activated tubulin dimer, and we have determined the effect of zampanolide on tubulin association and the binding of tubulin ligands at other binding sites. Tubulin activation by zampanolide does not affect its longitudinal oligomerization but does alter its lateral association properties. The covalent binding of zampanolide to ß-tubulin affects both the colchicine site, causing a change of the quantum yield of the bound ligand, and the exchangeable nucleotide binding site, reducing the affinity for the nucleotide. While these global effects do not change the binding affinity of 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one (MTC) (a reversible binder of the colchicine site), the binding affinity of a fluorescent analogue of GTP (Mant-GTP) at the nucleotide E-site is reduced from 12 ± 2 × 105 M-1 in the case of unmodified tubulin to 1.4 ± 0.3 × 105 M-1 in the case of the zampanolide tubulin adduct, indicating signal transmission between the taxane site and the colchicine and nucleotide sites of ß-tubulin.
Asunto(s)
Sitios de Unión/fisiología , Hidrocarburos Aromáticos con Puentes/metabolismo , Colchicina/metabolismo , Macrólidos/metabolismo , Nucleótidos/metabolismo , Taxoides/metabolismo , Tubulina (Proteína)/metabolismo , Animales , Productos Biológicos/metabolismo , Bovinos , Humanos , Ligandos , Microtúbulos/metabolismoRESUMEN
The oligomeric complex transport protein particle I (TRAPPI) mediates nucleotide exchange on the RAB GTPase RAB1/Ypt1. TRAPPII is composed of TRAPPI plus three additional subunits, Trs120, Trs130, and Trs65. Unclear is whether TRAPPII mediates nucleotide exchange on RAB1/Ypt1, RAB11/Ypt31, or both. In Aspergillus nidulans, RabO(RAB1) resides in the Golgi, RabE(RAB11) localizes to exocytic post-Golgi carriers undergoing transport to the apex, and hypA encodes Trs120. RabE(RAB11), but not RabO(RAB1), immunoprecipitates contain Trs120/Trs130/Trs65, demonstrating specific association of TRAPPII with RabE(RAB11) in vivo. hypA1(ts) rapidly shifts RabE(RAB11), but not RabO(RAB1), to the cytosol, consistent with HypA(Trs120) being specifically required for RabE(RAB11) activation. Missense mutations rescuing hypA1(ts) at 42 °C mapped to rabE, affecting seven residues. Substitutions in six, of which four resulted in 7- to 36-fold accelerated GDP release, rescued lethality associated to TRAPPII deficiency, whereas equivalent substitutions in RabO(RAB1) did not, establishing that the essential role of TRAPPII is facilitating RabE(RAB11) nucleotide exchange. In vitro, TRAPPII purified with HypA(Trs120)-S-tag accelerates nucleotide exchange on RabE(RAB11) and, paradoxically, to a lesser yet substantial extent, on RabO(RAB1). Evidence obtained by exploiting hypA1-mediated destabilization of HypA(Trs120)/HypC(Trs130)/Trs65 assembly onto the TRAPPI core indicates that these subunits sculpt a second RAB binding site on TRAPP apparently independent from that for RabO(RAB1), which would explain TRAPPII in vitro activity on two RABs. Using A. nidulans in vivo microscopy, we show that HypA(Trs120) colocalizes with RabE(RAB11), arriving at late Golgi cisternae as they dissipate into exocytic carriers. Thus, TRAPPII marks, and possibly determines, the Golgi-to-post-Golgi transition.
Asunto(s)
Aspergillus nidulans/genética , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Aparato de Golgi/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Aspergillus nidulans/metabolismo , Sitios de Unión , Citosol/metabolismo , Escherichia coli/metabolismo , Exocitosis , Proteínas Fúngicas/genética , Glutatión Transferasa/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Guanosina Difosfato/metabolismo , Microscopía Fluorescente , Mutación , Mutación Missense , Fenotipo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Temperatura , Proteínas de Transporte Vesicular/genética , Proteínas de Unión al GTP rab/genéticaRESUMEN
FtsZ is a self-assembling GTPase that forms, below the inner membrane, the mid-cell Z-ring guiding bacterial division. FtsZ monomers polymerize head to tail forming tubulin-like dynamic protofilaments, whose organization in the Z-ring is an unresolved problem. Rather than forming a well-defined structure, FtsZ protofilaments laterally associate in vitro into polymorphic condensates typically imaged on surfaces. We describe here nanoscale self-organizing properties of FtsZ assemblies in solution that underlie Z-ring assembly, employing time-resolved x-ray scattering and cryo-electron microscopy. We find that FtsZ forms bundles made of loosely bound filaments of variable length and curvature. Individual FtsZ protofilaments further bend upon nucleotide hydrolysis, highlighted by the observation of some large circular structures with 2.5-5° curvature angles between subunits, followed by disassembly end-products consisting of highly curved oligomers and 16-subunit -220 Å diameter mini-rings, here observed by cryo-electron microscopy. Neighbor FtsZ filaments in bundles are laterally spaced 70 Å, leaving a gap in between. In contrast, close contact between filament core structures (â¼50 Å spacing) is observed in straight polymers of FtsZ constructs lacking the C-terminal tail, which is known to provide a flexible tether essential for FtsZ functions in cell division. Changing the length of the intrinsically disordered C-tail linker modifies the interfilament spacing. We propose that the linker prevents dynamic FtsZ protofilaments in bundles from sticking to one another, holding them apart at a distance similar to the lateral spacing observed by electron cryotomography in several bacteria and liposomes. According to this model, weak interactions between curved polar FtsZ protofilaments through their the C-tails may facilitate the coherent treadmilling dynamics of membrane-associated FtsZ bundles in reconstituted systems, as well as the recently discovered movement of FtsZ clusters around bacterial Z-rings that is powered by GTP hydrolysis and guides correct septal cell wall synthesis and cell division.
Asunto(s)
Proteínas Arqueales/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Arqueales/química , Bacillus subtilis , Proteínas Bacterianas/química , Microscopía por Crioelectrón , Proteínas del Citoesqueleto/química , Escherichia coli , Hidrólisis , Methanocaldococcus , Modelos Moleculares , Polímeros , Dominios Proteicos , Multimerización de Proteína , Dispersión del Ángulo Pequeño , Soluciones/química , Difracción de Rayos XRESUMEN
The recent success of antibody-drug conjugates (ADCs) in the treatment of cancer has led to a revived interest in microtubule-destabilizing agents. Here, we determined the high-resolution crystal structure of the complex between tubulin and maytansine, which is part of an ADC that is approved by the US Food and Drug Administration (FDA) for the treatment of advanced breast cancer. We found that the drug binds to a site on ß-tubulin that is distinct from the vinca domain and that blocks the formation of longitudinal tubulin interactions in microtubules. We also solved crystal structures of tubulin in complex with both a variant of rhizoxin and the phase 1 drug PM060184. Consistent with biochemical and mutagenesis data, we found that the two compounds bound to the same site as maytansine and that the structures revealed a common pharmacophore for the three ligands. Our results delineate a distinct molecular mechanism of action for the inhibition of microtubule assembly by clinically relevant agents. They further provide a structural basis for the rational design of potent microtubule-destabilizing agents, thus opening opportunities for the development of next-generation ADCs for the treatment of cancer.
Asunto(s)
Antibióticos Antineoplásicos/química , Antineoplásicos Fitogénicos/química , Macrólidos/química , Maitansina/química , Microtúbulos/química , Policétidos/química , Pironas/química , Moduladores de Tubulina/química , Tubulina (Proteína)/química , Animales , Antibióticos Antineoplásicos/uso terapéutico , Antineoplásicos Fitogénicos/uso terapéutico , Sitios de Unión , Neoplasias de la Mama/tratamiento farmacológico , Bovinos , Ensayos Clínicos Fase I como Asunto , Cristalografía por Rayos X , Femenino , Humanos , Macrólidos/uso terapéutico , Maitansina/uso terapéutico , Policétidos/uso terapéutico , Pironas/uso terapéutico , Moduladores de Tubulina/uso terapéuticoRESUMEN
Zampanolide, first discovered in a sponge extract in 1996 and later identified as a microtubule-stabilizing agent in 2009, is a covalent binding secondary metabolite with potent, low nanomolar activity in mammalian cells. Zampanolide was not susceptible to single amino acid mutations at the taxoid site of ß-tubulin in human ovarian cancer 1A9 cells, despite evidence that it selectively binds to the taxoid site. As expected, it did not synergize with other taxoid site microtubule-stabilizing agents (paclitaxel, ixabepilone, discodermolide), but surprisingly also did not synergize in 1A9 cells with laulimalide/peloruside binding site agents either. Efforts to generate a zampanolide-resistant cell line were unsuccessful. Using a standard wound scratch assay in cell culture, it was an effective inhibitor of migration of human umbilical vein endothelial cells (HUVEC) and fibroblast cells (D551). These properties of covalent binding, the ability to inhibit cell growth in paclitaxel and epothilone resistant cells, and the ability to inhibit cell migration suggest that it would be of interest to investigate zampanolide in preclinical animal models to determine if it is effective in vivo at preventing tumor growth and metastasis.
Asunto(s)
Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Macrólidos/farmacología , Moduladores de Tubulina/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Línea Celular Tumoral , Femenino , Fibroblastos/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Humanos , Lactonas/farmacología , Microtúbulos/metabolismo , Taxoides/metabolismo , Tubulina (Proteína)/metabolismoRESUMEN
Quinolin-6-yloxyacetamides (QAs) are a chemical class of tubulin polymerization inhibitors that were initially identified as fungicides. Here, we report that QAs are potent anti-proliferative agents against human cancer cells including ones that are drug-resistant. QAs act by disrupting the microtubule cytoskeleton and by causing severe mitotic defects. We further demonstrate that QAs inhibit tubulin polymerization in vitro. The high resolution crystal structure of the tubulin-QA complex revealed that QAs bind to the colchicine site on tubulin, which is targeted by microtubule-destabilizing agents such as colchicine and nocodazole. Together, our data establish QAs as colchicine-site ligands and explain the molecular mechanism of microtubule destabilization by this class of compounds. They further extend our structural knowledge on antitubulin agents and thus should aid in the development of new strategies for the rational design of ligands against multidrug-resistant cancer cells.
Asunto(s)
Antineoplásicos/farmacología , Proliferación Celular/efectos de los fármacos , Quinolinas/farmacología , Moduladores de Tubulina/farmacología , Tubulina (Proteína)/metabolismo , Animales , Antineoplásicos/química , Bovinos , Línea Celular Tumoral , Colchicina/metabolismo , Humanos , Simulación del Acoplamiento Molecular , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Quinolinas/química , Moduladores de Tubulina/químicaRESUMEN
Four natural analogues of podophyllotoxin obtained from the Mexican medicinal plant Bursera fagaroides, namely, acetyl podophyllotoxin (2), 5'-desmethoxy-ß-peltatin A methyl ether (3), 7',8'-dehydro acetyl podophyllotoxin (4), and burseranin (5), have been characterized, and their interactions with tubulin have been investigated. Cytotoxic activity measurements, followed by immunofluorescence microscopy and flow cytometry studies, demonstrated that these compounds disrupt microtubule networks in cells and cause cell cycle arrest in the G2/M phase in the A549 cell line. A tubulin binding assay showed that compounds 1-4 were potent assembly inhibitors, displaying binding to the colchicine site with Kb values ranging from 11.75 to 185.0 × 10(5) M(-1). In contrast, burseranin (5) was not able to inhibit tubulin assembly. From the structural perspective, the ligand-binding epitopes of compounds 1-3 have been mapped using STD-NMR, showing that B and E rings are the major points for interaction with the protein. The obtained results indicate that the inhibition of tubulin assembly of this family of compounds is more effective when there are at least two methoxyl groups at the E ring, along with a trans configuration of the lactone ring in the aryltetralin lignan core.
Asunto(s)
Bursera/química , Podofilotoxina/farmacología , Tubulina (Proteína)/metabolismo , Ciclo Celular/efectos de los fármacos , Colchicina/farmacología , Humanos , Lactonas/química , Lactonas/farmacología , Lignanos/farmacología , Microtúbulos/efectos de los fármacos , Estructura Molecular , Podofilotoxina/análogos & derivados , Podofilotoxina/química , Unión Proteica , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologíaRESUMEN
12-Aza-epothilones (azathilones) incorporating quinoline side chains and bearing different N12-substituents have been synthesized via highly efficient RCM-based macrocyclizations. Quinoline-based azathilones with the side chain N-atom in the meta-position to the C15 atom in the macrocycle are highly potent inhibitors of cancer cell growth in vitro. In contrast, shifting the quinoline nitrogen to the position para to C15 leads to a ca. 1000-fold loss in potency. Likewise, the desaturation of the C9-C10 bond in the macrocycle to an E double bond produces a substantial reduction in antiproliferative activity. This is in stark contrast to the effect exerted by the same modification in the natural epothilone macrocycle. The conformation of a representative azathilone bound to α/ß-tubulin heterodimers was determined based on TR-NOE measurements and a model for the posture of the compound in its binding site on ß-tubulin was deduced through a combination of STD measurements and CORCEMA-ST calculations. The tubulin-bound, bioactive conformation of azathilones was found to be overall similar to that of epothilones A and B.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Epotilonas/síntesis química , Epotilonas/farmacología , Tubulina (Proteína)/metabolismo , Células A549 , Antineoplásicos/química , Sitios de Unión , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclización , Ensayos de Selección de Medicamentos Antitumorales , Epotilonas/química , Células Hep G2 , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Conformación Proteica , Tubulina (Proteína)/químicaRESUMEN
The total synthesis of (-)-amphidinolide K (1) based on asymmetric addition of allylsilane C1-C8 to enal C9-C22 is reported. The 1,9,18-tris-O-TBDPS ether was converted into the desired 9,18-dihydroxy acid. Its macrolactonization was accomplished by the Shiina method. Compound 1 together with some of its stereoisomers and analogues were subjected to evaluation of the possible disruption of the α,ß-tubulin-microtubule and/or G-actin-F-actin equilibria. Compound 1 behaves as a stabilizer of actin filaments (F-actin) in vitro.
Asunto(s)
Actinas/química , Antibacterianos/síntesis química , Macrólidos/síntesis química , Tubulina (Proteína)/química , Antibacterianos/química , Macrólidos/química , Estructura Molecular , EstereoisomerismoRESUMEN
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 ß-tubulin binding site or by overexpression of the highly dynamic ßIII-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 ßIII 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.
Asunto(s)
Antineoplásicos/farmacología , Hidrocarburos Aromáticos con Puentes/farmacología , Resistencia a Antineoplásicos/efectos de los fármacos , Taxoides/farmacología , Tubulina (Proteína)/metabolismo , Animales , Antineoplásicos/química , Sitios de Unión/efectos de los fármacos , Hidrocarburos Aromáticos con Puentes/química , Bovinos , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Citoesqueleto/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Microtúbulos/química , Microtúbulos/efectos de los fármacos , Microtúbulos/metabolismo , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Relación Estructura-Actividad , Taxoides/química , TermodinámicaRESUMEN
Targeting microtubules is the most effective wide-spectrum pharmacological strategy in antitumoral chemotherapy, and current research focuses on reducing main drawbacks: neurotoxicity and resistance. PM534 is a novel synthetic compound derived from the Structure-Activity-Relationship study on the natural molecule PM742, isolated from the sponge of the order Lithistida, family Theonellidae, genus Discodermia (du Bocage 1869). PM534 targets the entire colchicine binding domain of tubulin, covering four of the five centers of the pharmacophore model. Its nanomolar affinity and high retention time modulate a strikingly high antitumor activity that efficiently overrides two resistance mechanisms in cells (detoxification pumps and tubulin ßIII isotype overexpression). Furthermore, PM534 induces significant inhibition of tumor growth in mouse xenograft models of human non-small cell lung cancer. Our results present PM534, a highly effective new compound in the preclinical evaluation that is currently in its first human Phase I clinical trial.