RESUMO
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.
Assuntos
Sítios de Ligação/fisiologia , Hidrocarbonetos Aromáticos com Pontes/metabolismo , Colchicina/metabolismo , Macrolídeos/metabolismo , Nucleotídeos/metabolismo , Taxoides/metabolismo , Tubulina (Proteína)/metabolismo , Animais , Produtos Biológicos/metabolismo , Bovinos , Humanos , Ligantes , Microtúbulos/metabolismoRESUMO
Microtubule-targeting agents (MTAs) are some of the clinically most successful anti-cancer drugs. Unfortunately, instances of multidrug resistances to MTA have been reported, which highlights the need for developing MTAs with different mechanistic properties. One less explored class of MTAs are [1,2,4]triazolo[1,5-a]pyrimidines (TPs). These cytotoxic compounds are microtubule-stabilizing agents that inexplicably bind to vinblastine binding site on tubulin, which is typically targeted by microtubule-destabilizing agents. Here we used cellular, biochemical, and structural biology approaches to address this apparent discrepancy. Our results establish TPs as vinca-site microtubule-stabilizing agents that promote longitudinal tubulin contacts in microtubules, in contrast to classical microtubule-stabilizing agents that primarily promote lateral contacts. Additionally we observe that TPs studied here are not affected by p-glycoprotein overexpression, and suggest that TPs are promising ligands against multidrug-resistant cancer cells.
Assuntos
Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Pirimidinas/farmacologia , Triazóis/farmacologia , Tubulina (Proteína)/metabolismo , Alcaloides de Vinca/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , Humanos , Ligantes , Modelos Moleculares , Multimerização Proteica/efeitos dos fármacos , Estrutura Quaternária de Proteína , Tubulina (Proteína)/químicaRESUMO
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.
Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Quinolinas/farmacologia , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos/química , Bovinos , Linhagem Celular Tumoral , Colchicina/metabolismo , Humanos , Simulação de Acoplamento Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Quinolinas/química , Moduladores de Tubulina/químicaRESUMO
We investigated the microtubule-destabilizing, vascular-targeting, anti-tumor and anti-metastatic activities of a new series of chalcones, whose prototype compound is (E)-3-(3''-amino-4''-methoxyphenyl)-1-(5'-methoxy-3',4'-methylendioxyphenyl)-2-methylprop-2-en-1-one (TUB091). X-ray crystallography showed that these chalcones bind to the colchicine site of tubulin and therefore prevent the curved-to-straight structural transition of tubulin, which is required for microtubule formation. Accordingly, TUB091 inhibited cancer and endothelial cell growth, induced G2/M phase arrest and apoptosis at 1-10 nM. In addition, TUB091 displayed vascular disrupting effects in vitro and in the chicken chorioallantoic membrane (CAM) assay at low nanomolar concentrations. A water-soluble L-Lys-L-Pro derivative of TUB091 (i.e. TUB099) showed potent antitumor activity in melanoma and breast cancer xenograft models by causing rapid intratumoral vascular shutdown and massive tumor necrosis. TUB099 also displayed anti-metastatic activity similar to that of combretastatin A4-phosphate. Our data indicate that this novel class of chalcones represents interesting lead molecules for the design of vascular disrupting agents (VDAs). Moreover, we provide evidence that our prodrug approach may be valuable for the development of anti-cancer drugs.
Assuntos
Inibidores da Angiogênese/farmacologia , Antineoplásicos/farmacologia , Benzodioxóis/farmacologia , Neoplasias da Mama/tratamento farmacológico , Chalcona/farmacologia , Chalconas/farmacologia , Dipeptídeos/farmacologia , Endotélio Vascular/efeitos dos fármacos , Melanoma Experimental/tratamento farmacológico , Tubulina (Proteína)/metabolismo , Animais , Apoptose/efeitos dos fármacos , Benzodioxóis/síntese química , Sítios de Ligação , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/secundário , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Chalconas/síntese química , Cristalografia por Raios X , Dipeptídeos/síntese química , Endotélio Vascular/patologia , Feminino , Humanos , Melanoma Experimental/irrigação sanguínea , Melanoma Experimental/patologia , Camundongos , Camundongos SCID , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Pró-Fármacos/farmacologia , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade , Tubulina (Proteína)/química , Moduladores de Tubulina/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
To probe the exact role of the oxetane D ring in both tubulin binding and cytotoxicity of taxanes, novel D-seco taxanes bearing a C4 ether substituent have been prepared from paclitaxel 1a. Among them, 20-hydroxymethyl-4-allyloxy D-seco taxane 5e is the most active in both tubulin and cytotoxicity assays. It is only slightly less potent than 1a on tubulin polymerization promotion in vitro and the most cytotoxic among all D-seco taxanes known to date. The reason for the loss and restoration of bioactivity for these D-seco taxanes is also discussed with the assistance of NMR and molecular modeling studies. From these results, we draw a conclusion that the intact D ring of taxanes is not strictly necessary for their binding to tubulin and cytotoxic effects.
Assuntos
Microtúbulos/metabolismo , Paclitaxel/farmacologia , Éteres Cíclicos/química , Modelos Moleculares , Estrutura Molecular , Ressonância Magnética Nuclear Biomolecular , Paclitaxel/química , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
Vascular disrupting agents (VDAs) constitute an innovative anticancer therapy that targets the tumor endothelium, leading to tumor necrosis. Our approach for the identification of new VDAs has relied on a ligand 3-D shape similarity virtual screening (VS) approach using the ROCS program as the VS tool and as query colchicine and TN-16, which both bind the α,ß-tubulin dimer. One of the hits identified, using TN-16 as query, has been explored by the synthesis of its structural analogues, leading to 2-(1-((2-methoxyphenyl)amino)ethylidene)-5-phenylcyclohexane-1,3-dione (compound 16c) with an IC50 = 0.09 ± 0.01 µM in HMEC-1 and BAEC, being 100-fold more potent than the initial hit. Compound 16c caused cell cycle arrest in the G2/M phase and interacted with the colchicine-binding site in tubulin, as confirmed by a competition assay with N,N'-ethylenebis(iodoacetamide) and by fluorescence spectroscopy. Moreover, 16c destroyed an established endothelial tubular network at 1 µM and inhibited the migration and invasion of human breast carcinoma cells at 0.4 µM. In conclusion, our approach has led to a new chemotype of promising antiproliferative compounds with antimitotic and potential VDA properties.