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1.
Biochemistry ; 52(13): 2328-36, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23473345

RESUMO

Paclitaxel (PTX) is a microtubule-stabilizing agent that is widely used in cancer chemotherapy. This structurally complex natural product acts by binding to ß-tubulin in assembled microtubules. The 2'-hydroxyl group in the flexible side chain of PTX is an absolute requirement for activity, but its precise role in the drug-receptor interaction has not been specifically investigated. The contribution of the 2'-OH group to the affinity and tubulin-assembly efficacy of PTX has been evaluated through quantitative analysis of PTX derivatives possessing side chain deletions: 2'-deoxy-PTX, N-debenzoyl-2'-deoxy-PTX, and baccatin III. The affinity of 2'-deoxy-PTX for stabilized microtubules was more than 100-fold lower than that of PTX and only ~3-fold greater than the microtubule affinity of baccatin III. No microtubule binding activity was detected for the analogue N-debenzoyl-2'-deoxy-PTX. The tubulin-assembly efficacy of each ligand was consistent with the microtubule binding affinity, as was the trend in cytotoxicities. Molecular dynamics simulations revealed that the 2'-OH group of PTX can form a persistent hydrogen bond with D26 within the microtubule binding site. The absence of this interaction between 2'-deoxy-PTX and the receptor can account for the difference in binding free energy. Computational analyses also provide a possible explanation for why N-debenzoyl-2'-deoxy-PTX is inactive, in spite of the fact that it is essentially a substituted baccatin III. We propose that the hydrogen bonding interaction between the 2'-OH group and D26 is the most important stabilizing interaction that PTX forms with tubulin in the region of the C-13 side chain. We further hypothesize that the substituents at the 3'-position function to orient the 2'-OH group for a productive hydrogen bonding interaction with the protein.


Assuntos
Microtúbulos/metabolismo , Paclitaxel/química , Paclitaxel/farmacologia , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Sítios de Ligação , Bovinos , Linhagem Celular , Humanos , Modelos Moleculares , Paclitaxel/análogos & derivados , Ligação Proteica , Taxoides/farmacologia
2.
Chemistry ; 17(52): 14792-804, 2011 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-22127984

RESUMO

A series of conformationally restrained epothilone analogues with a short bridge between the methyl groups at C6 and C8 was designed to mimic the binding pose assigned to our recently reported EpoA-microtubule binding model. A versatile synthetic route to these bridged epothilone analogues has been successfully devised and implemented. Biological evaluation of the compounds against A2780 human ovarian cancer and PC3 prostate cancer cell lines suggested that the introduction of a bridge between C6-C8 reduced potency by 25-1000 fold in comparison with natural epothilone D. Tubulin assembly measurements indicate these bridged epothilone analogues to be mildly active, but without significant microtubule stabilization capacity. Molecular mechanics and DFT energy evaluations suggest the mild activity of the bridged epo-analogues may be due to internal conformational strain.


Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Epotilonas/química , Epotilonas/síntese química , Epotilonas/farmacologia , Neoplasias Ovarianas/tratamento farmacológico , Linhagem Celular Tumoral , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Modelos Moleculares , Ligação Proteica , Relação Estrutura-Atividade
3.
Mol Pharm ; 7(6): 2216-23, 2010 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-20863081

RESUMO

Taxanes are a critical component of chemotherapy for breast, prostate, lung and other cancers. Initial or acquired tumor resistance to taxanes is therefore one of the most important issues in oncology. Survivin is a prosurvival gene whose expression is a poor prognostic feature. Survivin is induced acutely upon exposure to taxanes and coordinates resistance to taxane-mediated cell death, although the exact mechanism of taxane-mediated survivin induction is not clear. Here, we describe the synthesis of a series of novel taxanes, with modifications on the 7- or 10-position of the taxane backbone, as well as the side chain. We found that the novel taxanes with modifications at the 10-position have robust tubulin binding and tubulin polymerization activity. Gene expression profiling and quantitative PCR of cells treated with the 10-position conjugates reveals that the effect of treatment with a subset of these novel taxanes lacks a gene expression signature, including survivin induction, which is characteristically induced with paclitaxel treatment. Furthermore, we show that this gene expression signature is not due to differences in G2/M arrest. Cell sensitivity studies suggest that the inability to induce survivin is associated with increased drug cytotoxicity and apoptosis. This work suggests that taxanes that effectively bind tubulin need not invariably induce survivin as a mechanism of drug resistance.


Assuntos
Antineoplásicos/farmacologia , Proteínas Associadas aos Microtúbulos/genética , Taxoides/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Ligação Competitiva/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Proteínas Inibidoras de Apoptose , Proteínas Associadas aos Microtúbulos/metabolismo , Estrutura Molecular , Poli(ADP-Ribose) Polimerases/metabolismo , Reação em Cadeia da Polimerase , Estereoisomerismo , Survivina , Taxoides/síntese química , Taxoides/química , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas
4.
Chem Commun (Camb) ; 46(12): 2019-21, 2010 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-20221478

RESUMO

An unusual reaction with Grubbs' catalyst during the synthesis of bridged epothilones yielded five-membered internal lactones instead of the expected metathesis products. Three of the lactones have activities comparable to epothilone D.


Assuntos
Epotilonas/química , Lactonas/química , Ciclização , Espectroscopia de Ressonância Magnética , Modelos Moleculares
5.
Biochemistry ; 49(13): 2932-42, 2010 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-20178367

RESUMO

Tubulin, the basic component of microtubules, is present in most eukaryotic cells as multiple gene products, called isotypes. The major tubulin isotypes are highly conserved in terms of structure and drug binding capabilities. Tubulin isotype betaVI, however, is significantly divergent from the other isotypes in sequence, assembly properties, and function. It is the major beta-tubulin isotype of hematopoietic tissue and forms the microtubules of platelet marginal bands. The interaction of the major tubulin isotypes betaI, betaII, betaIII, and betaIotaV with antimicrotubule drugs has been widely studied, but little is known about the drug binding properties of tubulin isotype betaVI. In this investigation, we characterize the activity of various colchicine site ligands with tubulin isolated from Gallus gallus erythrocytes (CeTb), which is approximately 95% betaVI. Colchicine binding is thought to be a universal property of higher eukaryotic tubulin; however, we were unable to detect colchicine binding to CeTb under any experimental conditions. Podophyllotoxin and nocodazole, other colchicine site ligands with divergent structures, were able to inhibit paclitaxel-induced CeTb assembly. Surprisingly, the colchicine isomer allocolchicine also inhibited CeTb assembly and displayed measurable, moderate affinity for CeTb (K(a) = 0.18 x 10(5) M(-1) vs 5.0 x 10(5) M(-1) for bovine brain tubulin). Since allocolchicine and colchicine differ in their C ring structures, the two C ring colchicine analogues were also tested for CeTb binding. Kinetic experiments indicate that thiocolchicine and chlorocolchicine bind to CeTb, but very slowly and with low affinity. Molecular modeling of CeTb identified five divergent amino acid residues within 6 A of the colchicine binding site compared to betaI, betaII, and betaIV; three of these amino acids are also altered in betaIII-tubulin. Interestingly, the altered amino acids are in the vicinity of the A ring region of the colchicine binding site rather than the C ring region. We propose that the amino acid differences in the binding site constrict the A ring binding domain in CeTb, which interferes with the positioning of the trimethoxyphenyl A ring and prevents C ring binding site interactions from efficiently occurring. Allocolchicine is able to accommodate the altered binding mode because of its smaller ring size and more flexible C ring substituents. The sequence of the colchicine binding domain of CeTb isotype betaVI is almost identical to that of its human hematopoietic counterpart. Thus, through analysis of the interactions of ligands with CeTb, it may be possible to discover colchicine site ligands that specifically target tubulin in human hematopoietic cells.


Assuntos
Colchicina/metabolismo , Tubulina (Proteína)/metabolismo , Aminoácidos , Animais , Sítios de Ligação , Galinhas , Eritrócitos/química , Ligantes , Ligação Proteica , Isoformas de Proteínas , Tubulina (Proteína)/química
6.
Bioorg Med Chem Lett ; 19(10): 2884-7, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19359169

RESUMO

A knowledge of the bioactive tubulin-binding conformation of paclitaxel (Taxol()) is crucial to a full understanding of the bioactivity of this important anticancer drug, and potentially also to the design of simplified analogs. The bioactive conformation has been shown to be best approximated by the T-Taxol conformation. As a further test of this conclusion, the paclitaxel analog 4 was designed as a compound which has all the chemical functionality necessary for activity, but which cannot adopt the T-Taxol conformation. The synthesis and bioassay of 4 confirmed its lack of activity, and thus provided further support for the T-Taxol conformation as the bioactive tubulin-binding conformation.


Assuntos
Antineoplásicos Fitogênicos/síntese química , Carbamatos/síntese química , Paclitaxel/análogos & derivados , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Carbamatos/química , Carbamatos/farmacologia , Linhagem Celular Tumoral , Humanos , Paclitaxel/síntese química , Paclitaxel/farmacologia , Tubulina (Proteína)/química
7.
Anal Biochem ; 360(1): 56-62, 2007 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-17097592

RESUMO

Promotion or inhibition of tubulin assembly into microtubules is the standard in vitro assay for evaluating potential antimicrotubule agents. Many agents to be tested are poorly soluble in aqueous solution and require a cosolvent such as dimethyl sulfoxide (DMSO). However, DMSO itself can promote tubulin assembly, and its inclusion in assays for compounds that induce tubulin assembly complicates interpretation of the results. Substituting GDP for GTP in the exchangeable nucleotide binding site of tubulin produces a less active form of the protein, tubulin-GDP. Here it is shown that tubulin-GDP can be assembled into normal microtubules in DMSO concentrations up to 15% (v/v), and polymerization assays performed under these conditions can be compared with assays run under more standard conditions. Assays for measuring the effective concentration of a ligand for promotion of tubulin assembly (EC(50)), measuring the concentration for inhibition of tubulin assembly (IC(50)) by a colchicine site ligand, and measuring tubulin critical concentrations in the presence of poorly soluble taxol derivatives are illustrated.


Assuntos
Paclitaxel/análogos & derivados , Tubulina (Proteína)/química , Biopolímeros , Cromatografia Líquida de Alta Pressão , Dimetil Sulfóxido/química , Guanosina Difosfato/química , Guanosina Trifosfato/química , Microscopia Eletrônica , Paclitaxel/química , Podofilotoxina/química , Solubilidade
8.
Org Lett ; 8(18): 3983-6, 2006 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-16928054

RESUMO

The synthesis of the bridged A-nor-paclitaxel 4 has been achieved from paclitaxel in a key test of the T-Taxol conformational hypothesis. Although the unbridged A-nor-paclitaxel 3 is essentially noncytotoxic, the bridged analogue 4 is strongly cytotoxic. This result provides strong evidence for the T-Taxol conformation as the bioactive tubulin-binding conformation of paclitaxel.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Paclitaxel/análogos & derivados , Paclitaxel/síntese química , Animais , Linhagem Celular , Modelos Moleculares , Estrutura Molecular , Paclitaxel/farmacologia , Ligação Proteica , Ratos , Tubulina (Proteína)/química
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