RESUMO
We report a mass-spectrometry-based metabolomics study of a laboratory-cultured strain of Microcystis aeruginosa (UTEX LB2385), which has led to the discovery of five peptides (1-5) belonging to the microginin class of linear cyanopeptides. The structures and configurations of these peptides were determined by spectroscopic analyses and chemical derivitization. The microginin peptides described herein are the first reported derivatives containing N-methyl methionine (1, 5) and N-methyl methionine sulfoxide (2-4). The two tripeptide microginin analogues (4, 5) identified represent the smallest members of this peptide family. Their angiotensin-converting enzyme (ACE) inhibitory activity was also investigated. Microginin 527 (4) was the most potent of the group, with an IC50 of 31 µM.
Assuntos
Proteínas de Bactérias/metabolismo , Cianobactérias/metabolismo , Microcystis/metabolismo , Peptídeos/metabolismo , Inibidores da Enzima Conversora de Angiotensina/metabolismo , Espectrometria de Massas/métodos , Metabolômica/métodos , Metionina/análogos & derivados , Metionina/metabolismoRESUMO
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)/químicaRESUMO
The important anticancer drug paclitaxel binds to the beta-subunit of the alphabeta-tubulin dimer in the microtubule in a stoichiometric ratio, promoting microtubule polymerization and stability. The conformation of microtubule-bound drug has been the subject of intense study, and various suggestions have been proposed. In previous work we presented experimental and theoretical evidence that paclitaxel adopts a T-shaped conformation when it is bound to tubulin. In this study we report additional experimental data and calculations that delineate the allowable parameters for effective paclitaxel-tubulin interactions.
Assuntos
Antineoplásicos/síntese química , Paclitaxel/análogos & derivados , Paclitaxel/síntese química , Tubulina (Proteína)/química , Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes/síntese química , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Lactamas Macrocíclicas/síntese química , Lactamas Macrocíclicas/farmacologia , Modelos Moleculares , Conformação Molecular , Paclitaxel/farmacologia , Soluções , Estereoisomerismo , Relação Estrutura-AtividadeRESUMO
Drugs that affect microtubule dynamics are among the most effective anticancer agents in routine clinical use. The standard assay for antimicrotubule agents observes the ability of a particular substance to affect in vitro microtubule assembly. We have modified these procedures so that they can be performed in 96-well plates using a standard fluorescence plate reader. Two different protocols are provided in this chapter. One of these protocols is for ligands that inhibit microtubule polymerization, such as colchicine and related molecules. The second is for ligands that promote in vitro microtubule assembly, such as Taxol.
Assuntos
Colchicina/química , Microtúbulos/química , Paclitaxel/química , Animais , Encéfalo/ultraestrutura , Bovinos , Colchicina/farmacologia , Avaliação Pré-Clínica de Medicamentos/métodos , Fluorescência , Ligantes , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Relação Estrutura-Atividade , Termodinâmica , Fatores de Tempo , Tubulina (Proteína)/química , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
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)/químicaRESUMO
A fluorescent probe has been attached to the carboxy terminus of the alpha-subunit of alpha,beta-tubulin by an enzymatic reaction followed by a chemical reaction. The unnatural amino acid 3-formyltyrosine is attached to the carboxy terminus of alpha-tubulin through the use of the enzyme tubulin tyrosine ligase. The aromatic aldehyde of the unnatural amino acid serves as an orthogonal electrophile that specifically reacts with a fluorophore containing an aromatic hydrazine functional group, which in this case is 7-hydrazino-4-methyl coumarin. Conditions for covalent bond formation between the unnatural amino acid and the fluorophore are mild, allowing fluorescently labeled tubulin to retain its ability to assemble into microtubules. A key feature of the labeling reaction is that it produces a red shift in the fluorophore's absorption and emission maxima, accompanied by an increase in its quantum yield; thus, fluorescently labeled protein can be observed in the presence of unreacted fluorophore. Both the enzymatic and coupling reaction can occur in living cells. The approach presented here should be applicable to a wide variety of in vitro systems.
Assuntos
Química Encefálica , Coloração e Rotulagem/métodos , Tubulina (Proteína)/química , Animais , Bovinos , Fluorescência , Humanos , Peptídeo Sintases/metabolismo , Tubulina (Proteína)/metabolismo , Tirosina/metabolismoRESUMO
A series of 2,5-diaryl-3-methylpyrimido[4,5-c]quinolin-1(2H)-ones (7-30), variously substituted at the 2- and 5-phenyl moieties, were synthesized and evaluated for their in vitro cytotoxic activity against a PC3 cancer cell line. Cytotoxicity data revealed that the type of substituent as well as substitution pattern have variable influence on cytotoxic activity. Among the compounds tested, compounds (9), (13), (18), (19), and (23) demonstrated appreciable cytotoxic activity with mean IC(50) values of 2.0, 1.4, 1.6, 2.2, and 1.9microM, respectively. Methyl substitution at the 2-phenyl ring was found to yield the least active compounds. Two of the most potent compounds (13) and (18) were further investigated for inhibition of tubulin polymerization and found to have no activity at the concentrations used in the assay.
Assuntos
Anticarcinógenos/farmacologia , Pirimidinas/química , Quinolinas/síntese química , Animais , Anticarcinógenos/síntese química , Anticarcinógenos/química , Encéfalo/efeitos dos fármacos , Bovinos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Microtúbulos/efeitos dos fármacos , Estrutura Molecular , Quinolinas/química , Quinolinas/farmacologia , Relação Estrutura-Atividade , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
The important anticancer drug Taxol (paclitaxel, PTX) owes its unique activity to its ability to bind to tubulin in a stoichiometric ratio and promote its assembly into microtubules. The conformation of the microtubule-bound drug has been the focus of numerous research efforts, since the inability of polymerized tubulin to form crystals precludes structure proof by X-ray crystallography. Likewise, although the alpha,beta-tubulin dimer structure has been solved by electron crystallography, the 3.7 A resolution is too low to permit direct determination of either ligand conformation or binding pose. In this article, we present experimental results from 2H{19F} REDOR NMR that provide direct confirmation that paclitaxel adopts a T-shaped conformation when it is bound to tubulin.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Paclitaxel/química , Tubulina (Proteína)/química , Animais , Sítios de Ligação , Bovinos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Ligantes , Modelos Moleculares , Conformação Molecular , Paclitaxel/síntese química , Paclitaxel/farmacologia , Conformação Proteica , Rotação , Sensibilidade e Especificidade , Estereoisomerismo , Relação Estrutura-Atividade , Fatores de TempoRESUMO
A series of modified colchicine and isocolchicine analogs (C-7 substituent) were synthesized and evaluated in vitro against a PC3 cancer cell line and for inhibition of microtubule polymerization. The colchicine analogs all displayed strong inhibition of tubulin polymerization, while compounds 6 and 20 also possessed an increased cytotoxic activity as compared to colchicine. More importantly, isocolchicine analogs 7, 15, and 17 showed inhibition of microtubule polymerization with IC(50) values ranging from 58 to 68muM. In addition, 7 displayed strong cytotoxic activity with an IC(50)=93nM which was more potent than colchicine analog 12.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Colchicina/síntese química , Colchicina/farmacologia , Linhagem Celular Tumoral , HumanosRESUMO
Five macrocyclic paclitaxel bis-lactones and their corresponding open chain taxoids were synthesized as models of the tubulin-binding conformation of paclitaxel. Macrocyclic lactones with a 19-21-membered ring underwent isomerization to form smaller rings. The lactones were evaluated for cytotoxicity and tubulin-polymerization ability. All five macrocyclic paclitaxel lactones were active, but less so than paclitaxel, while the rearranged macrocyclic lactones and the corresponding open-chain taxoids were much less active or inactive.
Assuntos
Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Hidrocarbonetos Aromáticos com Pontes , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Isomerismo , Lactonas/síntese química , Lactonas/farmacologia , Compostos Macrocíclicos/química , Conformação Molecular , Paclitaxel/síntese química , Paclitaxel/farmacologia , Relação Estrutura-Atividade , Taxoides/síntese química , Taxoides/farmacologia , Tubulina (Proteína)/metabolismo , Moduladores de TubulinaRESUMO
Vinblastine is an antimitotic agent that has been used extensively in cancer chemotherapy. The biological effects of the drug are believed to be the result of its interaction with tubulin, the major component of cellular microtubules. Fluorescence spectroscopy is a powerful and versatile technique for studying drug-tubulin interactions, but it rarely has been applied to studies involving vinca alkaloids. We have prepared a new fluorescent derivative of vinblastine designed to retain high affinity for tubulin while possessing a fluorophore that absorbs and emits visible light. A coumarin derivative of vinblastine, 17-deacetyl-O-(3-carbonylamino-7-diethylaminocoumarin) vinblastine (F-VLB), was prepared by reaction of 17-deacetylvinblastine with 7-diethylaminocoumarin-3-carbonyl azide. F-VLB was a potent inhibitor of in vitro microtubule assembly (IC(50) = 0.5 microM). F-VLB binding to tubulin was inhibited by vinblastine. Tubulin binding induced an increase in the F-VLB emission intensity and shifted the emission maximum to higher energy (from 500 to 480 nm). The Stokes shift of tubulin-bound F-VLB was about the same as the Stokes shift of the molecule in ethanol, indicating that the tubulin-bound fluorophore is probably on the exterior of the vinblastine binding site. Unlike vinblastine, F-VLB failed to induce self-assembly of tubulin that could be detected by light scattering or electron microscopy, although some self-association could be detected by analytical ultracentrifugation. Equilibrium binding parameters were quantitatively determined by monitoring the change in fluorescence anisotropy of F-VLB upon tubulin binding. The apparent equilibrium constant for F-VLB binding to tubulin [K(a)(app) = (7.7 +/- 0.5) x 10(4) M(-1) at 25 degrees C] was identical to the equilibrium constant for vinblastine binding to 2 microM tubulin (K(1)) measured under similar buffer and temperature conditions using ultracentrifugation [Vulevic, B., Lobert, S., and Correia, J. J. (1997) Biochemistry 36, 12828-12835]. Binding allocolchicine to tubulin did not significantly affect F-VLB's affinity for the protein [K(a)(app) = (9.1 +/- 0.4) x 10(4) M(-1) at 25 degrees C]. Analysis of the steady-state emission spectra yielded a distance between the colchicine and vinca binding sites on tubulin of approximately 40 A. F-VLB bound to paclitaxel- and glutaraldehyde-stabilized microtubules, with approximately equal affinity. We conclude that F-VLB can be used to obtain information about the vinblastine binding site on tubulin under equilibrium conditions.
Assuntos
Corantes Fluorescentes , Tubulina (Proteína)/química , Vimblastina/análogos & derivados , Animais , Cumarínicos , Reagentes de Ligações Cruzadas , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Cinética , Microtúbulos/efeitos dos fármacos , Microtúbulos/ultraestrutura , Espectrometria de Fluorescência , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/metabolismo , Vimblastina/síntese química , Vimblastina/química , Vimblastina/farmacologiaRESUMO
With the advent of combinatorial chemistry and the extensive libraries of potential drugs produced from it, there is a growing need for rapid sensitive, high-throughput screening for drug potency. Microtubules are important targets for anticancer agents, and new antimicrotubule compounds are of continued interest in drug development. The in vitro potency of antimicrotubule drugs may be evaluated by measuring the extent of tubulin assembly. The extent of polymerization is proportional to the turbidity of the solution, which usually has been measured as apparent absorption. The turbidity method has inherent problems that hinder its adaptation to a high-throughput format, such as a requirement for high protein concentrations and a high coefficient of variation. We present here a high-throughput assay for antimicrotubule activity in which fluorescence is used to monitor microtubule assembly. Both assembly-inhibiting and assembly-promoting compounds can be evaluated. The assay is rapid and easy to perform, and the data are reliable, with good accuracy and reproducibility.
Assuntos
Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Microtúbulos/efeitos dos fármacos , Química Farmacêutica/métodos , Relação Dose-Resposta a Droga , Microtúbulos/metabolismo , Paclitaxel/química , Paclitaxel/farmacologia , Espectrometria de Fluorescência/métodos , Temperatura , Fatores de Tempo , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismoRESUMO
Concurrent modifications on the C-3'NH/C-10, and C-2/C-10 positions on paclitaxel were carried out as a way of investigating possible synergistic effects. The biological activities of these analogues were evaluated in both a microtubule assembly assay and human ovarian cancer (A2780) and prostate cancer (PC3) cytotoxicity assay. In some cases the doubly modified analogues were more active than would have been predicted based on the activity of the singly modified analogues, indicating probable synergistic effects.
Assuntos
Antineoplásicos Fitogênicos/síntese química , Antineoplásicos Fitogênicos/farmacologia , Paclitaxel/análogos & derivados , Paclitaxel/síntese química , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Paclitaxel/farmacologia , Relação Estrutura-Atividade , Tubulina (Proteína)/biossíntese , Células Tumorais CultivadasRESUMO
The important anticancer drug Taxol (paclitaxel) binds to tubulin in a stoichiometric ratio and promotes its assembly into microtubules. The conformation of microtubule-bound drug has been the subject of intense study, and various suggestions have been made. In this work we present experimental and theoretical evidence that Taxol adopts a T-shaped conformation when it is bound to tubulin.