RESUMO
The CXC chemokine receptor 4 (CXCR4) is involved in chemotaxis and serves as a coreceptor for T-tropic HIV-1 viral entry, thus making this receptor an attractive drug target. Recently, crystal structures of CXCR4 were reported as complexes with the small molecule IT1t and the CVX15 peptide. Follow-up efforts to model different antagonists into the small molecule CXCR4:IT1t crystal structure did not generate poses consistent with either the X-ray crystal structure or site-directed mutagenesis (SDM). Here, we compare the binding pockets of the two CXCR4 crystal structures, revealing differences in helices IV, V, VI, and VII, with major differences for the His203 residue buried in the binding pocket. The small molecule antagonist AMD11070 was docked into both CXCR4 crystal structures. An AMD11070 pose identified from the CXCR4:CVX15 model presented interactions with Asp171, Glu288, Trp94, and Asp97, consistent with published SDM data, thus suggesting it is the bioactive pose. A CXCR4 receptor model was optimized around this pose of AMD11070, and the resulting model correlated HIV-1 inhibition with MM-GBSA docking scores for a congeneric AMD11070-like series. Subsequent NAMFIS NMR results successfully linked the proposed binding pose to an independent experimental structure. These results strongly suggest that not all small molecules will bind to CXCR4 in a similar manner as IT1t. Instead, the CXCR4:CVX15 crystal structure may provide a binding locus for small organic molecules that is more suitable than the secondary IT1t site. This work is expected to provide modeling insights useful for future CXCR4 antagonist and X4-tropic HIV-1 based drug design efforts.
Assuntos
Fármacos Anti-HIV/farmacologia , Compostos Heterocíclicos com 1 Anel/farmacologia , Peptídeos/antagonistas & inibidores , Receptores CXCR4/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Aminoquinolinas , Fármacos Anti-HIV/química , Benzimidazóis , Sítios de Ligação/efeitos dos fármacos , Butilaminas , Cristalografia por Raios X , Compostos Heterocíclicos com 1 Anel/química , Modelos Moleculares , Estrutura Molecular , Peptídeos/química , Peptídeos/metabolismo , Receptores CXCR4/química , Receptores CXCR4/metabolismo , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-AtividadeRESUMO
Dictyostatin (DCT, 1) is a complex, flexible polyketide macrolide that demonstrates potent microtubule-polymerization activity. Both a solution structure (2a) and a possible binding mode for DCT (Conf-1) have been proposed by earlier NMR experiments. In the present study, the conformational landscape of DCT in DMSO-d(6) and methanol-d(4) was explored using extensive force-field-based conformational searches combined with geometric parameters derived from solution NMR data. The results portray a diversity of conformations for dictyostatin that illustrates the molecule's flexibility and excludes the previously suggested dominant solution conformation 2a. One conformation present in DMSO-d(6) with a 7% population (Conf-2, 0.6 kcal/mol above the global minimum at 298°) also satisfies the TR-NOESY NMR parameters of Canales et al. that characterize the taxane binding-site interaction between DCT and assembled microtubules in water. Application of several docking methods (Glide, Autodock, and RosettaLigand) has identified a low-energy binding model of the DCT/ß-tubulin complex (Pose-2/Conf-2) that is gratifyingly compatible with the emerging DCT structure-activity data.
Assuntos
Hidrocarbonetos Aromáticos com Pontes/química , Macrolídeos/química , Tubulina (Proteína)/química , Sítios de Ligação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , SoluçõesRESUMO
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)/químicaRESUMO
Nearly 35 years after its discovery and 11 years after FDA approval of paclitaxel (PTX) as a breakthrough anticancer drug, the 3-D structure of the agent bound to its beta-tubulin target was proposed to be T-Taxol. The latter bioactive form has recently been challenged by the Ojima group with a structure, "PTX-NY" ("REDOR Taxol"), in which the C-13 side chain is proposed to adopt a different conformation and an alternative hydrogen-bonding pattern in the tubulin binding site. Previously, the two conformers were compared to show that only T-Taxol fits the PTX-derived electron crystallographic density. That work has been extended by molecular mechanics and quantum chemical methods to reveal that the PTX-NY conformation is relatively less stable, on average, by 10-11 kcal/mol. In agreement with NMR studies, an 11 ns molecular dynamics treatment for PTX in an explicit water pool locates T-Taxol along the trajectory, but not PTX-NY. Docking of various PTX conformers into the electron crystallographic binding site of tubulin demonstrates that PTX-NY cannot be accommodated unless the pocket is reorganized in violation of the experimental constraints. Finally, analysis of the structures of T-Taxol and PTX-NY for their capacity to predict the existence of superpotent PTX analogues discloses that only the former forecasts such analogues, as now established by the T-Taxol-inspired synthesis of bridged taxanes. In sum, all empirical criteria support T-Taxol as the bound conformation of PTX on beta-tubulin in microtubules.
Assuntos
Paclitaxel/análogos & derivados , Paclitaxel/química , Tubulina (Proteína)/química , Conformação Molecular , Estrutura Molecular , Estereoisomerismo , Relação Estrutura-Atividade , Taxoides/químicaRESUMO
The rationale for the structural and mechanistic basis of a tetrahydroisoquinoline (THIQ) based series of CXCR4 antagonists is presented. Using the previously reported crystal structures which reveal two distinct binding sites of CXCR4 defined as the small molecule (IT1t or minor) binding pocket and peptide (CVX15 or major) binding pocket, we hypothesized our THIQ small molecule series could bind like either molecule in these respective receptor configurations (IT1t versus CVX15 based poses). To this end, a thorough investigation was performed through a combination of receptor mutation studies, medicinal chemistry, biological testing, conformational analysis, and flexible docking. Our findings showed that the CVX15 peptide-based CXCR4 receptor complexes (red pose) were consistently favored over the small molecule IT1t based CXCR4 receptor configurations (blue pose) to correctly explain the computational and mutational studies as well as key structural components of activity for these small molecules.
RESUMO
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
T-Taxol is a proposal for the bioactive conformation of paclitaxel (PTX) derived from fitting ligand conformations to the electron crystallographic (EC) density. Although confirmed by a number of studies, some structural ambiguities based on the interpretation of two solid-state REDOR (13)C-(19)F distances in a fluorinated PTX derivative remain. An evaluation of the static and dynamic properties of the PTX-tubulin complex shows that small 6-12 degrees variations in calculated torsions and a justifiable increase of the REDOR distance error to > or = +/-0.7 A readily resolves key discrepancies around T-Taxol's service as the bioactive conformation. In addition, conformational analysis reveals a range of (13)C-(19)F separations compatible with the REDOR measurements suggesting that the present PTX REDOR distances may not provide a precise model for bioactive, tubulin-bound bridged taxanes. In addition, we show that New York-Taxol (PTX-NY), a recently proposed alternative to T-Taxol, is incompatible with both the EC density and the activity of a highly active series of bridged taxanes.
Assuntos
Paclitaxel/química , Sítios de Ligação , Cristalografia , Docetaxel , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Paclitaxel/farmacologia , Estrutura Terciária de Proteína , Estereoisomerismo , Relação Estrutura-Atividade , Taxoides/química , Taxoides/farmacologia , Tubulina (Proteína)/química , Tubulina (Proteína)/efeitos dos fármacosRESUMO
The diastereomers of dibromo-7-epi-10-deacetylcephalomannine (6 and 7) have been isolated and characterized. Cytotoxicity and microtubule assembly assays demonstrate that cephalomannine analogue 6 possesses a potency profile very similar to that of Taxol, while isomer 7 is slightly less active. Solid state, solution, and tubulin-bound conformations of the two diastereomers were probed by using X-ray crystallography, 2-D NMR experiments in conjunction with the NAMFIS analysis, and the Glide docking protocol. In the crystal, isomer 7 exhibits an intermolecular halogen bond that may contribute to self-assembly. Neither crystal structure appears in the NAMFIS solution analysis, but both diastereomers are represented in solution by a T-shaped Taxol conformer. Glide docking demonstrates the latter to best fill the tubulin binding pocket, as has been shown for the parent Taxol drug. Each model of the bound complexes for 6 and 7 presents a single well-defined halogen bond from one of the ligand's bromines to Glu22 or Asp26 near the N-terminus of beta-tubulin, respectively. This first report of a halogen bond between taxanes and tubulin may prove useful in guiding the design and synthesis of other microtubule-stabilizing agents with a similar capacity.
Assuntos
Antineoplásicos/química , Bromo/química , Modelos Moleculares , Taxoides/química , Antineoplásicos/farmacologia , Sítios de Ligação , Biopolímeros , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Paclitaxel/química , Estereoisomerismo , Relação Estrutura-Atividade , Taxoides/farmacologia , Termodinâmica , Tubulina (Proteína)/químicaRESUMO
[chemical structure: see text]. T-Taxol has been proposed as the bioactive conformation on beta-tubulin and subsequently utilized in the design of a series of highly active bridged taxane analogues. A modified T-form with a reversed C-13 side chain orientation has recently been proposed as an equally plausible bioactive shape. A comparison of the two spatial alternatives within the tubulin binding site electron crystallographic density suggests strongly that T-Taxol is the bioactive conformation.
Assuntos
Modelos Moleculares , Paclitaxel/análogos & derivados , Paclitaxel/química , Tubulina (Proteína)/química , Sítios de Ligação , Conformação Molecular , Estrutura MolecularRESUMO
A de novo hit-to-lead effort involving the redesign of benzimidazole-containing antagonists of the CXCR4 receptor resulted in the discovery of a novel series of 1,2,3,4-tetrahydroisoquinoline (TIQ) analogues. In general, this series of compounds show good potencies (3-650 nM) in assays involving CXCR4 function, including both inhibition of attachment of X4 HIV-1IIIB virus in MAGI-CCR5/CXCR4 cells and inhibition of calcium release in Chem-1 cells. Series profiling permitted the identification of TIQ-(R)-stereoisomer 15 as a potent and selective CXCR4 antagonist lead candidate with a promising in vitro profile. The drug-like properties of 15 were determined in ADME in vitro studies, revealing low metabolic liability potential. Further in vivo evaluations included pharmacokinetic experiments in rats and mice, where 15 was shown to have oral bioavailability (F = 63%) and resulted in the mobilization of white blood cells (WBCs) in a dose-dependent manner.
RESUMO
Taxanes and other microtubule-targeting drugs (MTDs) represent one of the most effective classes of cancer chemotherapeutics. However, ultimately their utility is limited due to drug-induced myelosuppression. Here we identify 2-Methoxyestradiol (2ME2) as the first MTD able to specifically target tumor cells while sparing the bone marrow from dose-limiting, life-threatening toxicities. Following drug selection with 2ME2, epithelial cancer cells acquired a tubulin mutation at Vbeta236I that impaired the 2ME2-tubulin interaction and rendered cells resistant to 2ME2. We further show that the hematopoietic-specific Hbeta1 tubulin isotype naturally encodes Ibeta236 and is insensitive to 2ME2. Systemic administration of 2ME2 in C57BL6 mice revealed that there was no effect on bone marrow microtubules, in contrast to the taxane or Vinca alkaloid induced toxicities. Similar results were obtained upon drug treatment of human bone marrow and CD34-positive stem/progenitor cells. Herein, we describe the first isotype-targeted chemotherapeutic, setting a new paradigm for the entire class of MTDs, and providing a model that could allow the design of new tubulin inhibitors devoid of myelosuppression. The ability to design a drug with minimal side-effects would significantly augment the chances of clinical success by allowing the use of a truly therapeutic dose rather than the maximally tolerated.
Assuntos
Estradiol/análogos & derivados , Moduladores de Tubulina/farmacologia , Tubulina (Proteína)/metabolismo , 2-Metoxiestradiol , Sequência de Aminoácidos , Animais , Antineoplásicos/toxicidade , Hidrocarbonetos Aromáticos com Pontes/toxicidade , Estradiol/farmacologia , Hematopoese , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microtúbulos/efeitos dos fármacos , Dados de Sequência Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Células-Tronco/efeitos dos fármacos , Taxoides/toxicidade , Tubulina (Proteína)/químicaRESUMO
Microtubule binding and tubulin assembly promotion by a series of conformationally restricted paclitaxel (PTX) derivatives was investigated. In these derivatives, the C-4 acetate of the taxane is tethered to the C-3' phenyl at ortho and meta positions with different length linkers. The apparent affinity of these derivatives for GMPCPP-stabilized microtubules was assessed by a competition assay, and their influence on microtubule polymerization was evaluated by measuring the critical concentration of GDP-tubulin in the presence of the respective molecule. In general, taxane derivatives with higher apparent affinity for microtubules induced tubulin assembly more efficiently. Among the derivatives, molecules with the shortest tether display the strongest affinity for microtubules. These derivatives exhibited enhanced microtubule stabilization properties and efficiently induced GDP-tubulin assembly into microtubules at low temperature of 12 degrees C and in the absence of Mg2+ ions in 0.1 M PIPES. Based on molecular dynamics simulations, we propose that the enhanced ability to assemble microtubules by these taxane derivatives is linked to their ability to effectively shape the conformation of the M-loop of tubulin for cross-protofilament interaction.
Assuntos
Microtúbulos/metabolismo , Paclitaxel/análogos & derivados , Paclitaxel/química , Tubulina (Proteína)/metabolismo , Animais , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/farmacologia , Ligação Competitiva , Encéfalo , Bovinos , Cinética , Modelos Moleculares , Conformação Molecular , Paclitaxel/farmacologia , Relação Estrutura-AtividadeRESUMO
In combination with chemical modifications, bioassays, and computational simulation techniques, C-2 benzoylthio, and benzylthio taxoids were synthesized, biologically evaluated, and their binding conformations rationalized, in order to probe the interaction of taxane molecule with beta-tubulin.
Assuntos
Paclitaxel/análogos & derivados , Enxofre/química , Taxoides/química , Moduladores de Tubulina/química , Tubulina (Proteína)/química , Humanos , Conformação Molecular , Conformação Proteica , Relação Estrutura-Atividade , Taxoides/síntese química , Moduladores de Tubulina/síntese químicaRESUMO
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 strategy for the design and synthesis of simplified paclitaxel analogs based on the T-Taxol conformation is presented. The resulting compounds have both cytotoxic and tubulin polymerization activities, although less so than those of paclitaxel itself.