RESUMO
Gene therapy could offer improvement in the treatment of glaucoma compared to the current standard of lowering intraocular pressure. We have developed and characterized non-viral gemini surfactant-phospholipid nanoparticles (GL-NPs) for intravitreal and topical administration. Optimized GL-NPs (size range 150-180 nm) were biocompatible with rat retinal ganglion (RGC-5) cells with >95% viability by PrestoBlue™ assay. GL-NPs carrying Cy5-labeled plasmid DNA demonstrated distinct trafficking behavior and biodisposition within the eye in vivo after intravitreal or topical application with respect to pathways of movement and physicochemical stability. After intravitreal injection in mice, GL-NPs localized within the nerve fiber layer of the retina, whereas after topical application, GL-NPs were located in several anterior chamber tissues, including the limbus, iris and conjunctiva. GL-NPs were thermodynamically stable in the vitreous and tear fluid and were trafficked as single, non-aggregated particles after both types of administration. FROM THE CLINICAL EDITOR: In this paper, the development and characterization of non-viral gemini surfactant-phospholipid nanoparticles is reported with the goal of establishing a gene delivery system that addresses glaucoma in a non-invasive fashion. The authors found that after topical application, the concentration of these nanoparticles was higher in anterior chamber-related components of the eye, whereas intra-vitreal administration resulted in accumulation in the retinal nerve fibre layer.
Assuntos
Olho/metabolismo , Terapia Genética/métodos , Glaucoma/terapia , Nanopartículas/química , Administração Tópica , Animais , Técnicas de Transferência de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The human Aurora kinase-A (AK-A) is an essential mitotic regulator that is frequently overexpressed in several cancers. The recent development of several novel AK-A inhibitors has been driven by the well-established association of this target with cancer development and progression. However, resistance and cross-reactivity with similar kinases demands an improvement in our understanding of key molecular interactions between the Aurora kinase-A substrate binding pocket and potential inhibitors. Here, we describe the implementation of state-of-the-art virtual screening techniques to discover a novel set of Aurora kinase-A ligands that are predicted to strongly bind not only to the wild type protein, but also to the T217D mutation that exhibits resistance to existing inhibitors. Furthermore, a subset of these computationally screened ligands was shown to be more selective toward the mutant variant over the wild type protein. The description of these selective subsets of ligands provides a unique pharmacological tool for the design of new drug regimens aimed at overcoming both kinase cross-reactivity and drug resistance associated with the Aurora kinase-A T217D mutation.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Aurora Quinase A/genética , Resistência a Medicamentos/efeitos dos fármacos , Mutação/genética , Inibidores de Proteínas Quinases/química , Simulação por Computador , Humanos , LigantesRESUMO
The outermost layer of the skin, known as the stratum corneum (SC), is composed of dead corneocytes embedded in an intercellular lipid matrix consisting of ceramides, free fatty acids, and cholesterol. The high level of organization within this matrix protects the body by limiting the permeation of most compounds through the skin. While essential for its protective functions, the SC poses a significant barrier for the delivery of topically applied pharmaceutical agents. Chemical permeation enhancers (CPEs) can increase delivery of small drug compounds into the skin by interacting with the intercellular lipids through physical processes including extraction, fluidization, increased disorder, and phase separation. However, it is not clear whether these same mechanisms are involved in delivery of biotherapeutic macromolecules, such as proteins. Here we describe the effect of three categories of CPEs {solvents [ethanol, propylene glycol, diethylene glycol monoethyl ether (transcutol), oleic acid], terpenes [menthol, nerol, camphor, methyl salicylate], and surfactants [Tween 80, SDS, benzalkonium chloride, polyoxyl 40 hydrogenated castor oil (Cremophor RH40), didecyldimethylammonium bromide (DDAB), didecyltrimethylammonium bromide (DTAB)]} on the lipid organizational structure of human SC as determined by X-ray scattering studies. Small- and wide-angle X-ray scattering studies were conducted to correlate the degree of structural changes and hydrocarbon chain packing in SC lipids caused by these various classes of CPEs to the extent of permeation of interferon alpha-2b (IFNα), a 19 kDa protein drug, into human skin. With the exception of solvents, propylene glycol and ethanol, all classes of CPEs caused increased disordering of lamellar and lateral packing of lipids. We observed that the highest degree of SC lipid disordering was caused by surfactants (especially SDS, DDAB, and DTAB) followed by terpenes, such as nerol. Interestingly, in vitro skin permeation studies indicated that, in most cases, absorption of IFNα was low and that an increase in SC lipid disorder does not correspond to an increase in IFNα absorption.
Assuntos
Interferon-alfa/metabolismo , Mama/metabolismo , Feminino , Humanos , Técnicas In Vitro , Microscopia Confocal , Absorção Cutânea/fisiologiaRESUMO
Several isotypes of the structural protein tubulin have been characterized. Their expression offers a plausible explanation for differences regarding microtubule function. Although sequence variation between tubulin isotypes occurs throughout the entire protein, it is the extreme carboxy-terminal tails (CTTs) that exhibit the greatest concentration of differences. In humans, the CTTs range in length from 9 to 25 residues and because of a considerable number of glutamic acid residues, contain over 1/3 of tubulin's total electrostatic charge. The CTTs are believed to be highly disordered and their precise function has yet to be determined. However, their absence has been shown to result in altered microtubule stability and a reduction in the interaction with several microtubule-associated proteins (MAPs). To characterize the role that CTTs play in microtubule function, we examined the global conformational differences within a set of nine human beta-tubulin isotypes using replica exchange molecular dynamics simulations. Through the analysis of the resulting configuration ensembles, we quantified differences such as the CTTs sequence influence on overall flexibility and average secondary structure. Although only minor variations between each CTT were observed, we suggest that these differences may be significant enough to affect interactions with MAPs, thereby influencing important properties such as microtubule assembly and stability.
Assuntos
Tubulina (Proteína)/química , Algoritmos , Análise por Conglomerados , Biologia Computacional/métodos , Computadores , Cristalografia por Raios X/métodos , Humanos , Microtúbulos/metabolismo , Conformação Molecular , Peptídeos/química , Análise de Componente Principal , Conformação Proteica , Isoformas de Proteínas , Estrutura Secundária de Proteína , Estrutura Terciária de ProteínaRESUMO
Numerous isotypes of the structural protein tubulin have now been characterized in various organisms and their expression offers a plausible explanation for observed differences affecting microtubule function in vivo. While this is an attractive hypothesis, there are only a handful of studies demonstrating a direct influence of tubulin isotype composition on the dynamic properties of microtubules. Here, we present the results of experimental assays on the assembly of microtubules from bovine brain tubulin using purified isotypes at various controlled relative concentrations. A novel data analysis is developed using recursive maps which are shown to be related to the master equation formalism. We have found striking similarities between the three isotypes of bovine tubulin studied in regard to their dynamic instability properties, except for subtle differences in their catastrophe frequencies. When mixtures of tubulin isotypes are analyzed, their nonlinear concentration dependence is modeled and interpreted in terms of lower affinities of tubulin dimers belonging to the same isotype than those that represent different isotypes indicating hitherto unsuspected influences of tubulin dimers on each other within a microtubule. Finally, we investigate the fluctuations in microtubule assembly and disassembly rates and conclude that the inherent rate variability may signify differences in the guanosine-5'-triphosphate composition of the growing and shortening microtubule tips. It is the main objective of this article to develop a quantitative model of tubulin polymerization for individual isotypes and their mixtures. The possible biological significance of the observed differences is addressed.
Assuntos
Microtúbulos/química , Microtúbulos/ultraestrutura , Modelos Químicos , Modelos Moleculares , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestrutura , Misturas Complexas/química , Simulação por Computador , Complexos Multiproteicos/química , Complexos Multiproteicos/ultraestrutura , Conformação ProteicaRESUMO
Over the past decade the application of gene therapy of retinal diseases such as glaucoma has produced promising results. However, optic nerve regeneration and restoration of vision in patients with glaucoma is still far from reality. Neuroprotective approaches in the form of gene therapy may provide significant advantages, but are still limited by many factors both at the organ and cellular levels. In general, gene delivery systems for eye diseases range from simple eye drops and ointments to more advanced bio- and nanotechnology-based systems such as muco-adhesive systems, polymers, liposomes and ocular inserts. Most of these technologies were developed for front-of-the-eye ophthalmic therapies and are not applicable as back-of-the-eye delivery systems. Currently, only the invasive intravitreal injections are capable of successfully delivering genes to the retina. Here we review the challenges and possible strategies for the noninvasive gene therapy of glaucoma including the barriers in the eye and in neural cells, and present a cross-sectional view of gene delivery as it pertains to the prevention and treatment of glaucoma.
Assuntos
Técnicas de Transferência de Genes , Terapia Genética/métodos , Glaucoma/genética , Glaucoma/terapia , Animais , Sistemas de Liberação de Medicamentos/métodos , Olho/metabolismo , Olho/patologia , Glaucoma/patologia , HumanosRESUMO
BACKGROUND: Carbon nanotubes (CNTs) are novel materials with considerable potential in many areas related to nanomedicine. However, a major limitation in the development of CNT-based therapeutic nanomaterials is a lack of reliable and reproducible data describing their chemical and structural composition. Knowledge of properties including purity, structural quality, dispersion state, and concentration are essential before CNTs see widespread use in in vitro and in vivo experiments. In this work, we describe the characterization of several commercially available and two in-house-produced CNT samples and discuss the physicochemical profiles that will support their use in nanomedicine. METHODS: Eighteen single-walled and multi-walled CNT raw materials were characterized using established analytical techniques. Solid CNT powders were analyzed for purity and structural quality using thermogravimetric analysis and Raman spectroscopy. Extinction coefficients for each CNT sample were determined by ultraviolet-visible near infrared absorption spectroscopy. Standard curves for each CNT sample were generated in the 0-5 µg/mL concentration range for dispersions prepared in 1,2-dichlorobenzene. RESULTS: Raman spectroscopy and thermogravimetric analysis results demonstrated that CNT purity and overall quality differed substantially between samples and manufacturer sources, and were not always in agreement with purity levels claimed by suppliers. Absorbance values for individual dispersions were found to have significant variation between individual single-walled CNTs and multi-walled CNTs and sources supplying the same type of CNT. Significant differences (P < 0.01) in extinction coefficients were observed between and within single-walled CNTs (24.9-53.1 mL·cm(-1)·mg(-1)) and multi-walled CNTs (49.0-68.3 mL·cm(-1)·mg(-1)). The results described here suggest a considerable role for impurities and structural inhomogeneities within individual CNT preparations and the resulting spectroscopic properties of their dispersions. CONCLUSION: Raw CNT materials require thorough analytical workup before they can be used as nanoexcipients. This applies especially to the determination of CNT purity, structure, and concentration. The results presented here clearly demonstrate that extinction coefficients must be determined for individual CNT preparations prior to their use.
Assuntos
Nanotubos de Carbono/química , Análise de Variância , Clorobenzenos/química , Excipientes/química , Nanomedicina , Espectrofotometria Ultravioleta , Espectroscopia de Luz Próxima ao Infravermelho , Análise Espectral Raman , TermogravimetriaRESUMO
Cytoskeletal proteins, such as tubulin, are a primary target for many successful anti-cancer drugs. The expression of several beta-tubulin isotypes in normal and cancerous cells provides a platform upon which to construct chemotherapeutic agents capable of differentiating between them. To test this hypothesis, we have previously designed several colchicine derivatives and computationally probed them for affinity to the beta-tubulin isotypes. Subsequent synthesis and cytotoxicity assays produced a small set of promising compounds exhibiting IC(50) values approximately 30 fold lower than values previously reported for colchicine. Here we describe the creation and testing of these first-generation colchicine derivatives and discuss the subsequent design and preliminary computational screening of a novel set of second-generation derivatives using the most promising first-generation derivatives as scaffolds.
Assuntos
Colchicina/química , Antineoplásicos/farmacologia , Sítios de Ligação , Linhagem Celular Tumoral , Citoesqueleto/metabolismo , Células HeLa , Humanos , Concentração Inibidora 50 , Modelos Químicos , Isoformas de Proteínas , Software , Termodinâmica , Tubulina (Proteína)/química , Moduladores de Tubulina/químicaRESUMO
Conventional approaches to site mapping have so far failed to identify the laulimalide binding site on microtubules. Using mass shift perturbation analysis and data-directed docking, we demonstrate that laulimalide binds to the exterior of the microtubule on beta-tubulin, in a region previously unknown to support ligand binding and well removed from the paclitaxel site. Shift maps for docetaxel and laulimalide are otherwise identical, indicating a common state of microtubule stability induced by occupancy of the distinct sites. The preferred binding mode highlights the penetration of the laulimalide side chain into a deep, narrow cavity through a unique conformation not strongly populated in solution, akin to a "striking cobra." This mode supports the development of a pharmacophore model and reveals the importance of the C1-C15 axis in the macrocycle.
Assuntos
Antineoplásicos/metabolismo , Macrolídeos/metabolismo , Espectrometria de Massas/métodos , Microtúbulos/metabolismo , Regulação Alostérica , Amidas/química , Animais , Antineoplásicos/química , Bovinos , Medição da Troca de Deutério , Ligantes , Macrolídeos/química , Simulação de Dinâmica Molecular , Conformação ProteicaRESUMO
Tubulin, the primary subunit of microtubules, is remarkable for the variety of small molecules to which it binds. Many of these are very useful or promising agents in cancer chemotherapy. One of the most useful of these is paclitaxel. The tubulin molecule is itself an alpha/beta heterodimer, both alpha- and beta-tubulin monomers existing as multiple isotypes. Despite the success of paclitaxel as an anticancer drug, resistance often occurs in cancer cells and has been associated with variations in tubulin isotype expression, most notably with the increased expression of betaIII-tubulin. Paclitaxel is thought to reach its binding site on beta-tubulin by diffusion through nanopores in the microtubule wall. It has been suggested that a transitional step in this process may be the binding of paclitaxel to an intermediate site within a nanopore, from which it moves directly to its binding site in the microtubule interior facing the lumen. To test this hypothesis, we have computationally docked paclitaxel within a microtubule nanopore and simulated its passage to the intermediate binding site. Targeted molecular dynamics was then used to test the hypothesis that paclitaxel utilizes the H6/H7 loop as a hinge to move directly from this intermediate binding site to its final position in the luminal binding site. We observed that this motion appears to be stabilized by the formation of a hydrogen bond involving serine 275 in beta-tubulin isotypes I, IIa, IIb, IVa, IVb, V, VII, and VIII. Interestingly, this residue is replaced by alanine in the betaIII and VI isotypes. This observation raises the possibility that the observed isotype difference in paclitaxel binding may be a kinetic effect arising from the isotype difference at this residue. We are now able to suggest derivatives of paclitaxel that may reverse the isotype-specificity or lead to an alternate stabilizing hydrogen-bond interaction with tubulin, thus increasing the rate of passage to the luminal binding site and hopefully offering a therapeutic advantage in paclitaxel resistant cases.
Assuntos
Antineoplásicos/metabolismo , Microtúbulos/metabolismo , Paclitaxel/metabolismo , Antineoplásicos/química , Sítios de Ligação , Modelos Moleculares , Paclitaxel/químicaRESUMO
Tubulin is the target for numerous small molecule ligands which alter microtubule dynamics leading to cell cycle arrest and apoptosis. Many of these ligands are currently used clinically for the treatment of several types of cancer, and they bind to one of three distinct binding sites within beta-tubulin (paclitaxel, vinca, and colchicine), all of which have been identified crystallographically. Unfortunately, serious side effects always accompany chemotherapy since these drugs bind to tubulin indiscriminately, leading to the death of both cancerous and healthy cells. However, the existence and distribution of divergent tubulin isoforms provide a platform upon which we may build novel chemotherapeutic drugs that can differentiate between different cell types and therefore reduce undesirable side effects. We report results of computational analysis that aims at predicting differences between the binding energies of a family of colchicine derivatives against 10 human alpha/beta-tubulin isoforms. Free energy perturbation method has been used in our calculations and the results provide a proof of principle by indicating significant differences both among the derivatives and between tubulin isoforms.
Assuntos
Colchicina/análogos & derivados , Colchicina/química , Modelos Químicos , Termodinâmica , Tubulina (Proteína)/química , Sítios de Ligação/efeitos dos fármacos , Colchicina/farmacologia , Simulação por Computador , Modelos Moleculares , Estrutura Molecular , Isoformas de Proteínas/química , Isoformas de Proteínas/efeitos dos fármacos , Tubulina (Proteína)/efeitos dos fármacosRESUMO
Microtubules are involved in numerous cellular processes including chromosome segregation during mitosis and, as a result, their constituent protein, tubulin, has become a successful target of several chemotherapeutic drugs. In general, these drugs bind indiscriminately to tubulin within both cancerous and healthy cells, resulting in unwanted side effects. However, differences between beta-tubulin isotypes expressed in a wide range of cell types may aid in the development of anti-tubulin drugs having increased specificity for only certain types of cells. Here, we describe a digital signal processing (DSP) method that is capable of predicting hot spots for the tubulin family of proteins as well as determining relative differences in binding affinities to these hot spots based only on the primary sequence of 10 human tubulin isotypes. Due to the fact that several drug binding sites have already been characterized within beta-tubulin, we are able to correlate hot spots with the binding sites for known chemotherapy drugs. We have also verified the accuracy of this method using the correlation between the binding affinities of characterized drugs and the tubulin isotypes. Additionally, the DSP method enables the rapid estimation of relative differences in binding affinities within the binding sites of tubulin isotypes that are yet to be experimentally determined.
Assuntos
Preparações Farmacêuticas , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Sítios de Ligação , Simulação por Computador , Humanos , Ligantes , Modelos Moleculares , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , Tubulina (Proteína)/classificaçãoRESUMO
Microtubules are significant therapeutic targets for the treatment of cancer, where suppression of microtubule dynamicity by drugs such as paclitaxel forms the basis of clinical efficacy. Peloruside A, a macrolide isolated from New Zealand marine sponge Mycale hentscheli, is a microtubule-stabilizing agent that synergizes with taxoid drugs through a unique site and is an attractive lead compound in the development of combination therapies. We report here unique allosteric properties of microtubule stabilization via peloruside A and present a structural model of the peloruside-binding site. Using a strategy involving comparative hydrogen-deuterium exchange mass spectrometry of different microtubule-stabilizing agents, we suggest that taxoid-site ligands epothilone A and docetaxel stabilize microtubules primarily through improved longitudinal interactions centered on the interdimer interface, with no observable contributions from lateral interactions between protofilaments. The mode by which peloruside A achieves microtubule stabilization also involves the interdimer interface, but includes contributions from the alpha/beta-tubulin intradimer interface and protofilament contacts, both in the form of destabilizations. Using data-directed molecular docking simulations, we propose that peloruside A binds within a pocket on the exterior of beta-tubulin at a previously unknown ligand site, rather than on alpha-tubulin as suggested in earlier studies.
Assuntos
Antineoplásicos Fitogênicos/química , Compostos Bicíclicos Heterocíclicos com Pontes/metabolismo , Lactonas/metabolismo , Microtúbulos/metabolismo , Estrutura Quaternária de Proteína , Regulação Alostérica , Sequência de Aminoácidos , Animais , Antineoplásicos Fitogênicos/metabolismo , Sítios de Ligação , Compostos Bicíclicos Heterocíclicos com Pontes/química , Bovinos , Dimerização , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/química , Lactonas/química , Ligantes , Espectrometria de Massas , Microtúbulos/química , Modelos Moleculares , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Alinhamento de Sequência , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/metabolismoRESUMO
The antitumor drug paclitaxel stabilizes microtubules and reduces their dynamicity, promoting mitotic arrest and eventually apoptosis. Upon assembly of the alpha/beta-tubulin heterodimer, GTP becomes bound to both the alpha and beta-tubulin monomers. During microtubule assembly, the GTP bound to beta-tubulin is hydrolyzed to GDP, eventually reaching steady-state equilibrium between free tubulin dimers and those polymerized into microtubules. Tubulin-binding drugs such as paclitaxel interact with beta-tubulin, resulting in the disruption of this equilibrium. In spite of several crystal structures of tubulin, there is little biochemical insight into the mechanism by which anti-tubulin drugs target microtubules and alter their normal behavior. The mechanism of drug action is further complicated, as the description of altered beta-tubulin isotype expression and/or mutations in tubulin genes may lead to drug resistance as has been described in the literature. Because of the relationship between beta-tubulin isotype expression and mutations within beta-tubulin, both leading to resistance, we examined the properties of altered residues within the taxane, colchicine and Vinca binding sites. The amount of data now available, allows us to investigate common patterns that lead to microtubule disruption and may provide a guide to the rational design of novel compounds that can inhibit microtubule dynamics for specific tubulin isotypes or, indeed resistant cell lines. Because of the vast amount of data published to date, we will only provide a broad overview of the mutational results and how these correlate with differences between tubulin isotypes. We also note that clinical studies describe a number of predictive factors for the response to anti-tubulin drugs and attempt to develop an understanding of the features within tubulin that may help explain how they may affect both microtubule assembly and stability.
RESUMO
Within the ubiquitin degradation pathway, the canonical signal is a lysine 48-linked polyubiquitin chain that is assembled upon an internal lysine residue of a substrate protein. Once constructed, this ubiquitin chain becomes the principle signal for recognition and target degradation by the 26S proteasome. The mechanism by which polyubiquitin chains are assembled on a substrate protein, however, has yet to be clearly defined. In an in vitro model system, purified E2-ubiquitin thiolester was unable to catalyze the formation of polyubiquitin chains in the absence of the ubiquitin-activating enzyme E1. Mutagenesis of key residues within the E1 active site revealed that its conserved catalytic cysteine residue is essential for the formation of these chains. Moreover, inactivation of the E2 active site had no effect on the ability of E1 to catalyze ubiquitin chain formation. These findings strongly suggest E1 is responsible for not only the activation of ubiquitin but also for the direct catalytic extension of a lysine 48-linked polyubiquitin chain.
Assuntos
Lisina/química , Poliubiquitina/química , Enzimas Ativadoras de Ubiquitina/química , Sítios de Ligação , Catálise , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Ligases/química , Mutagênese , Processamento de Proteína Pós-Traducional , Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/químicaRESUMO
Using comparative modeling, we have generated structural models of 475 alpha and beta tubulins. Using these models, we observed a global, structural similarity between the tubulin isotypes. However, a number of subtle differences in the isotypes physical properties, including net electric charges, solvent accessible surface areas, and electric dipole moments were also apparent. In order to examine the roles that these properties may play in microtubule (MT) assembly and stability, we have created a model to evaluate the dipole-dipole interaction energies of varying MT lattice conformations, using human tubulin isotypes as particularly important examples. We conclude that the dipole moments of each tubulin isotype may influence their functional characteristics within the cell, resulting in differences for MT assembly kinetics and stability.