RESUMEN
Maurocalcine (MCa) is the first natural cell penetrating peptide to be discovered in animal venom. In addition to the fact that it represents a potent vector for the cell penetration of structurally diverse therapeutic compounds, MCa also displays several distinguishing features that make it a potential peptide of choice for clinical and biotechnological applications. The aim of the present study was to gain new information about the properties of MCa in vivo in order to delineate the future potential applications of this vector. For this purpose, two analogues of this peptide with (Tyr-MCa) and without (Lin-Tyr-MCa) disulfide bridges were synthesized, radiolabeled with (125)I, and their in vitro stabilities were first evaluated in mouse blood. The results indicated that (125)I-Tyr-MCa was stable in vitro and that the disulfide bridges conferred a competitive advantage for the stability of peptide. Following in vivo injection in mice, (125)I-Tyr-MCa targeted peripheral organs with interesting quantitative differences and the main route of peptide elimination was renal.
Asunto(s)
Péptidos de Penetración Celular/farmacocinética , Venenos de Escorpión/farmacocinética , Animales , Péptidos de Penetración Celular/administración & dosificación , Péptidos de Penetración Celular/síntesis química , Cromatografía Líquida de Alta Presión , Estabilidad de Medicamentos , Marcaje Isotópico , Ratones , Tomografía de Emisión de Positrones , Venenos de Escorpión/administración & dosificación , Venenos de Escorpión/síntesis química , Distribución Tisular , Microtomografía por Rayos XRESUMEN
Maurocalcine is the first demonstrated example of an animal toxin peptide with efficient cell penetration properties. Although it is a highly competitive cell-penetrating peptide (CPP), its relatively large size of 33 amino acids and the presence of three internal disulfide bridges may hamper its development for in vitro and in vivo applications. Here, we demonstrate that several efficient CPPs can be derived from maurocalcine by replacing Cys residues by isosteric 2-aminobutyric acid residues and sequence truncation down to peptides of up to 9 residues in length. A surprising finding is that all of the truncated maurocalcine analogues possessed cell penetration properties, indicating that the maurocalcine is a highly specialized CPP. Careful examination of the cell penetration properties of the truncated analogues indicates that several maurocalcine-derived peptides should be of great interest for cell delivery applications where peptide size matters.
Asunto(s)
Péptidos de Penetración Celular/farmacología , Venenos de Escorpión/farmacología , Animales , Células CHO , Péptidos de Penetración Celular/síntesis química , Péptidos de Penetración Celular/química , Cricetinae , Cricetulus , Venenos de Escorpión/síntesis química , Venenos de Escorpión/químicaRESUMEN
T-type calcium channels represent a key pathway for Ca(2+) entry near the resting membrane potential. Increasing evidence supports a unique role of these channels in fast and low-threshold exocytosis in an action potential-independent manner, but the underlying molecular mechanisms have remained unknown. Here, we report the existence of a syntaxin-1A/Ca(v)3.2 T-type calcium channel signaling complex that relies on molecular determinants that are distinct from the synaptic protein interaction site (synprint) found in synaptic high voltage-activated calcium channels. This interaction potently modulated Ca(v)3.2 channel activity, by reducing channel availability. Other members of the T-type calcium channel family were also regulated by syntaxin-1A, but to a smaller extent. Overexpression of Ca(v)3.2 channels in MPC 9/3L-AH chromaffin cells induced low-threshold secretion that could be prevented by uncoupling the channels from syntaxin-1A. Altogether, our findings provide compelling evidence for the existence of a syntaxin-1A/T-type Ca(2+) channel signaling complex and provide new insights into the molecular mechanism by which these channels control low-threshold exocytosis.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Exocitosis/fisiología , Complejos Multiproteicos/metabolismo , Transducción de Señal/fisiología , Sintaxina 1/metabolismo , Canales de Calcio Tipo T/genética , Línea Celular , Humanos , Complejos Multiproteicos/genética , Sintaxina 1/genéticaRESUMEN
Maurocalcine has been the first demonstrated animal toxin acting as a cell-penetrating peptide. Although it possesses competitive advantages, its use as a cell-penetrating peptide (CPP) requires that analogues be developed that lack its characteristic pharmacological activity on ryanodine-sensitive calcium channels without affecting its cell-penetrating and vector efficiencies. Here, we present the synthesis, three-dimensional (1)H NMR structure, and activity of D-maurocalcine. We demonstrate that it possesses all of the desired features for an excellent CPP: preserved structure, lack of pharmacological action, conserved vector properties, and absence of cell toxicity. This is the first report of a folded/oxidized animal toxin in its D-diastereomer conformation for use as a CPP. The protease resistance of this new peptide analogue, combined with its efficient cell penetration at concentrations devoid of cell toxicity, suggests that D-maurocalcine should be an excellent vector for in vivo applications.
Asunto(s)
Péptidos/química , Venenos de Escorpión/química , Animales , Células CHO , Canales de Calcio/química , Membrana Celular/metabolismo , Dicroismo Circular , Cricetinae , Cricetulus , Fluoresceínas/química , Espectroscopía de Resonancia Magnética/métodos , Microscopía Confocal/métodos , Péptido Hidrolasas/química , Rianodina/química , Venenos de Escorpión/farmacología , Sales de Tetrazolio/farmacología , Tiazoles/farmacologíaRESUMEN
The interest of the scientific community for cell penetrating peptides (CPP) has been growing exponentially for these last years, and the list of novel CPP is increasing. These peptides are powerful tools for the delivery of cargoes to their site of action. Indeed, several drugs that cannot translocate through the cell plasma membrane have been successfully delivered into cells when grafted to a CPP. Various cargoes have been linked to CPP, such as oligonucleotides, pharmacologically active drugs, contrast agents for imaging, or nanoparticles as platforms for multigrafting purposes This review illustrates the fabulous potential of CPP and the diversity of their use, but their most interesting application appears their future clinical use for the treatment of various pathological conditions.
Asunto(s)
Péptidos de Penetración Celular/administración & dosificación , Portadores de Fármacos/administración & dosificación , Sistemas de Liberación de Medicamentos , Secuencia de Aminoácidos , Animales , Antineoplásicos/administración & dosificación , Transporte Biológico , Proteínas Portadoras/administración & dosificación , Proteínas Portadoras/farmacocinética , Permeabilidad de la Membrana Celular , Péptidos de Penetración Celular/farmacocinética , Portadores de Fármacos/farmacocinética , Endocitosis , Colorantes Fluorescentes/administración & dosificación , Productos del Gen tat/administración & dosificación , Productos del Gen tat/farmacocinética , Humanos , Modelos Biológicos , Imagen Molecular/métodos , Datos de Secuencia Molecular , Nanopartículas/administración & dosificación , Péptidos/administración & dosificación , Péptidos/farmacocinética , Venenos de Escorpión/administración & dosificación , Venenos de Escorpión/farmacocinéticaRESUMEN
Quantum dots (QDs) are ideal scaffolds for the development of multimodal imaging agents, but their application in clinical diagnostics is limited by the toxicity of classical CdSe QDs. A new bimodal MRI/optical nanosized contrast agent with high gadolinium payload has been prepared through direct covalent attachment of up to 80 Gd(III) chelates on fluorescent nontoxic InP/ZnS QDs. It shows a high relaxivity of 900 mM(-1) s(-1) (13 mM(-1 )s(-1) per Gd ion) at 35 MHz (0.81 T) and 298 K, while the bright luminescence of the QDs is preserved. Eu(III) and Tb(III) chelates were also successfully grafted to the InP/ZnS QDs. The absence of energy transfer between the QD and lanthanide emitting centers results in a multicolor system. Using this convenient direct grafting strategy additional targeting ligands can be included on the QD. Here a cell-penetrating peptide has been co-grafted in a one-pot reaction to afford a cell-permeable multimodal multimeric MRI contrast agent that reports cellular localization by fluorescence and provides high relaxivity and increased tissue retention with respect to commercial contrast agents.
Asunto(s)
Quelantes/química , Indio/química , Indio/metabolismo , Elementos de la Serie de los Lantanoides/química , Imagen Molecular/métodos , Fosfinas/química , Fosfinas/metabolismo , Puntos Cuánticos , Animales , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Gadolinio/química , Imagen por Resonancia Magnética , Fenómenos Ópticos , Compuestos Organometálicos/química , Permeabilidad , Ratas , Espectrometría de Fluorescencia , Sulfuros/química , Propiedades de Superficie , Compuestos de Zinc/químicaRESUMEN
PURPOSE: Synchrotron microbeam radiation therapy (MRT) relies on spatial fractionation of the incident photon beam into parallel micron-wide beams. Our aim was to analyze the effects of MRT on normal brain and 9L gliosarcoma tissues, particularly on blood vessels. METHODS AND MATERIALS: Responses to MRT (two arrays, one lateral, one anteroposterior (2 × 400 Gy), intersecting orthogonally in the tumor region) were studied during 6 weeks using MRI, immunohistochemistry, and vascular endothelial growth factor Western blot. RESULTS: MRT increased the median survival time of irradiated rats (×3.25), significantly increased blood vessel permeability, and inhibited tumor growth; a cytotoxic effect on 9L cells was detected 5 days after irradiation. Significant decreases in tumoral blood volume fraction and vessel diameter were measured from 8 days after irradiation, due to loss of endothelial cells in tumors as detected by immunochemistry. Edema was observed in the normal brain exposed to both crossfired arrays about 6 weeks after irradiation. This edema was associated with changes in blood vessel morphology and an overexpression of vascular endothelial growth factor. Conversely, vascular parameters and vessel morphology in brain regions exposed to one of the two arrays were not damaged, and there was no loss of vascular endothelia. CONCLUSIONS: We show for the first time that preferential damage of MRT to tumor vessels versus preservation of radioresistant normal brain vessels contributes to the efficient palliation of 9L gliosarcomas in rats. Molecular pathways of repair mechanisms in normal and tumoral vascular networks after MRT may be essential for the improvement of such differential effects on the vasculature.