RESUMEN
Cross-priming allows dendritic cells (DCs) to induce cytotoxic T cell (CTL) responses to extracellular antigens. DCs require cognate 'licensing' for cross-priming, classically by helper T cells. Here we demonstrate an alternative mechanism for cognate licensing by natural killer T (NKT) cells recognizing microbial or synthetic glycolipid antigens. Such licensing caused cross-priming CD8alpha(+) DCs to produce the chemokine CCL17, which attracted naive CTLs expressing the chemokine receptor CCR4. In contrast, DCs licensed by helper T cells recruited CTLs using CCR5 ligands. Thus, depending on the type of antigen they encounter, DCs can be licensed for cross-priming by NKT cells or helper T cells and use at least two independent chemokine pathways to attract naive CTLs. Because these chemokines acted synergistically, this can potentially be exploited to improve vaccinations.
Asunto(s)
Quimiocina CCL17/inmunología , Reactividad Cruzada/inmunología , Células Dendríticas/inmunología , Células T Asesinas Naturales/inmunología , Receptores CCR4/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Presentación de Antígeno/inmunología , Movimiento Celular/inmunología , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
PURPOSE: Drug-eluting balloon catheters (DEBc) coated with paclitaxel (PTX) have been associated with potential safety concerns. An efficacious but less toxic balloon coating may reduce these outcomes. We evaluated a novel DEBc, Epi-Solve, coated with metacept-3 (MCT-3), a member of the histone deacetylase inhibitor (HDACi) class of epigenetic agents, in a large animal model of neointimal hyperplasia (NIH). METHODS: Plain balloon angioplasty (PABA) catheters were ultrasonically coated with MCT-3 to generate Epi-Solve DEBc. An ovine model of NIH formation was established utilising partial left common carotid artery (LCA) ligation. Twenty-eight days post neointima (NI) induction, PABA, Epi-Solve or PTX-coated DEBc were deployed at the site of induced NI formation. Twenty-eight days post-intervention, ligated vessels were evaluated for attenuation of NI formation, gene expression profiles and immunohistochemical analysis. RESULTS: Epi-Solve DEBc demonstrated attenuation of NIH over no intervention and a trend to inhibition of NIH over PABA. Gene expression analysis and immunohistochemical studies identified significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA. CONCLUSIONS: Epi-Solve is a novel HDACi-coated DEBc which demonstrates significant anti-proliferative and anti-inflammatory signatures and reduced vascular endothelial cell activation compared to PABA in an ovine model and may afford endothelial protection.
Asunto(s)
Angioplastia de Balón/instrumentación , Enfermedades de las Arterias Carótidas/terapia , Arteria Carótida Común/patología , Materiales Biocompatibles Revestidos , Epigénesis Genética/efectos de los fármacos , Inhibidores de Histona Desacetilasas/administración & dosificación , Neointima , Dispositivos de Acceso Vascular , Animales , Enfermedades de las Arterias Carótidas/genética , Enfermedades de las Arterias Carótidas/metabolismo , Enfermedades de las Arterias Carótidas/patología , Arteria Carótida Común/metabolismo , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Modelos Animales de Enfermedad , Diseño de Equipo , Femenino , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Mediadores de Inflamación/metabolismo , Paclitaxel/administración & dosificación , Oveja Doméstica , Factores de TiempoRESUMEN
ß(3)-amino acid based polypeptides offer a unique starting material for the design of self-assembled nanostructures such as fibres and hierarchical dendritic assemblies, due to their well-defined helical geometry in which the peptide side chains align at 120° due to the 3.0-3.1 residue pitch of the helix. In a previous work we have described the head-to-tail self-assembly of N-terminal acetylated ß(3)-peptides into infinite helical nanorods that was achieved by designing a bioinspired supramolecular self-assembly motif. Here we describe the effect of consecutively more polar side chains on the self-assembly characteristics of ß(3)-tetrapeptides Ac-ß (3)Ala-ß(3)Leu-ß(3)Ile-ß(3)Ala (Ac-ß(3)[ALIA]), Ac-ß(3)Ser-ß(3)Leu-ß(3)Ile-ß(3)Ala (Ac-ß(3)[SLIA]) and Ac-ß (3)Lys-ß (3)Leu-ß(3)Ile-ß (3)Glu (Ac-ß(3)[KLIE]). ß(3)-tetrapeptides complete 1 1/3 turns of the helix: thus in the oligomeric form the side chain positions shift 120° with each added monomer, forming a regular periodic pattern along the nanorod. Dynamic light scattering (DLS) measurements confirmed that these peptides self-assemble even in highly polar solvents such as water and DMSO, while diffusion-ordered NMR spectroscopy revealed the presence of a substantial monomeric population. Temperature dependence of the size distribution in DLS measurements suggests a dynamic equilibrium between monomers and oligomers. Solution casting produced distinct fibrillar deposits after evaporating the solvent. In the case of the apolar Ac-ß(3)[ALIA] the longitudinal helix morphology gives rise to geometrically defined (â¼70°) junctions between fibres, forming a mesh that opens up possibilities for applications e.g. in tissue scaffolding. The deposits of polar Ac-ß(3)[SLIA] and Ac-ß(3)[KLIE] exhibit fibres in regular parallel alignment over surface areas in the order of 10 µm.
Asunto(s)
Nanoestructuras/química , Oligopéptidos/química , Dispersión Dinámica de Luz , Microscopía de Fuerza Atómica , Modelos Moleculares , Conformación Molecular , Solventes , TemperaturaRESUMEN
We have previously shown that individual ß-amino acid substitution in angiotensin (Ang) II reduced Ang II type 1 receptor (AT1R) but not Ang II type 2 receptor (AT2R)-binding and that the heptapeptide Ang III exhibited greater AT2R:AT1R selectivity than Ang II. Therefore, we hypothesized that ß-amino-acid-substituted Ang III peptide analogues would yield highly selective AT2R ligands, which we have tested in binding and functional vascular assays. In competition binding experiments using either AT1R- or AT2R-transfected human embryonic kidney (HEK)-293 cells, novel ß-substituted Ang III analogues lacked appreciable AT1R affinity, whereas most compounds could fully displace (125)I-Sar(1)Ile(8) Ang II from AT2R. The rank order of affinity at AT2R was CGP42112 > Ang III > ß-Pro(7) Ang III=Ang II > ß-Tyr(4) Ang III ≥ PD123319 >> ß-Phe(8) Ang III >> ß Arg(2) Ang III=ß-Val(3) Ang III >> ß-Ile(5) Ang III. The novel analogue ß-Pro(7) Ang III was the most selective AT2R ligand tested, which was >20,000-fold more selective for AT2R than AT1R. IC50 values at AT2R from binding studies correlated with maximum vasorelaxation in mouse aortic rings. Given that ß-Pro(7) Ang III was an AT2R agonist, we compared ß-Pro(7) Ang III and native Ang III for their ability to reduce blood pressure in separate groups of conscious spontaneously hypertensive rats. Whereas Ang III alone increased mean arterial pressure (MAP), ß-Pro(7) Ang III had no effect. During low-level AT1R blockade, both Ang III and ß-Pro(7) Ang III, but not Ang II, lowered MAP (by â¼30 mmHg) at equimolar infusions (150 pmol/kg/min for 4 h) and these depressor effects were abolished by the co-administration of the AT2R antagonist PD123319. Thus, ß-Pro(7) Ang III has remarkable AT2R selectivity determined in binding and functional studies and will be a valuable research tool for insight into AT2R function and for future drug development.
Asunto(s)
Bloqueadores del Receptor Tipo 2 de Angiotensina II/farmacología , Angiotensina III/análogos & derivados , Hipertensión/tratamiento farmacológico , Imidazoles/farmacología , Piridinas/farmacología , Vasoconstrictores/farmacología , Secuencia de Aminoácidos , Análisis de Varianza , Angiotensina III/sangre , Angiotensina III/farmacología , Animales , Aorta Torácica/efectos de los fármacos , Aorta Torácica/fisiopatología , Bencimidazoles/metabolismo , Unión Competitiva , Compuestos de Bifenilo , Estabilidad de Medicamentos , Células HEK293 , Humanos , Técnicas In Vitro , Concentración 50 Inhibidora , Contracción Isométrica/efectos de los fármacos , Masculino , Ratones , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/fisiopatología , Ratas , Receptores de Angiotensina/química , Receptores de Angiotensina/metabolismo , Tetrazoles/metabolismo , Vasodilatación/efectos de los fármacosRESUMEN
A putative antibacterial and antifungal compound, (R)-2-methylheptyl isonicotinate, was synthesized via reductive lactone alkylation of (R)-4-methyldihydrofuran-2(3H)-one. Structural characterisation data as well as bioassay results (with Bacillus subtilis or Escherichia coli) contradict those previously reported.
Asunto(s)
Antibacterianos/síntesis química , Antibacterianos/farmacología , Ácidos Isonicotínicos/síntesis química , Ácidos Isonicotínicos/farmacología , Ampicilina/farmacología , Antibacterianos/química , Bacillus subtilis/efectos de los fármacos , Escherichia coli/efectos de los fármacos , Ácidos Isonicotínicos/química , Estructura MolecularRESUMEN
Three highly aprotic bis(trifluoromethylsulfonyl)amide (NTf2(-)) based ionic liquids (ILs) containing the cations trihexyl(tetradecyl)phosphonium (P6,6,6,14(+)), N-butyl-N-methylpyrrolidinium (Pyrr4,1(+)), and (trimethylamine)(dimethylethylammine)dihydroborate ((N111)(N112)BH2(+)) have been examined as media for room temperature voltammetric detection of highly basic stilbene dianions electrochemically generated by the reduction of trans-stilbene (t-Stb) and its derivatives (4-methoxy-, 2-methoxy-, 4,4'-dimethyl-, and 4-chloromethyl-). Transient and steady-state data in the ILs were compared with results obtained in the molecular solvent acetonitrile. In all media examined, the t-Stb(0/â¢-) process is chemically and electrochemically reversible with a heterogeneous charge transfer rate constant in CH3CN of 1.5 cm s(-1), as determined by Fourier transformed AC voltammetry. However, further reduction to the dianion was always irreversible in this molecular but weakly acidic solvent. On the other hand, a substantial level of chemical reversibility for the reduction of t-Stb(â¢-) to t-Stb(2-) on the time scale of cyclic voltammetry is achieved when the concentration of trans-stilbene, [t-Stb], appreciably exceeds the concentration of adventitious water or other proton sources. In particular, these conditions are met when [t-Stb] ≥ 0.1 M in thoroughly dehydrated and purified ILs, while in the presence of CH3CN, t-Stb(2-) still suffers fast irreversible protonation under these stilbene concentration conditions. The E0/â¢-(0) values (vs Fc(0/+)) for substituted trans-stilbenes in acetonitrile and (N111)(N112)BH2-NTf2 do not differ substantially, nor do the E0/â¢-(0) and Eâ¢-/2-(0) differences or other aspects of the voltammetric behavior.
RESUMEN
Grb7 is an adapter protein involved in the propagation of signals in cancer cell migration and proliferation, and is thus a target for the development of novel anti-cancer agents. An 11-residue thioether-cyclized peptide known as G7-18NATE has previously been developed, that inhibits Grb7 via specific interactions with its SH2 domain with micromolar affinity. Here we explore whether the peptide binding is enhanced by the addition of a second linkage designed to restrain the peptide in its bound conformation and thus reduce the entropic loss upon binding. The use of an O-ally ser covalent linkage between residue positions 1 and 8 successfully enhanced the affinity, and ITC showed that the entropic loss was reduced. A peptide with thioether-cyclization exchanged for an amide linkage showed reduce affinity, though the formation of a disulfide bond between positions 1 and 8 in this peptide enhanced its binding. This study paves the way for improving the G7-18NATE scaffold for second generation inhibitors of Grb7.
Asunto(s)
Proteína Adaptadora GRB7 , Dominios Homologos src , Antineoplásicos/farmacología , Movimiento Celular/efectos de los fármacos , Proteína Adaptadora GRB7/metabolismo , Humanos , Péptidos/farmacología , Unión ProteicaRESUMEN
The title compound, C28H40N2O2S2, was obtained as a minor product from an anti-aldol reaction between the corresponding N-propionyl-thiol-actam and benzaldehyde. The asymmetric unit contains one half-molecule, which is completed by inversion symmetry. The molecule displays a nearly eclipsed conformation along the central C-C bond with a C-C-C-C- torsion angle of 20.4â (3)°.
RESUMEN
A library of 14-helical hexa ß(3)-peptides was synthesized in order to determine the influence of sequence variation as well as staple size and location on conformational stability. From this study we show that appropriately stapled hexa-ß(3)-peptides can allow for a number of variations without significant perturbation of the 14-helix.
Asunto(s)
Biblioteca de Péptidos , Alquenos/química , Dicroismo Circular , Conformación Molecular , Estabilidad ProteicaRESUMEN
In spite of the important role of angiotensin converting enzyme 2 (ACE2) in the cardiovascular system, little is known about the substrate structural requirements of the AngII-ACE2 interaction. Here we investigate how changes in angiotensin II (AngII) structure affect binding and cleavage by ACE2. A series of C3 ß-amino acid AngII analogs were generated and their secondary structure, ACE2 inhibition, and proteolytic stability assessed by circular dichroism (CD), quenched fluorescence substrate (QFS) assay, and LC-MS analysis, respectively. The ß-amino acid-substituted AngII analogs showed differences in secondary structure, ACE2 binding and proteolytic stability. In particular, three different subsets of structure-activity profiles were observed corresponding to substitutions in the N-terminus, the central region and the C-terminal region of AngII. The results show that ß-substitution can dramatically alter the structure of AngII and changes in structure correlated with ACE2 inhibition and/or substrate cleavage. ß-amino acid substitution in the N-terminal region of AngII caused little change in structure or substrate cleavage, while substitution in the central region of AngII lead to increased ß-turn structure and enhanced substrate cleavage. ß-amino acid substitution in the C-terminal region significantly diminished both secondary structure and proteolytic processing by ACE2. The ß-AngII analogs with enhanced or decreased proteolytic stability have potential application for therapeutic intervention in cardiovascular disease.
Asunto(s)
Sustitución de Aminoácidos , Angiotensina II/análogos & derivados , Angiotensina II/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Secuencia de Aminoácidos , Angiotensina II/química , Enzima Convertidora de Angiotensina 2 , Dicroismo Circular , Datos de Secuencia MolecularRESUMEN
A new method for in-gel sample processing and tryptic digestion of proteins is described. Sample preparation, rehydration, in situ digestion and peptide extraction from gel slices are dramatically accelerated by treating the gel slice with surface acoustic waves (SAWs). Only 30 minutes total workflow time is required for this new method to produce base peak chromatograms (BPCs) of similar coverage and intensity to those observed for traditional processing and overnight digestion. Simple set up, good reproducibility, excellent peptide recoveries, rapid turnover of samples and high confidence protein identifications put this technology at the fore-front of the next generation of proteomics sample processing tools.
Asunto(s)
Acústica , Métodos Analíticos de la Preparación de la Muestra/instrumentación , Proteínas/metabolismo , Animales , Bovinos , Geles , Humanos , Técnicas Analíticas Microfluídicas , Modelos Moleculares , Conformación Proteica , Proteínas/química , Propiedades de Superficie , Factores de TiempoRESUMEN
Grb7 is an adapter protein that is involved in signalling pathways that mediate eukaryotic cell proliferation and migration. Its overexpression in several cancer types has implicated it in cancer progression and led to the development of the G7-18NATE cyclic peptide inhibitor. Here, the preparation of crystals of G7-18NATE in complex with its Grb7 SH2 domain target is reported. Crystals of the complex were grown by the hanging-drop vapour-diffusion method using PEG 3350 as the precipitant at room temperature. X-ray diffraction data were collected from crystals to 2.4â Å resolution using synchrotron X-ray radiation at 100â K. The diffraction was consistent with space group P2(1), with unit-cell parameters a=52.7, b=79.1, c=54.7â Å, α=γ=90.0, ß=104.4°. The structure of the G7-18NATE peptide in complex with its target will facilitate the rational development of Grb7-targeted cancer therapeutics.
Asunto(s)
Proteína Adaptadora GRB7/antagonistas & inhibidores , Proteína Adaptadora GRB7/química , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Dominios Homologos src , Cristalización , Cristalografía por Rayos X , Humanos , Fosforilación/efectos de los fármacosRESUMEN
A new modality for chemical synthesis on a drop scale which employs a piezoelectric chip as the reactor and surface acoustic waves (SAWs) as the source of energy (and consequent heating) is described.
RESUMEN
The key tricyclic intermediate 3a, for the total synthesis of the C(20)-nor analogue of salvinorin A, was prepared in seven steps from 3-furaldehyde. Key steps involved a highly regio- and diastereoselective Lewis acid assisted Diels-Alder reaction followed by base-promoted epimerization and a completely stereoselective conjugate reduction.
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Diterpenos de Tipo Clerodano/síntesis química , Compuestos Heterocíclicos con 3 Anillos/síntesis química , Furaldehído/química , EstereoisomerismoRESUMEN
A mild, general method for the [3,3]-sigmatropic rearrangement of N,O-diacylhydroxylamines, employing a combination of mild base and Lewis acid, is described. Employing stoichiometric amounts of reagents with respect to substrate provides alpha-acyloxyamides, whereas an excess of reagents favors formation of cyclic orthoamides.
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Hidroxilaminas/química , Estructura Molecular , EstereoisomerismoRESUMEN
Peptides comprised entirely of ß3-amino acids, commonly referred to as ß-foldamers, have been shown to self-assemble into a range of materials. Previously, ß-foldamers have been functionalised via various side chain chemistries to introduce function to these materials without perturbation of the self-assembly motif. Here, we show that insertion of both rigid and flexible molecules into the backbone structure of the ß-foldamer did not disturb the self-assembly, provided that the molecule is positioned between two ß3-tripeptides. These hybrid ß3-peptide flanked molecules self-assembled into a range of structures. α-Arginlyglycylaspartic acid (RGD), a commonly used cell attachment motif derived from fibronectin in the extracellular matrix, was incorporated into the peptide sequence in order to form a biomimetic scaffold that would support neuronal cell growth. The RGD-containing sequence formed the desired mesh-like scaffold but did not encourage neuronal growth, possibly due to over-stimulation with RGD. Mixing the RGD peptide with a ß-foldamer without the RGD sequence produced a well-defined scaffold that successfully encouraged the growth of neurons and enabled neuronal electrical functionality. These results indicate that ß3-tripeptides can form distinct self-assembly units separated by a linker and can form fibrous assemblies. The linkers within the peptide sequence can be composed of a bioactive α-peptide and tuned to provide a biocompatible scaffold.
RESUMEN
Grb7 is a signaling protein with critical roles in tumor cell proliferation and migration and an established cancer therapeutic target. Here we explore chemical space to develop a new bicyclic peptide inhibitor, incorporating thioether and lactam linkers that binds with affinity (KD = 1.1 µM) and specificity to the Grb7-SH2 domain. Structural analysis of the Grb7-SH2/peptide complex revealed an unexpected binding orientation underlying the binding selectivity by this new scaffold. We further incorporated carboxymethylphenylalanine and carboxyphenylalanine phosphotyrosine mimetics and arrived at an optimized inhibitor that potently binds Grb7-SH2 (KD = 0.13 µM) under physiological conditions. X-ray crystal structures of these Grb7-SH2/peptide complexes reveal the structural basis for the most potent and specific inhibitors of Grb7 developed to date. Finally, we demonstrate that cell permeable versions of these peptides successfully block Grb7 mediated interactions in a breast cancer cell line, establishing the potential of these peptides in the development of novel therapeutics targeted to Grb7.
Asunto(s)
Proteína Adaptadora GRB7/antagonistas & inhibidores , Péptidos Cíclicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Descubrimiento de Drogas , Quinasa 1 de Adhesión Focal/metabolismo , Proteína Adaptadora GRB7/química , Proteína Adaptadora GRB7/metabolismo , Humanos , Lactamas/síntesis química , Lactamas/química , Lactamas/farmacología , Ligandos , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/química , Fosfatos/química , Conformación Proteica , Receptor ErbB-2/metabolismo , Proteínas Adaptadoras de la Señalización Shc/metabolismo , Dominios Homologos srcRESUMEN
We have described a new class of hydrogelator based on helical ß(3)-peptides carrying a bioactive payload. The ß(3)-peptides self-assemble in aqueous solution to form a nanofibrous mesh resulting in a stable hydrogel. The simple design provides the versatility for attaching different functional payloads to the ß(3)-peptide scaffold to develop new materials.
Asunto(s)
Hidrogeles/síntesis química , Lipopéptidos/síntesis química , Oligopéptidos/síntesis química , Animales , Adhesión Celular/efectos de los fármacos , Línea Celular , Elasticidad , Hidrogeles/farmacología , Lipopéptidos/farmacología , Ratones , Oligopéptidos/farmacología , Reología , ViscosidadRESUMEN
The design of potent and specific peptide inhibitors to therapeutic targets is of enormous utility for both proof-of-concept studies and for the development of potential new therapeutics. Grb7 is a key signaling molecule in the progression of HER2 positive and triple negative breast cancers. Here we report the crystal structure of a stapled bicyclic peptide inhibitor G7-B1 in complex with the Grb7-SH2 domain. This revealed an unexpected binding mode of the peptide, in which the staple forms an alternative contact with the surface of the target protein. Based on this structural information, we designed a new series of bicyclic G7 peptides that progressively constrain the starting peptide, to arrive at the G7-B4 peptide that binds with an approximately 2-fold enhanced affinity to the Grb7-SH2 domain (KD = 0.83 µM) compared to G7-B1 and shows low affinity binding to Grb2-, Grb10- and Grb14-SH2 domains (KD > 100 µM). Furthermore, we determined the structure of the G7-B4 bicyclic peptide in complex with the Grb7-SH2 domain, both before and after ring closing metathesis to show that the closed staple is essential to the target interaction. The G7-B4 peptide represents an advance in the development of Grb7 inhibitors and is a classical example of structure aided inhibitor development.