RESUMO
Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited cardiac arrhythmia syndrome that often leads to sudden cardiac death. The most common form of CPVT is caused by autosomal-dominant variants in the cardiac ryanodine receptor type-2 (RYR2) gene. Mutations in RYR2 promote calcium (Ca2+ ) leak from the sarcoplasmic reticulum (SR), triggering lethal arrhythmias. Recently, it was demonstrated that tetracaine derivative EL20 specifically inhibits mutant RyR2, normalizes Ca2+ handling and suppresses arrhythmias in a CPVT mouse model. The objective of this study was to determine whether EL20 normalizes SR Ca2+ handling and arrhythmic events in induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) from a CPVT patient. Blood samples from a child carrying RyR2 variant RyR2 variant Arg-176-Glu (R176Q) and a mutation-negative relative were reprogrammed into iPSCs using a Sendai virus system. iPSC-CMs were derived using the StemdiffTM kit. Confocal Ca2+ imaging was used to quantify RyR2 activity in the absence and presence of EL20. iPSC-CMs harbouring the R176Q variant demonstrated spontaneous SR Ca2+ release events, whereas administration of EL20 diminished these abnormal events at low nanomolar concentrations (IC50 = 82 nM). Importantly, treatment with EL20 did not have any adverse effects on systolic Ca2+ handling in control iPSC-CMs. Our results show for the first time that tetracaine derivative EL20 normalized SR Ca2+ handling and suppresses arrhythmogenic activity in iPSC-CMs derived from a CPVT patient. Hence, this study confirms that this RyR2-inhibitor represents a promising therapeutic candidate for treatment of CPVT.
RESUMO
The current five-year survival rate of <5% for pancreatic ductal adenocarcinoma (PDAC) is compounded by late diagnosis, a lack of PDAC-specific intraoperative guidance to ensure complete resection, and the ineffectiveness of current therapies. Previously, utilizing compound 1, a fluorophore with inherent PDAC selectivity, PDAC was visualized both in vivo and ex vivo in a murine model. In the current study, human PDAC tissue is targeted. Compound 1 selectively stains ducts of the adenocarcinoma versus the surrounding stroma, enabling the imaging of PDAC in frozen tissue sections with high contrast. To enhance the potential of 1 for intraoperative applications, the ex vivo staining protocol was optimized for rapid margin assessment, with a final staining time ofâ¯~15â¯min. To measure diagnostic performance, the area under a receiver operating characteristic (ROC) curve was measured for the identification of ductal adenocarcinoma vs. stroma. The bright fluorescence contrast enabled quantitative determination of PDAC (or precancerous PanIN lesions) versus healthy pancreas tissue in human tissue array samples.
Assuntos
Carcinoma Ductal Pancreático/diagnóstico por imagem , Imagem Óptica/métodos , Neoplasias Pancreáticas/diagnóstico por imagem , Animais , Humanos , CamundongosRESUMO
Fluorescent small molecules enable researchers and clinicians to visualize biological events in living cells, tissues, and organs in real time. Herein, the focus is on the structure and properties of the relatively rare benzo[ a]xanthenes that exhibit enhanced steric and electronic interactions due to their annulated structures. Three types of fluorophores were synthesized: (i) pH- and solvent-dependent seminaphthorhodafluors, (ii) pH- and solvent-independent seminaphthorhodafluors, and (iii) pH-independent but solvent-sensitive seminaphthorhodamines. The probes exhibited promising far-red to near-infrared (NIR) emission, large Stoke shifts, broad full width at half-maximum (fwhm), relatively high quantum yields, and utility in immunofluorescence staining. Deviation of the π-system from planarity due to changes in the fluorophore ionization state resulted in fluorescence properties that are atypical of common xanthene dyes.
Assuntos
Corantes Fluorescentes/química , Xantenos/química , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Imunofluorescência , Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacocinética , Humanos , Concentração de Íons de Hidrogênio , Modelos Moleculares , Conformação Molecular , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Rodaminas/síntese química , Rodaminas/química , Espectroscopia de Luz Próxima ao Infravermelho/métodos , Eletricidade Estática , Relação Estrutura-Atividade , Xantenos/síntese química , Xantenos/farmacocinéticaRESUMO
Regioselective functionalization of 2,3,5,6,8-pentachloro-BODIPY 1 produced unsymmetric BODIPY 5, bearing an isothiocyanate group suitable for conjugation, in only four steps. The X-ray structure of 5 reveals a nearly planar BODIPY core with aryl dihedral angles in the range 47.4-62.9°. Conjugation of 5 to two EGFR-targeting pegylated peptides, 3PEG-LARLLT (6) and 3PEG-GYHWYGYTPQNVI (7), under mild conditions (30 min at room temperature), afforded BODIPY conjugates 8 and 9 in 50-80% isolated yields. These conjugates showed red-shifted absorption and emission spectra compared with 5, in the near-IR region, and were evaluated as potential fluorescence imaging agents for EGFR overexpressing cells. SPR and docking investigations suggested that conjugate 8 bearing the LARLLT sequence binds to EGFR more effectively than 9 bearing the GYHWYGYTPQNVI peptide, in part due to the lower solubility of 9, and its tendency for aggregation at concentrations above 10 µM. Studies in human carcinoma HEp2 cells overexpressing EGFR demonstrated low dark and photo cytotoxicities for BODIPY 5 and the two peptide conjugates, and remarkably high cellular uptake for both conjugates 8 and 9, up to 90-fold compared with BODIPY 5 after 1 h. Fluorescence imaging studies in HEp2 cells revealed subcellular localization of the BODIPY-peptide conjugates mainly in the Golgi apparatus and the cell lysosomes. The low cytotoxicity of the new conjugates and their remarkably high uptake into EGFR overexpressing cells renders them promising imaging agents for cancers overexpressing EGFR.
Assuntos
Compostos de Boro/química , Carcinoma de Células Escamosas/patologia , Receptores ErbB/química , Fragmentos de Peptídeos/síntese química , Carcinoma de Células Escamosas/metabolismo , Sobrevivência Celular , Receptores ErbB/metabolismo , Fluorescência , Humanos , Modelos Moleculares , Imagem Óptica/métodos , Células Tumorais CultivadasRESUMO
Cisplatin is widely used to treat a variety of cancers. However, ototoxicity and nephrotoxicity remain serious side effects of cisplatin-based chemotherapy. In order to inform the study of cisplatin's off-target effects, a new drug-fluorophore conjugate was synthesized that exhibited utility as a tracer to determine the cellular uptake and in vivo distribution of cisplatin. This probe will serve as a useful tool to facilitate investigations into the kinetics and biodistribution of cisplatin and its associated side effects in preclinical models after systemic administration.
Assuntos
Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Cisplatino/administração & dosagem , Cisplatino/uso terapêutico , Animais , Relação Dose-Resposta a Droga , Humanos , Neoplasias/tratamento farmacológicoRESUMO
The first solid phase extraction materials for selective lysophosphatidic acid (LPA) enrichment from human plasma are described. Molecularly imprinted polymers were designed, synthesized and evaluated as cartridge fillings. They enabled a relatively rapid and simple extraction protocol for LPA without any need for multiple liquid-liquid extraction steps. The five major subspecies of lysophosphatidic acid are readily separated from all other native plasma phospholipids, including those well-known to interfere with LPA quantitation, such as phosphatidylcholine and lysophosphatidylcholine. Outstanding LPA purity is obtained via these solid phase materials in a tandem extraction setup.
Assuntos
Lisofosfolipídeos/sangue , Lisofosfolipídeos/isolamento & purificação , Impressão Molecular/métodos , Polímeros/química , Extração em Fase Sólida/métodos , Cromatografia Líquida de Alta Pressão , Humanos , Extração Líquido-Líquido , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
A simple and highly efficient technique for the analysis of lysophosphatidic acid (LPA) subspecies in human plasma is described. The streamlined sample preparation protocol furnishes the five major LPA subspecies with excellent recoveries. Extensive analysis of the enriched sample reveals only trace levels of other phospholipids. This level of purity not only improves MS analyses, but enables HPLC post-column detection in the visible region with a commercially available fluorescent phospholipids probe. Human plasma samples from different donors were analyzed using the above method and validated by LC-ESI/MS/MS.
Assuntos
Lisofosfolipídeos/sangue , Cromatografia Líquida de Alta Pressão , Humanos , Limite de Detecção , Lisofosfolipídeos/químicaRESUMO
Elevated levels of homocysteine are associated with several major diseases. However, it is not clear whether homocysteine is a marker or a causative agent. The majority (ca. 80%) of the homocysteine present in humans is protein bound. The study of the posttranslational modification of proteins by homocysteine and its cyclic congener, homocysteine thiolactone, is emerging as an area of great current interest for unraveling the ongoing "mediator/marker controversy" [Jacobsen DW (2009) Clin Chem 55:1-2]. Interestingly, many of the pathologies associated with homocysteine are also linked to oxidative stress. In the current study, chemical evidence for a causal relationship between homocysteine-bound proteins and oxidative damage is presented. For example, a reproducible increase in protein carbonyl functionality occurs as a consequence of the reaction of human serum albumin with homocysteine thiolactone. This occurs at physiological temperature upon exposure to air without any added oxidants or free-radical initiators. Alpha-amino acid carbon-centered radicals, well-known precursors of protein carbonyls, are shown to form via a hydrogen atom transfer process involving thiolactone-derived homocystamides. Model peptides in buffer as well as native proteins in human blood plasma additionally exhibit properties in keeping with the homocystamide-facilitated hydrogen atom transfer and resultant carbon-centered radicals.
Assuntos
Homocisteína/metabolismo , Doença de Alzheimer/metabolismo , Doenças Cardiovasculares/metabolismo , Anormalidades Congênitas/metabolismo , Radicais Livres/metabolismo , Homocisteína/sangue , Humanos , Ligação de Hidrogênio , Cinética , Lisina/metabolismo , Osteoporose/metabolismo , Estresse Oxidativo , Processamento de Proteína Pós-Traducional , Insuficiência Renal/metabolismo , Albumina Sérica/metabolismoRESUMO
There is ongoing interest in near-infrared (NIR) absorbing and emitting dyes for a variety of biomedical and materials applications. Simple and efficient synthetic procedures enable the judicious tuning of through-space polar (field) effects as well as low barrier hydrogen bonding to modulate the HOMO-LUMO gap in xanthene dyes. This affords unique NIR-absorbing xanthene chromophores.
Assuntos
Corantes Fluorescentes/química , Xantenos/química , Ligação de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Espectroscopia de Luz Próxima ao InfravermelhoRESUMO
Red-shifted and near-infrared (NIR)-active rhodamine analogs and their boronic acid derivatives were synthesized and studied. These latter compounds function as fluorogenic NIR active substrates for sugar sensing. The effects of benzannulation and boronic acid functionalization on fluorophore optical and sensing properties are described.
RESUMO
A readily accessible new class of near infrared (NIR) molecular probes has been synthesized and evaluated. Specific fluorophores in this unique xanthene based regioisomeric seminaphthofluorone dye series exhibit a combination of desirable characteristics including (i) low molecular weight (339 amu), (ii) aqueous solubility, and (iii) dual excitation and emission from their fluorescent neutral and anionic forms. Importantly, systematic changes in the regiochemistry of benzannulation and the ionizable moieties afford (iv) tunable deep-red to NIR emission from anionic species and (v) enhanced Stokes shifts. Anionic SNAFR-6, exhibiting an unusually large Stokes shift of approximately 200 nm (5,014 cm(-1)) in aqueous buffer, embodies an unprecedented fluorophore that emits NIR fluorescence when excited in the blue/green wavelength region. The successful use of SNAFR-6 in cellular imaging studies demonstrates proof-of-concept that this class of dyes possesses photophysical characteristics that allow their use in practical applications. Notably, each of the new fluorophores described is a minimal template structure for evaluation of their basic spectral properties, which may be further functionalized and optimized yielding concomitant improvements in their photophysical properties.
RESUMO
Porphyrin-peptide conjugates bearing multiple nuclear localization sequences (NLS) could show increased tumor cell uptake and affinity for nuclear receptors, and consequently increased photodynamic activity. Previous studies suggest that an increase number of NLS might enhance the nuclear uptake of proteins and other macromolecules. We report the syntheses and investigation of a series of multimeric porphyrin-NLS conjugates bearing two, three or four peptides with the minimum sequence PKKKRKV, linked via PEG or 5-carbon linkers, and with different distributions at the porphyrin periphery. Our results show that the tumor cell uptake and phototoxicity of these conjugates is mainly determined by their amphiphilic character, and not the number of NLS residues per molecule, contrary to previous studies. The mono- and di-substituted photosensitizers bearing one or two PEG linkers and up to three peptide sequences were found to be the most phototoxic toward human carcinoma HEp2 cells, while the tetra-NLS conjugates symmetrically substituted around the porphyrin ring accumulated the least within cells and were non-phototoxic. All conjugates localized intracellularly within endosomal vesicles and lysosomes, probably as a result of an endocytic mechanism of uptake; as a consequence no nuclear uptake was detected by fluorescence microscopy.
Assuntos
Carcinoma/tratamento farmacológico , Peptídeos/química , Peptídeos/farmacologia , Fármacos Fotossensibilizantes/química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/química , Porfirinas/farmacologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Dados de Sequência Molecular , Peptídeos/síntese química , Peptídeos/farmacocinética , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/farmacocinética , Polietilenoglicóis/química , Porfirinas/síntese química , Porfirinas/farmacocinéticaRESUMO
Three BODIPY-peptide conjugates designed to target the epidermal growth factor receptor (EGFR) at the extracellular domain were synthesized, and their specificity for binding to EGFR was investigated. Peptide sequences containing seven amino acids, GLARLLT (2) and KLARLLT (4), and 13 amino acids, GYHWYGYTPQNVI (3), were conjugated to carboxyl BODIPY dye (1) by amide bond formation in up to 73% yields. The BODIPY-peptide conjugates and their "parent" peptides were determined to bind to EGFR experimentally using SPR analysis and were further investigated using computational methods (AutoDock). Results of SPR, competitive binding and docking studies propose that conjugate 6 including the GYHWYGYTPQNVI sequence binds to EGFR more effectively than conjugates 5 and 7, bearing the smaller peptide sequences. Findings in human carcinoma HEp2 cells overexpressing EGFR showed nontoxic behavior in the presence of activated light (1.5 J cm-2 ) and in the absence of light for all BODIPYs. Furthermore, conjugate 6 showed about five-fold higher accumulation within HEp2 cells compared with conjugates 5 and 7, localizing preferentially in the cell ER and lysosomes. Our findings suggest that BODIPY-peptide conjugate 6 is a promising contrast agent for detection of colorectal cancer and potentially other EGFR-overexpressing cancers.
Assuntos
Amidas/química , Compostos de Boro/química , Neoplasias Colorretais/metabolismo , Receptores ErbB/metabolismo , Peptídeos/química , Sequência de Aminoácidos , Ligação Competitiva , Linhagem Celular Tumoral , Neoplasias Colorretais/patologia , Humanos , Microscopia de Fluorescência , Ressonância de Plasmônio de SuperfícieRESUMO
The colorimetric reaction of homocysteine (HCy) with a series of viologen salts suggests a linear correlation between the mid-point reduction potential of Hcy-derived alpha-carbon radicals and pH.
Assuntos
Carbono/química , Cisteína/química , Homocisteína/química , Viologênios/química , Radicais Livres/química , Concentração de Íons de Hidrogênio , OxirreduçãoRESUMO
Synthetic dyes have been widely used in supramolecular chemistry not only to probe fundamental chemical interactions but also as components of functional materials. Most current efforts in this regard are directed at designing new host systems for the dyes. Herein we report on the study of a versatile new organic fluorophore. We describe a synthesis which affords improved yields in a convenient one pot procedure. Moreover, a simple method for predicting and controlling the dye's responses to external stimuli affords a potentially practical method for achieving red-green-blue and concomitant white light generation.
RESUMO
A series of four porphyrin-peptide conjugates bearing one linear bifunctional sequence containing a cell penetrating peptide (CPP) and a nuclear localization signal (NLS) were synthesized and their in vitro biological and stability properties investigated. All conjugates accumulated within human HEp2 cells to a significantly higher extent than their porphyrin-PEG precursor, and the extent of their uptake and cytotoxicity depends on the nature and sequence of the amino acids. Conjugates 2 and 5 bearing a NLS-CPP accumulated the most within cells and were the most phototoxic (IC50 approximately 7 microM at 1 J/cm2). All conjugates localized preferentially within the cell lysosomes, and in addition, conjugate 2 was also found in the ER. All conjugates were highly stable under nonenzymatic conditions, but their peptide sequences were cleaved to some extent (ca. 50% after 24 h) by proteolytic enzymes, such as cathepsin B, cathepsin D, prolidase, and plasmin.
Assuntos
Peptídeos/química , Fármacos Fotossensibilizantes/química , Porfirinas/química , Sinais Direcionadores de Proteínas , Linhagem Celular , Linhagem Celular Tumoral , Dicroísmo Circular , Humanos , Luz , Microscopia de Fluorescência , Sinais de Localização Nuclear , Proteínas Nucleares/química , Nucleoplasminas , Oligopeptídeos/química , Peptídeos/farmacologia , Fosfoproteínas/química , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/síntese química , Porfirinas/farmacologia , Conformação Proteica , Relação Estrutura-Atividade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/químicaRESUMO
We describe the total synthesis and biological properties of a new carboranyl-containing chlorin (TPFC) that might find application as a dual sensitizer in the PDT and BNCT treatment of cancer. TPFC was found to be non-toxic in the dark but showed extensive photosensitizing ability both in vitro and in vivo despite its relatively low singlet oxygen quantum yield. In particular, TPFC exhibited significant photosensitizing activity against highly pigmented melanotic melanoma tumors in mice.
Assuntos
Compostos de Boro/síntese química , Compostos de Boro/farmacologia , Fármacos Fotossensibilizantes/síntese química , Fármacos Fotossensibilizantes/farmacologia , Porfirinas/síntese química , Porfirinas/farmacologia , Animais , Compostos de Boro/metabolismo , Compostos de Boro/toxicidade , Terapia por Captura de Nêutron de Boro , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Espaço Intracelular/metabolismo , Camundongos , Fotoquímica , Fotoquimioterapia , Fármacos Fotossensibilizantes/metabolismo , Fármacos Fotossensibilizantes/toxicidade , Porfirinas/química , Porfirinas/metabolismo , Porfirinas/toxicidadeRESUMO
BACKGROUND: Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an arrhythmogenic disorder caused by mutations in the cardiac ryanodine receptor RyR2 that increase diastolic calcium cation (Ca2+) leak from the sarcoplasmic reticulum (SR). Calmodulin (CaM) dissociation from RyR2 has been associated with diastolic Ca2+ leak in heart failure. OBJECTIVE: Determine whether the tetracaine-derivative compound EL20 inhibits abnormal Ca2+ release from RyR2 in a CPVT model and investigate the underlying mechanism of inhibition. METHODS: Spontaneous Ca2+ sparks in cardiomyocytes and inducible ventricular tachycardia were assessed in a CPVT mouse model, which is heterozygous for the R176Q mutation in RyR2 (R176Q/+ mice) in the presence of EL20 or vehicle. Single-channel studies using sheep cardiac SR or purified RyR2 reconstituted into proteoliposomes with and without exogenous CaM were used to assess mechanisms of inhibition. RESULTS: EL20 potently inhibits abnormal Ca2+ release in R176Q/+ myocytes (half-maximal inhibitory concentration = 35.4 nM) and diminishes arrhythmia in R176Q/+ mice. EL20 inhibition of single-channel activity of purified RyR2 occurs in a similar range as seen in R176Q/+ myocytes (half-maximal inhibitory concentration = 8.2 nM). Inhibition of single-channel activity for cardiac SR or purified RyR2 supplemented with 100-nM or 1-µM CaM shows a 200- to 1000-fold reduction in potency. CONCLUSION: This work provides a potential therapeutic mechanism for the development of antiarrhythmic compounds that inhibit leaky RyR2 resulting from CaM dissociation, which is often associated with failing hearts. Our data also suggest that CaM dissociation may contribute to the pathogenesis of arrhythmias with the CPVT-linked R176Q mutation.
Assuntos
Antiarrítmicos/farmacologia , Calmodulina/deficiência , DNA/genética , Mutação , Miócitos Cardíacos/metabolismo , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Taquicardia Ventricular/genética , Animais , Cálcio/metabolismo , Análise Mutacional de DNA , Modelos Animais de Doenças , Camundongos , Camundongos Mutantes , Miócitos Cardíacos/patologia , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Retículo Sarcoplasmático , Ovinos , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/patologiaRESUMO
The total syntheses of four PEG-functionalized porphyrins, containing one to four low molecular weight PEG chains linked via amide bonds to the para-phenyl positions of meso-tetraphenylporphyrin, are reported. The hydrophobic character of the PEG-porphyrins decreases with the number of PEG chains linked to the porphyrin ring, while their tendency for aggregation in buffered aqueous solution increases. The porphyrins containing one or two PEG chains accumulated within human HEp2 cells to a much higher extent than those having three or four PEGs at the macrocycle periphery. All PEG-porphyrins were found to be non-toxic in the dark, and only those containing one or two PEG chains were phototoxic (IC(50)=2 microM at 1J/cm(2) light dose). The preferential sites of subcellular localization of the porphyrins containing one or two PEG chains were found to be the mitochondria and endoplasmic reticulum (ER), while those containing three or four PEG chains localize preferentially in the lysosomes.
Assuntos
Polietilenoglicóis , Porfirinas/síntese química , Porfirinas/farmacocinética , Animais , Linhagem Celular , Retículo Endoplasmático/metabolismo , Humanos , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Relação Estrutura-AtividadeRESUMO
Molecular probes that selectively highlight pancreatic cancer (PC) tissue have the potential to improve pancreatic ductal adenocarcinoma (PDAC) margin assessment through the selective highlighting of individual PC cells. Herein, we report a simple and unique family of systematically modified red and near-infrared fluorescent probes that exhibit a field-effect-derived redshift. Two of thirteen probes distributed to the normal mouse pancreas following systemic administration. One selectively accumulated in genetically modified mouse models of PDAC. The probe exhibited intracellular accumulation and enabled visualization of four levels of the structure, including the whole organ, resected tissue, individual cells, and subcellular organelles. In contrast to the small-molecule probes reported previously, it possesses an inherent affinity toward PDAC cells and thus does not require conjugation to any targeting agent. The fluorescent probe can thus promote new strategies not only for precision image-guided surgery, but also for PC detection, monitoring of therapeutic outcomes, and basic research.