RESUMEN
Human programmed cell death protein 1 (hPD-1) is an essential receptor in the immune checkpoint pathway. It has played an important role in cancer therapy. However, not all patients respond positively to the PD-1 antibody treatment, and the underlying mechanism remains unknown. PD-1 is a transmembrane glycoprotein, and its extracellular domain (ECD) is reported to be responsible for interactions and signal transduction. This domain contains 4 N-glycosylation sites and 25 potential O-glycosylation sites, which implicates the importance of glycosylation. The structure of hPD-1 has been intensively studied, but the glycosylation of this protein, especially the glycan on each glycosylation site, has not been comprehensively illustrated. In this study, hPD-1 ECD expressed by human embryonic kidney 293 (HEK 293) and Chinese hamster ovary (CHO) cells was analyzed; not only N- and O-glycosylation sites but also the glycans on these sites were comprehensively analyzed using mass spectrometry. In addition, hPD-1 ECD binding to different anti-hPD-1 antibodies was tested, and N-glycans were found functioned differently. All of this glycan information will be beneficial for future PD-1 studies.
RESUMEN
Many cancer-driving protein targets remain undruggable due to a lack of binding molecular scaffolds. In this regard, octahedral metal complexes with unique and versatile three-dimensional structures have rarely been explored as inhibitors of undruggable protein targets. Here, we describe antitumor iridium(III) pyridinium-N-heterocyclic carbene complex 1a, which profoundly reduces the viability of lung and breast cancer cells as well as cancer patient-derived organoids at low micromolar concentrations. Compound 1a effectively inhibits the growth of non-small-cell lung cancer and triple-negative breast cancer xenograft tumors, impedes the metastatic spread of breast cancer cells, and can be modified into an antibody-drug conjugate payload to achieve precise tumor delivery in mice. Identified by thermal proteome profiling, an important molecular target of 1a in cellulo is Girdin, a multifunctional adaptor protein that is overexpressed in cancer cells and unequivocally serves as a signaling hub for multiple pivotal oncogenic pathways. However, specific small-molecule inhibitors of Girdin have not yet been developed. Notably, 1a exhibits high binding affinity to Girdin with a Kd of 1.3 µM and targets the Girdin-linked EGFR/AKT/mTOR/STAT3 cancer-driving pathway, inhibiting cancer cell proliferation and metastatic activity. Our study reveals a potent Girdin-targeting anticancer compound and demonstrates that octahedral metal complexes constitute an untapped library of small-molecule inhibitors that can fit into the ligand-binding pockets of key oncoproteins.
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Antineoplásicos , Iridio , Metano , Animales , Humanos , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/patología , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Complejos de Coordinación/farmacología , Complejos de Coordinación/química , Iridio/química , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/secundario , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Metano/análogos & derivados , Metano/química , Metano/farmacología , Proteínas de Microfilamentos/metabolismo , Metástasis de la Neoplasia , Neoplasias de la Mama Triple Negativas/tratamiento farmacológico , Neoplasias de la Mama Triple Negativas/patología , Neoplasias de la Mama Triple Negativas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto , MasculinoRESUMEN
Herein, we discover the new reactivity of the 1,3,5-triazine moiety reacting with a phenol group and report the development of biocompatible and catalyst-free triazine-pyridine chemistry (TPC) for tyrosine labelling under physiological conditions and profiling in the whole proteome. TPC exhibited high tyrosine chemoselectivity in biological systems after cysteine blocking, displayed potential in tyrosine-guided protein labelling, and had bio-compatibility in live cells.
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Triazinas , Tirosina , Cisteína , Proteoma , Piridinas , Tirosina/metabolismoRESUMEN
Membrane proteins on the cell surface perform a myriad of biological functions; however, ligand discovery for membrane proteins is highly challenging, because a natural cellular environment is often necessary to maintain protein structure and function. DNA-encoded chemical libraries (DELs) have emerged as a powerful technology for ligand discovery, but they are mainly limited to purified proteins. Here we report a method that can specifically label membrane proteins with a DNA tag, and thereby enable target-specific DEL selections against endogenous membrane proteins on live cells without overexpression or any other genetic manipulation. We demonstrate the generality and performance of this method by screening a 30.42-million-compound DEL against the folate receptor, carbonic anhydrase 12 and the epidermal growth factor receptor on live cells, and identify and validate a series of novel ligands for these targets. Given the high therapeutic significance of membrane proteins and their intractability to traditional high-throughput screening approaches, this method has the potential to facilitate membrane-protein-based drug discovery by harnessing the power of DEL.
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Anhidrasas Carbónicas/química , ADN/química , Receptores ErbB/química , Receptores de Folato Anclados a GPI/química , Bibliotecas de Moléculas Pequeñas/química , Anticuerpos Inmovilizados/química , Anticuerpos Inmovilizados/inmunología , Anhidrasas Carbónicas/metabolismo , Receptores ErbB/inmunología , Receptores ErbB/metabolismo , Fluoresceína-5-Isotiocianato/química , Receptores de Folato Anclados a GPI/metabolismo , Células HeLa , Humanos , Ligandos , Microscopía Fluorescente , Bibliotecas de Moléculas Pequeñas/metabolismoRESUMEN
In hepatocellular carcinoma (HCC) patients with extrahepatic metastasis, the lung is the most frequent site of metastasis. However, how the lung microenvironment favors disseminated cells remains unclear. Here, it is found that nidogen 1 (NID1) in metastatic HCC cell-derived extracellular vesicles (EVs) promotes pre-metastatic niche formation in the lung by enhancing angiogenesis and pulmonary endothelial permeability to facilitate colonization of tumor cells and extrahepatic metastasis. EV-NID1 also activates fibroblasts, which secrete tumor necrosis factor receptor 1 (TNFR1), facilitate lung colonization of tumor cells, and augment HCC cell growth and motility. Administration of anti-TNFR1 antibody effectively diminishes lung metastasis induced by the metastatic HCC cell-derived EVs in mice. In the clinical perspective, analysis of serum EV-NID1 and TNFR1 in HCC patients reveals their positive correlation and association with tumor stages suggesting the potential of these molecules as noninvasive biomarkers for the early detection of HCC. In conclusion, these results demonstrate the interplay of HCC EVs and activated fibroblasts in pre-metastatic niche formation and how blockage of their functions inhibits distant metastasis to the lungs. This study offers promise for the new direction of HCC treatment by targeting oncogenic EV components and their mediated pathways.
RESUMEN
Cysteine thiols of many cancer-associated proteins are attractive targets of anticancer agents. Herein, we unequivocally demonstrate a distinct thiol-targeting property of gold(III) mesoporphyrin IX dimethyl ester (AuMesoIX) and its anticancer activities. While the binding of cysteine thiols with metal complexes usually occurs via M-S bond formation, AuMesoIX is unique in that the meso-carbon atom of the porphyrin ring is activated by the gold(III) ion to undergo nucleophilic aromatic substitution with thiols. AuMesoIX was shown to modify reactive cysteine residues and inhibit the activities of anticancer protein targets including thioredoxin, peroxiredoxin, and deubiquitinases. Treatment of cancer cells with AuMesoIX resulted in the formation of gold-bound sulfur-rich protein aggregates, oxidative stress-mediated cytotoxicity, and accumulation of ubiquitinated proteins. Importantly, AuMesoIX exhibited effective antitumor activity in mice. Our study has uncovered a gold(III)-induced ligand scaffold reactivity for thiol targeting that can be exploited for anticancer applications.
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Antineoplásicos/química , Cisteína/química , Oro/química , Mesoporfirinas/química , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/uso terapéutico , Enzimas Desubicuitinizantes/química , Enzimas Desubicuitinizantes/metabolismo , Células HCT116 , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Neoplasias Experimentales/tratamiento farmacológico , Peroxirredoxinas/química , Peroxirredoxinas/metabolismo , Unión Proteica , Tiorredoxinas/química , Tiorredoxinas/metabolismo , Distribución TisularRESUMEN
The complement system is involved in the immunosurveillance of pathogens and tumour cells. Proteomic profiling revealed that extracellular vesicles (EVs) released by metastatic hepatocellular carcinoma (HCC) cells contained a significant number of complement proteins. Complement Factor H (CFH), an abundant soluble serum protein that inhibits the alternative complement pathway, was found to be highly expressed in EVs of metastatic HCC cell lines. Here, we investigated the functional role of EV-CFH and explored the therapeutic efficacy of targeting EV-CFH with an anti-CFH antibody in HCC. The results showed that EVs that are enriched in CFH promoted HCC cell growth, migration, invasiveness and enhanced liver tumour formation in mice. EV-CFH also promoted metastasis, which was significantly abrogated when treated with an anti-CFH antibody. These findings demonstrate an unexplored function of EV-CFH in protecting HCC cells by evading complement attack, thereby facilitating tumorigenesis and metastasis. Lastly, we demonstrated the therapeutic efficacy of an anti-CFH antibody in suppressing tumour formation in a syngeneic mouse model. This study suggests a new therapeutic strategy for HCC, by inhibiting EV-CFH with a tumour specific anti-CFH antibody.
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Carcinogénesis/metabolismo , Carcinoma Hepatocelular/metabolismo , Vesículas Extracelulares/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Proteínas de Neoplasias/metabolismo , Animales , Carcinogénesis/patología , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Factor H de Complemento/metabolismo , Vesículas Extracelulares/patología , Humanos , Neoplasias Hepáticas Experimentales/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Metástasis de la NeoplasiaRESUMEN
BACKGROUND: Galectins are beta-galactose specific binding proteins. In human cancers, including hepatocellular carcinoma (HCC), galectin-1 (Gal-1) is often found to be overexpressed. In order to combat the dismal diagnosis and death rates of HCC, gene silencing and targeted inhibition of Gal-1 was investigated for its improved therapeutic potential. METHODS: Cellular and secretory Gal-1 levels were analyzed using HCC clinical samples. The study of Gal-1 was carried by both knockdown and overexpression approaches. The stable clones were tested by in vitro assays and in vivo experiments. Mass spectrometry was used to identify downstream targets of Gal-1. The upstream regulator of Gal-1, microRNA-22 (miR-22) was characterized by functional assays. The therapeutic effect of inhibiting Gal-1 was also analyzed. RESULTS: Gal-1 overexpression was observed in HCC and correlated with aggressive clinicopathological features and poorer survival. The loss of Gal-1 resulted in hindered cell migration, invasion and anchorage independent growth. This was also observed in the animal models, in that when Gal-1 was knocked down, there were fewer lung metastases. Proteomic profiling of control and Gal-1 knockdown cells identified that the level of retention in endoplasmic reticulum 1 (RER1) was suppressed when Gal-1 level was reduced. The cell motility of Gal-1 knockdown cells was enhanced upon the rescue of RER1 expression. In HCC tissues, Gal-1 and RER1 expressions displayed a significant positive correlation. The upstream regulator of Gal-1, miR-22 was observed to be underexpressed in HCC tissues and negatively correlated with Gal-1. Silencing of miR-22 resulted in the upregulation of Gal-1 and enhanced cell growth, migration and invasion. However, such enhancement was abolished in cells treated with OTX008, an inhibitor of Gal-1. Combinational treatment of OTX008 and sorafenib significantly reduced tumor growth and size. CONCLUSIONS: Gal-1 overexpression was detected in HCC and this played a role in promoting tumorigenic processes and metastasis. The function of Gal-1 was found to be mediated through RER1. The correlations between miR-22, Gal-1 and RER1 expressions demonstrated the importance of miR-22 regulation on Gal-1/RER1 oncogenic activity. Lastly, the combinational treatment of OTX008 and sorafenib proved to be an improved therapeutic option compared to when administering sorafenib alone.
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Calixarenos/uso terapéutico , Carcinoma Hepatocelular/genética , Galectina 1/efectos adversos , Neoplasias Hepáticas/genética , Sorafenib/uso terapéutico , Animales , Calixarenos/farmacología , Carcinoma Hepatocelular/patología , Humanos , Neoplasias Hepáticas/patología , Masculino , Ratones , Ratones Desnudos , Sorafenib/farmacología , TransfecciónRESUMEN
Auranofin (AuRF) has been reported to display anticancer activity and has entered several clinical trials; however, its mechanism of action remains largely unknown. In this work, the anticancer mechanism of auranofin was investigated using a proteomics strategy entailing subcellular fractionation prior to mass spectrometric analysis. Bioinformatics analysis of the nuclear sub-proteomes revealed that tumor suppressor p14ARF is a key regulator of transcription. Through independent analysis, we validated that up-regulation of p14ARF is associated with E2F-dependent transcription and increased p53 expression. Our analyses further reveal that 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), which is the rate-determining enzyme of the mevalonate pathway, is a novel target of auranofin with half maximal inhibitory concentration at micromolar levels. The auranofin-induced cancer cell death could be partially reverted by the addition of downstream products of the mevalonate pathway (mevalonolactone or geranyleranyl pyrophosphate (GGPP)), implying that auranofin may target the mevalonate pathway to exert its anticancer effect.
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Antineoplásicos/farmacología , Auranofina/farmacología , Hidroximetilglutaril-CoA Reductasas/metabolismo , Terapia Molecular Dirigida , Línea Celular Tumoral , Factores de Transcripción E2F/metabolismo , Oro/farmacología , Humanos , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Proteína p14ARF Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Histone posttranslational modifications (PTMs) regulate chromatin structure and dynamics during various DNA-associated processes. Here, we report that lysine glutarylation (Kglu) occurs at 27 lysine residues on human core histones. Using semi-synthetic glutarylated histones, we show that an evolutionarily conserved Kglu at histone H4K91 destabilizes nucleosome in vitro. In Saccharomyces cerevisiae, the replacement of H4K91 by glutamate that mimics Kglu influences chromatin structure and thereby results in a global upregulation of transcription and defects in cell-cycle progression, DNA damage repair, and telomere silencing. In mammalian cells, H4K91glu is mainly enriched at promoter regions of highly expressed genes. A downregulation of H4K91glu is tightly associated with chromatin condensation during mitosis and in response to DNA damage. The cellular dynamics of H4K91glu is controlled by Sirt7 as a deglutarylase and KAT2A as a glutaryltransferase. This study designates a new histone mark (Kglu) as a new regulatory mechanism for chromatin dynamics.
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Ensamble y Desensamble de Cromatina , Daño del ADN , Glutaratos/metabolismo , Histonas/metabolismo , Mitosis , Nucleosomas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Células HEK293 , Células HL-60 , Células HeLa , Células Hep G2 , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Lisina , Ratones , Nucleosomas/genética , Células RAW 264.7 , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crecimiento & desarrollo , Proteínas de Saccharomyces cerevisiae/genética , Sirtuinas/genética , Sirtuinas/metabolismo , Factores de TiempoRESUMEN
New anticancer platinum(II) compounds with distinctive modes of action are appealing alternatives to combat the drug resistance and improve the efficacy of clinically used platinum chemotherapy. Herein, we describe a rare example of an antitumor PtII complex targeting a tumor-associated protein, rather than DNA, under cellular conditions. Complex [(bis-NHC)Pt(bt)]PF6 (1 a; Hbt=1-(3-hydroxybenzo[b]thiophen-2-yl)ethanone) overcomes cisplatin resistance in cancer cells and displays significant tumor growth inhibition in mice with higher tolerable doses compared to cisplatin. The cellular Pt species shows little association with DNA, and localizes in the cytoplasm as revealed by nanoscale secondary ion mass spectrometry. An unbiased thermal proteome profiling experiment identified asparagine synthetase (ASNS) as a molecular target of 1 a. Accordingly, 1 a treatment reduced the cellular asparagine levels and inhibited cancer cell proliferation, which could be reversed by asparagine supplementation. A bis-NHC-ligated Pt species generated from the hydrolysis of 1 a forms adducts with thiols and appears to target an active-site cysteine of ASNS.
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Antineoplásicos/farmacología , Aspartatoamoníaco Ligasa/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Compuestos Organoplatinos/farmacología , Antineoplásicos/química , Aspartatoamoníaco Ligasa/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Cisplatino/química , Cisplatino/farmacología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Inhibidores Enzimáticos/química , Humanos , Ligandos , Estructura Molecular , Compuestos Organoplatinos/química , Relación Estructura-ActividadRESUMEN
Dehydroeffusol (DHE) is a phenanthrene isolated from the Chinese medicinal plant Juncus effusus. Biological evaluation of DHE reveals in vitro and in vivo anticancer effects. We performed a shotgun proteomic analysis using liquid chromatography-tandem mass spectrometry to investigate the changes in the protein profiles in cancer cells upon DHE treatment. DHE affected cancer-associated signaling pathways, including NF-κB, ß-catenin, and endoplasmic reticulum stress. Through quantitative pathway and key node analysis of the proteomics data, activating transcription factor 2 (ATF-2) and c-Jun kinase (JNK) were found to be the key components in DHE's modulated biological pathways. Based on the pathway analysis as well as chemical similarity to estradiol, DHE is proposed to be a phytoestrogen. The proteomic, bioinformatic, and chemoinformatic analyses were further verified with individual cell-based experiments. Our study demonstrates a workflow for identifying the mechanisms of action of DHE through shotgun proteomic analysis.
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Antineoplásicos/farmacología , Fenantrenos/farmacología , Fitoquímicos/farmacología , Factor de Transcripción Activador 2/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Neoplasias/patología , Fitoestrógenos , Poaceae/química , Proteómica/métodos , Transducción de Señal/efectos de los fármacosRESUMEN
Metal N-heterocyclic carbene (NHC) complexes are a promising class of anti-cancer agents displaying potent inâ vitro and inâ vivo activities. Taking a multi-faceted approach employing two clickable photoaffinity probes, herein we report the identification of multiple molecular targets for anti-cancer active pincer gold(III) NHC complexes. These complexes display potent and selective cytotoxicity against cultured cancer cells and inâ vivo anti-tumor activities in mice bearing xenografts of human cervical and lung cancers. Our experiments revealed the specific engagement of the gold(III) complexes with multiple cellular targets, including HSP60, vimentin, nucleophosmin, and YB-1, accompanied by expected downstream mechanisms of action. Additionally, PtII and PdII analogues can also bind the cellular proteins targeted by the gold(III) complexes, uncovering a distinct pincer cyclometalated metal-NHC scaffold in the design of anti-cancer metal medicines with multiple molecular targets.
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Antineoplásicos/farmacología , Compuestos Heterocíclicos/farmacología , Metano/análogos & derivados , Compuestos Orgánicos de Oro/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Peso Corporal/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Compuestos Heterocíclicos/química , Humanos , Ligandos , Metano/química , Metano/farmacología , Ratones , Estructura Molecular , Neoplasias Experimentales/tratamiento farmacológico , Neoplasias Experimentales/patología , Compuestos Orgánicos de Oro/química , Relación Estructura-ActividadRESUMEN
Sirtuins are NAD-dependent deacylases. Previous studies have established two important enzymatic intermediates in sirtuin-catalyzed deacylation, an alkylamidate intermediate I, which is then converted to a bicyclic intermediate II. However, how intermediate II is converted to products is unknown. Based on potent SIRT2-specific inhibitors we developed, here we report crystal structures of SIRT2 in complexes with a thiomyristoyl lysine peptide-based inhibitor and carba-NAD or NAD. Interestingly, by soaking crystals with NAD, we capture a distinct covalent catalytic intermediate (III) that is different from the previously established intermediates I and II. In this intermediate, the covalent bond between the S and the myristoyl carbonyl carbon is broken, and we believe this intermediate III to be the decomposition product of II en route to form the end products. MALDI-TOF data further support the intermediate III formation. This is the first time such an intermediate has been captured by X-ray crystallography and provides more mechanistic insights into sirtuin-catalyzed reactions.
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Ácidos Grasos Monoinsaturados/química , Sirtuina 2/metabolismo , Acilación , Biocatálisis , Cristalografía por Rayos X , Humanos , NAD/análogos & derivados , NAD/química , NAD/metabolismo , Péptidos/análisis , Péptidos/síntesis química , Péptidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Sirtuina 2/química , Sirtuina 2/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Palladium(II) complexes are generally reactive toward substitution/reduction, and their biological applications are seldom explored. A new series of palladium(II) N-heterocyclic carbene (NHC) complexes that are stable in the presence of biological thiols are reported. A representative complex, [Pd(C^N^N)(N,N'-nBu2 NHC)](CF3 SO3 ) (Pd1 d, HC^N^N=6-phenyl-2,2'-bipyridine, N,N'-nBu2 NHC=N,N'-di-n-butylimidazolylidene), displays potent killing activity toward cancer cell lines (IC50 =0.09-0.5â µm) but is less cytotoxic toward a normal human fibroblast cell line (CCD-19Lu, IC50 =11.8â µm). Inâ vivo anticancer studies revealed that Pd1 d significantly inhibited tumor growth in a nude mice model. Proteomics data and inâ vitro biochemical assays reveal that Pd1 d exerts anticancer effects, including inhibition of an epidermal growth factor receptor pathway, induction of mitochondrial dysfunction, and antiangiogenic activity to endothelial cells.
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Antineoplásicos/uso terapéutico , Complejos de Coordinación/uso terapéutico , Metano/análogos & derivados , Neoplasias/tratamiento farmacológico , Paladio/uso terapéutico , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Humanos , Metano/química , Metano/farmacología , Metano/uso terapéutico , Ratones Desnudos , Neoplasias/patología , Paladio/química , Paladio/farmacologíaRESUMEN
A panel of luminescent platinum(II) complexes containing bidentate N-heterocyclic carbene ligands selectively localize to the endoplasmic reticulum (ER) domain, induce ER stress and cell apoptosis. Some of them show potent photo-toxicity to cancer cells.
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Complejos de Coordinación/farmacología , Citotoxinas/farmacología , Compuestos Organoplatinos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/química , Citotoxinas/química , Retículo Endoplásmico/metabolismo , Estrés del Retículo Endoplásmico/efectos de los fármacos , Humanos , Ligandos , Luz , Luminiscencia , Metano/análogos & derivados , Metano/química , Compuestos Organoplatinos/químicaRESUMEN
Electron capture dissociation (ECD) of model peptides adducted with first row divalent transition metal ions, including Mn(2+), Fe(2+), Co(2+), Ni(2+), Cu(2+), and Zn(2+), were investigated. Model peptides with general sequence of ZGGGXGGGZ were used as probes to unveil the ECD mechanism of metalated peptides, where X is either V or W; and Z is either R or N. Peptides metalated with different divalent transition metal ions were found to generate different ECD tandem mass spectra. ECD spectra of peptides metalated by Mn(2+) and Zn(2+) were similar to those generated by ECD of peptides adducted with alkaline earth metal ions. Series of c-/z-type fragment ions with and without metal ions were observed. ECD of Fe(2+), Co(2+), and Ni(2+) adducted peptides yielded abundant metalated a-/y-type fragment ions; whereas ECD of Cu(2+) adducted peptides generated predominantly metalated b-/y-type fragment ions. From the present experimental results, it was postulated that electronic configuration of metal ions is an important factor in determining the ECD behavior of the metalated peptides. Due presumably to the stability of the electronic configuration, metal ions with fully-filled (i.e., Zn(2+)) and half filled (i.e., Mn(2+)) d-orbitals might not capture the incoming electron. Dissociation of the metal ions adducted peptides would proceed through the usual ECD channel(s) via "hot-hydrogen" or "superbase" intermediates, to form series of c-/z(â¢)- fragments. For other transition metal ions studied, reduction of the metal ions might occur preferentially. The energy liberated by the metal ion reduction would provide enough internal energy to generate the "slow-heating" type of fragment ions, i.e., metalated a-/y- fragments and metalated b-/y- fragments.