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1.
Neurosci Lett ; 750: 135766, 2021 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-33639221

RESUMEN

Ischemic stroke is one of the major diseases that cause mortality and morbidity of human beings, but there is still lack of effective treatment and prevention. We found that 2-(2-Benzofuranyl)-2-Imidazoline (2-BFI) is potently protective against stroke and acute inflammatory immune disease. Moreover, the mammalian target of rapamycin (mTOR) signaling contributes effectively to the modulation of post-stroke neuroinflammatory response. However, whether the protection of 2-BFI against ischemic injury is through mTOR-mediated neuroinflammatory response remains unestablished. Here, we used 2-BFI to treat ischemic rats induced by distal middle cerebral artery occlusion (dMCAO). We found that 2-BFI administration after dMCAO improved the neurological deficits and decreased the infarct volume. 2-BFI reduced phosphorylation of mTOR and p70S6, increased IL-10 and TGF-ß, and decreased IFN-γ levels in ischemic rats. Our results demonstrated that 2-BFI attenuates ischemic injury by inhibiting the activation of mTOR signaling and modulating neuroinflammation after stroke in rats.


Asunto(s)
Marcadores de Afinidad/uso terapéutico , Antiinflamatorios/uso terapéutico , Benzofuranos/uso terapéutico , Imidazoles/uso terapéutico , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Serina-Treonina Quinasas TOR/metabolismo , Marcadores de Afinidad/farmacología , Animales , Antiinflamatorios/farmacología , Benzofuranos/farmacología , Imidazoles/farmacología , Infarto de la Arteria Cerebral Media/metabolismo , Masculino , Ratas , Ratas Sprague-Dawley , Transducción de Señal
2.
Sci Rep ; 10(1): 18078, 2020 10 22.
Artículo en Inglés | MEDLINE | ID: mdl-33093565

RESUMEN

Lipopolysaccharide (LPS), a component of the outer membrane of gram-negative bacteria, disrupts the alveolar-capillary barrier, triggering pulmonary vascular leak thus inducing acute lung injury (ALI). Extracellular purines, adenosine and ATP, protected against ALI induced by purified LPS. In this study, we investigated whether these purines can impact vascular injury in more clinically-relevant E.coli (non-sterile LPS) murine ALI model. Mice were inoculated with live E. coli intratracheally (i.t.) with or without adenosine or a non-hydrolyzable ATP analog, adenosine 5'-(γ-thio)-triphosphate (ATPγS) added intravenously (i.v.). After 24 h of E. coli treatment, we found that injections of either adenosine or ATPγS 15 min prior or adenosine 3 h after E.coli insult significantly attenuated the E.coli-mediated increase in inflammatory responses. Furthermore, adenosine prevented weight loss, tachycardia, and compromised lung function in E. coli-exposed mice. Accordingly, treatment with adenosine or ATPγS increased oxygen saturation and reduced histopathological signs of lung injury in mice exposed to E. coli. Lastly, lung-targeting gene delivery of adenosine or ATPγS downstream effector, myosin phosphatase, significantly attenuated the E. coli-induced compromise of lung function. Collectively, our study has demonstrated that adenosine or ATPγS mitigates E. coli-induced ALI in mice and may be useful as an adjuvant therapy in future pre-clinical studies.


Asunto(s)
Lesión Pulmonar Aguda/prevención & control , Adenosina Trifosfato/análogos & derivados , Adenosina/farmacología , Escherichia coli/patogenicidad , Neumonía Bacteriana/complicaciones , Vasodilatadores/farmacología , Lesión Pulmonar Aguda/etiología , Adenosina Trifosfato/farmacología , Marcadores de Afinidad/farmacología , Animales , Masculino , Ratones , Ratones Endogámicos C57BL
3.
ACS Chem Biol ; 15(4): 952-961, 2020 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-32191434

RESUMEN

We synthesized affinity-based chemical probes of cytosine-adenosine bisubstrate analogs and identified several potential targets by proteomic analysis. The validation of the proteomic analysis identified the chemical probe as a specific inhibitor of glucose-regulated protein 94 (GRP94), a potential drug target for several types of cancers. Therefore, as a result of the use of bisubstrate-type chemical probes and a chemical-biology methodology, this work opens the way to the development of a new family of GRP94 inhibitors that could potentially be of therapeutic interest.


Asunto(s)
Adenosina/análogos & derivados , Adenosina/farmacología , Marcadores de Afinidad/farmacología , Citosina/análogos & derivados , Citosina/farmacología , Glicoproteínas de Membrana/antagonistas & inhibidores , Adenosina/efectos de la radiación , Marcadores de Afinidad/síntesis química , Marcadores de Afinidad/efectos de la radiación , Línea Celular Tumoral , Química Clic , Citosina/efectos de la radiación , Humanos , Glicoproteínas de Membrana/química , Proteoma/química , Proteómica , Rayos Ultravioleta
4.
J Bioenerg Biomembr ; 52(2): 103-111, 2020 04.
Artículo en Inglés | MEDLINE | ID: mdl-31960257

RESUMEN

Cancer cells apply the Warburg pathway to meet their increased metabolic demands caused by their rapid growth and proliferation and also creates an acidic environment to promote cancer cell invasion. 3-bromopyruvate (3-BrP) as an anti-cancer agent disrupts glycolytic pathway. Moreover, one of the mechanism of actions of Methyl Jasmonate (MJ) is interference in glycolysis. Hence, the aim of this study was to evaluate MJ and 3-BrP interaction. MTT assay was used to determine IC50 and synergistic concentrations. Combination index was applied to evaluate the drug- drug interaction. Human tumor xenograft breast cancer mice was used to evaluate drug efficacy in vivo. Tumor size was considered as a drug efficacy criterion. In addition to drug efficacy, probable side effects of these drugs including hepatotoxicity, renal failure, immunotoxicity, and losing weight were evaluated. Serum alanine aminotransferase and aspartate aminotransferase for hepatotoxicity, serum urea and creatinine level for the possibility of renal failure and changes in body weight were measured to evaluate drug toxicity. IL10 and TGFß secretion in supernatant of isolated splenocytes from treated mice were assessed to check immunotoxicity. 3-BrP synergistically augmented the efficacy of MJ in the specific concentrations. This polytherapy was more effective than monotherapy of 3-BrP, MJ, and also surprisingly cyclophosphamide as a routine treatment for breast cancer in the tumor bearing mice. These results have been shown by decrease in tumor volume and increase of tumor growth inhibition percentage. This combination therapy didn't have any noticeable side effects on kidney, liver, and immune system and body weight.


Asunto(s)
Acetatos/uso terapéutico , Marcadores de Afinidad/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Ciclopentanos/uso terapéutico , Oxilipinas/uso terapéutico , Reguladores del Crecimiento de las Plantas/química , Piruvatos/uso terapéutico , Acetatos/farmacología , Marcadores de Afinidad/farmacología , Animales , Línea Celular Tumoral , Proliferación Celular , Ciclopentanos/farmacología , Modelos Animales de Enfermedad , Femenino , Ratones , Oxilipinas/farmacología , Piruvatos/farmacología , Ensayos Antitumor por Modelo de Xenoinjerto
5.
Chem Commun (Camb) ; 55(67): 9919-9922, 2019 Aug 28.
Artículo en Inglés | MEDLINE | ID: mdl-31328197

RESUMEN

Reported herein is a relebactam-derived fluorogenic reagent for covalent labeling of serine ß-lactamases (SBLs), which are the major causes of bacterial resistance to ß-lactam antibiotics. This highly selective imaging reagent generates over 300-fold stronger near-infrared fluorescence signals upon covalently bonding to SBLs, allowing wash-free visualization of live antimicrobial-resistant bacteria.


Asunto(s)
Marcadores de Afinidad/farmacología , Compuestos de Azabiciclo/farmacología , Enterobacter cloacae/aislamiento & purificación , Colorantes Fluorescentes/farmacología , Inhibidores de beta-Lactamasas/farmacología , beta-Lactamasas/química , Marcadores de Afinidad/síntesis química , Marcadores de Afinidad/química , Compuestos de Azabiciclo/síntesis química , Compuestos de Azabiciclo/química , Enterobacter cloacae/enzimología , Fluoresceínas/síntesis química , Fluoresceínas/química , Fluoresceínas/farmacología , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Indoles/síntesis química , Indoles/química , Indoles/farmacología , Inhibidores de beta-Lactamasas/síntesis química , Inhibidores de beta-Lactamasas/química
6.
ACS Chem Biol ; 13(11): 3184-3192, 2018 11 16.
Artículo en Inglés | MEDLINE | ID: mdl-30289689

RESUMEN

Benzothiazinones (BTZ) are highly potent bactericidal inhibitors of mycobacteria and the lead compound, BTZ043, and the optimized drug candidate, PBTZ169, have potential for the treatment of tuberculosis. Here, we exploited the tractability of the BTZ scaffold by attaching a range of fluorophores to the 2-substituent of the BTZ ring via short linkers. We show by means of fluorescence imaging that the most advanced derivative, JN108, is capable of efficiently labeling its target, the essential flavoenzyme DprE1, both in cell-free extracts and after purification as well as in growing cells of different actinobacterial species. DprE1 displays a polar localization in Mycobacterium tuberculosis, M. marinum, M. smegmatis, and Nocardia farcinica but not in Corynebacterium glutamicum. Finally, mutation of the cysteine residue in DprE1 in these species, to which BTZ covalently binds, abolishes completely the interaction with JN108, thereby highlighting the specificity of this fluorescent probe.


Asunto(s)
Marcadores de Afinidad/farmacología , Oxidorreductasas de Alcohol/antagonistas & inhibidores , Antituberculosos/farmacología , Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Tiazinas/farmacología , Actinomycetales/efectos de los fármacos , Actinomycetales/enzimología , Marcadores de Afinidad/síntesis química , Oxidorreductasas de Alcohol/genética , Antituberculosos/síntesis química , Proteínas Bacterianas/genética , Membrana Celular/metabolismo , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Fluoresceínas/síntesis química , Fluoresceínas/farmacología , Fluorescencia , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Células Hep G2 , Humanos , Pruebas de Sensibilidad Microbiana , Microscopía Fluorescente/métodos , Mutación , Tiazinas/síntesis química
7.
Klin Onkol ; 31(Supplementum1): 140-144, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29808688

RESUMEN

BACKGROUND: Nucleoside analogues represent a relevant class of antimetabolites used for therapy of various types of cancer. However, their effectivity is limited by drug resistance. The nucleoside transport capability of tumour cells is considered to be a determinant of the clinical outcome of treatment regimens using antimetabolites. Due to hydrophilic properties of antimetabolites, their transport across the plasma membrane is mediated by two families of transmembrane proteins, the SLC28 family of cation-linked concentrative nucleoside transporters (hCNTs) and SLC29 family of energy-independent equilibrative nucleoside transporters (hENTs). Loss of functional nucleoside transporters has been associated with reduced efficacy of antimetabolites and their derivatives and treatment failure in diverse malignancies including solid tumours, such as pancreatic adenocarcinoma. MATERIAL AND METHODS: The effectivity and kinetics of antimetabolite uptake were analysed using control and docetaxel-resistant PC3 cells. For this purpose, fluorescent nucleoside analogue probe uridine-furane and inhibitor of nucleoside transporters, S-(4-nitrobenzyl) -6-thioinosine were exploited. Combination of flow cytometry, confocal microscopy and real-time quantitative polymerase chain reaction methodology were used for the analysis. RESULTS: Here we utilized flow cytometric assay for analysis of nucleoside transporters activity employing fluorescent nucleoside analogue, uridine-furane. We have determined the long-time kinetics of uridine-furane incorporation and quantified its levels in the parental prostate cancer cell line PC3 and its chemoresistant derivative. Finally, we have shown an association between the activity and mRNA expression of nucleoside transporters and sensitivity to various nucleoside analogues. CONCLUSION: Fluorescent techniques can serve as an effective tool for the detection of nucleoside transporter activity which has the potential for application in clinical oncology.Key words: nucleoside transporter proteins - drug resistance - prostatic neoplasm - chemotherapy.


Asunto(s)
Resistencia a Antineoplásicos/genética , Citometría de Flujo/métodos , Regulación Neoplásica de la Expresión Génica , Proteínas de Transporte de Nucleósidos/genética , Neoplasias de la Próstata/genética , Marcadores de Afinidad/farmacología , Antineoplásicos/farmacología , Docetaxel/farmacología , Colorantes Fluorescentes/farmacología , Humanos , Masculino , Células PC-3 , Neoplasias de la Próstata/tratamiento farmacológico , Tioinosina/análogos & derivados , Tioinosina/farmacología
8.
Anal Biochem ; 482: 25-31, 2015 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-25912417

RESUMEN

We studied the target proteins of artemisinin in Trypanosoma brucei brucei using the affinity-labeling method. We designed and synthesized four biotinylated probes of artemisinin for use as molecular tools. Their in vitro trypanocidal activities (data not shown) proved that they mimicked the biological action of artemisinin. We assessed the chemical stability for all of the probes in the parasite culture medium and lysate using reversed-phase high-performance liquid chromatography (HPLC). After 3-h incubations, the probes remained undecomposed in a range of 40 to 65% in the parasite culture medium, whereas approximately 80% of the probes remained stable in the parasite lysate. Using liquid chromatography mass spectrometry (LC-MS), we demonstrated that, with respect to all of the probes, uptakes into the parasite ranging from 81 to 96% occurred after 30-min incubations. In a competitive binding assay between artemisinin and the four biotinylated probes, we searched for the trypanosomal target protein of artemisinin. Consequently, we observed that only the diazirine-free probe 5 could provide the desired result with high affinity-labeling efficiency. Using the horseradish peroxidase-tagged streptavidin-biotin method, we showed that artemisinin could specifically bind to candidate target proteins of approximately 60, 40, and 39 kDa.


Asunto(s)
Marcadores de Afinidad/química , Antiprotozoarios/química , Artemisininas/química , Proteínas Protozoarias/metabolismo , Trypanosoma brucei brucei/metabolismo , Marcadores de Afinidad/farmacología , Animales , Antiprotozoarios/farmacología , Artemisininas/farmacología , Biotinilación , Western Blotting , Humanos , Terapia Molecular Dirigida , Trypanosoma brucei brucei/efectos de los fármacos , Tripanosomiasis Africana/tratamiento farmacológico , Tripanosomiasis Africana/parasitología
9.
Spectrochim Acta A Mol Biomol Spectrosc ; 143: 309-18, 2015 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-25766241

RESUMEN

The binding capabilities of a series of novel quinazolinone molecules were established and stated in a comprehensive computational methodology as well as by in vitro analysis. The main focus of this work was to achieve more insight of the interactions with crystal structure of PDB ID: 1M17 and predict their binding mode to EGFR. Three molecules were screened for further examination, which were synthesized and characterized using spectroscopic techniques. The persuasive affinity of these molecules towards EGFR inhibition (IC50 for QT=45nM) was established and validated from specific kinase assay including the cell viability spectrophotometric assay (QT=12nM). Drug likeliness property were also considered by analysing, the ADME of these molecules by using scintigraphic techniques. The result showed antitumour activity of QT (4.17 tumour/muscle at 4h). Further photo physical properties were also analysed to see in vitro HSA binding to QT.


Asunto(s)
Marcadores de Afinidad/química , Antineoplásicos/química , Receptores ErbB/antagonistas & inhibidores , Quinazolinonas/química , Marcadores de Afinidad/farmacocinética , Marcadores de Afinidad/farmacología , Animales , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Receptores ErbB/metabolismo , Células MCF-7 , Ratones Endogámicos BALB C , Simulación del Acoplamiento Molecular , Quinazolinonas/farmacocinética , Quinazolinonas/farmacología , Conejos , Distribución Tisular
10.
Food Environ Virol ; 6(4): 269-75, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25106777

RESUMEN

The damage to a viral capsid after low-pressure (LP) and medium-pressure (MP) UV irradiation was assessed, using the quantitative or quantitative reverse transcription PCR coupled with ethidium monoazide treatment (EMA-PCR). After UV irradiation, adenovirus 5 (Ad5) and poliovirus 1 (PV1) were subjected to a plaque assay, PCR, and EMA-PCR to investigate the effect of UV irradiation on viral infectivity, genome damage, and capsid damage, respectively. The effectiveness of UV wavelengths in a viral genome and capsid damage of both PV1 and Ad5 was also further investigated using a band-pass filter. It was found that an MPUV lamp was more effective than an LPUV lamp in inactivating Ad5, whereas there was no difference in the case of PV1. The results of viral reduction determined by PCR and EMA-PCR indicated that MP UV irradiation damaged Ad5 capsid. The damage to PV1 and Ad5 capsid was also not observed after LP UV irradiation. The investigation of effects of UV wavelengths suggested that UV wavelengths at 230-245 nm have greater effects on adenovirus capsid in addition to viral genome than UV wavelengths beyond 245 nm.


Asunto(s)
Adenovirus Humanos/efectos de la radiación , Marcadores de Afinidad/farmacología , Azidas/farmacología , Cápside/efectos de la radiación , Desinfección/métodos , Genoma Viral/efectos de la radiación , Poliovirus/efectos de la radiación , Adenovirus Humanos/crecimiento & desarrollo , Adenovirus Humanos/metabolismo , Adenovirus Humanos/patogenicidad , Animales , Cápside/metabolismo , Línea Celular , Chlorocebus aethiops , ADN Viral/metabolismo , ADN Viral/efectos de la radiación , Humanos , Poliovirus/crecimiento & desarrollo , Poliovirus/metabolismo , Poliovirus/patogenicidad , Presión , ARN Viral/metabolismo , ARN Viral/efectos de la radiación , Tolerancia a Radiación , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Rayos Ultravioleta , Ensayo de Placa Viral , Inactivación de Virus/efectos de la radiación
11.
Biochemistry ; 53(1): 135-42, 2014 Jan 14.
Artículo en Inglés | MEDLINE | ID: mdl-24341978

RESUMEN

Propofol, an intravenous general anesthetic, produces many of its anesthetic effects in vivo by potentiating the responses of GABA type A receptors (GABAAR), members of the superfamily of pentameric ligand-gated ion channels (pLGICs) that contain anion-selective channels. Propofol also inhibits pLGICs containing cation-selective channels, including nicotinic acetylcholine receptors and GLIC, a prokaryotic proton-gated homologue from Gloeobacter violaceus . In the structure of GLIC cocrystallized with propofol at pH 4 (presumed open/desensitized states), propofol was localized to an intrasubunit pocket at the extracellular end of the transmembrane domain within the bundle of transmembrane α-helices (Nury, H, et al. (2011) Nature 469, 428-431). To identify propofol binding sites in GLIC in solution, we used a recently developed photoreactive propofol analogue (2-isopropyl-5-[3-(trifluoromethyl)-3H-diazirin-3-yl]phenol or AziPm) that acts as an anesthetic in vivo and potentiates GABAAR in vitro. For GLIC expressed in Xenopus oocytes, propofol and AziPm inhibited current responses at pH 5.5 (EC20) with IC50 values of 20 and 50 µM, respectively. When [(3)H]AziPm (7 µM) was used to photolabel detergent-solubilized, affinity-purified GLIC at pH 4.4, protein microsequencing identified propofol-inhibitable photolabeling of three residues in the GLIC transmembrane domain: Met-205, Tyr-254, and Asn-307 in the M1, M3, and M4 transmembrane helices, respectively. Thus, for GLIC in solution, propofol and AziPm bind competitively to a site in proximity to these residues, which, in the GLIC crystal structure, are in contact with the propofol bound in the intrasubunit pocket.


Asunto(s)
Proteínas Bacterianas/química , Canales Iónicos/química , Propofol/química , Marcadores de Afinidad/farmacología , Secuencia de Aminoácidos , Proteínas Bacterianas/antagonistas & inhibidores , Sitios de Unión , Diazometano/análogos & derivados , Diazometano/química , Diazometano/farmacología , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos Activados por Ligandos , Modelos Moleculares , Propofol/análogos & derivados , Propofol/farmacología , Estructura Terciaria de Proteína , Receptores de GABA-A/metabolismo
12.
Transl Stroke Res ; 4(5): 533-45, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-24312160

RESUMEN

Increasing endogenous ciliary neurotrophic factor (CNTF) expression with a pharmacological agent might be beneficial after stroke as CNTF both promotes neurogenesis and, separately, is neuroprotective. P2X7 purinergic receptor inhibition is neuroprotective in rats and increases CNTF release in rat CMT1A Schwann cells. We, first, investigated the role of P2X7 in regulating CNTF and neurogenesis in adult mouse subventricular zone (SVZ). CNTF expression was increased by daily intravenous injections of the P2X7 antagonist Brilliant Blue G (BBG) in naïve C57BL/6 or Balb/c mice over 3 days. Despite the ∼40-60 % increase or decrease in CNTF with BBG or the agonist BzATP, respectively, the number of proliferated BrdU+SVZ nuclei did not change. BBG failed to increase FGF2, which is involved in CNTF-regulated neurogenesis, but induced IL-6, LIF, and EGF, which are known to reduce SVZ proliferation. Injections of IL-6 next to the SVZ induced CNTF and FGF2, but not proliferation, suggesting that IL-6 counteracts their neurogenesis-inducing effects. Following ischemic injury of the striatum by middle cerebral artery occlusion (MCAO), a 3-day BBG treatment increased CNTF in the medial penumbra containing the SVZ. BBG also induced CNTF and LIF, which are known to be protective following stroke, in the whole striatum after MCAO, but not GDNF or BDNF. However, BBG treatment did not reduce the lesion area or apoptosis in the penumbra. Even so, this study shows that P2X7 can be targeted with systemic drug treatments to differentially regulate neurotrophic factors in the brain following stroke.


Asunto(s)
Factor Neurotrófico Ciliar/biosíntesis , Infarto de la Arteria Cerebral Media/metabolismo , Antagonistas del Receptor Purinérgico P2X/farmacología , Receptores Purinérgicos P2X7/metabolismo , Accidente Cerebrovascular/metabolismo , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/farmacología , Marcadores de Afinidad/farmacología , Animales , Citocinas/metabolismo , Modelos Animales de Enfermedad , Indicadores y Reactivos/farmacología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Neurogénesis/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Colorantes de Rosanilina/farmacología
13.
Cancer Chemother Pharmacol ; 72(1): 189-99, 2013 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-23673445

RESUMEN

PURPOSE: Specific tyrosine kinase inhibitors were recently reported to modulate the activity of ABC transporters, leading to an increase in the intracellular concentration of their substrate drugs. In this study, we determine whether PD173074, a specific fibroblast growth factor receptor (FGFR) inhibitor, could reverse ABC transporter-mediated multidrug resistance. METHODS: 3-(4,5-Dimethylthiazol-yl)-2,5-diphenyllapatinibrazolium bromide assay was used to determine the effect of PD173074 on reversal of ABC transporter-mediated multidrug resistance (MDR). In addition, [³H]-paclitaxel accumulation/efflux assay, western blotting analysis, ATPase, and photoaffinity labeling assays were done to study the interaction of PD173074 on ABC transporters. RESULTS: PD173074 significantly sensitized both ABCB1-transfected and drug-selected cell lines overexpressing this transporter to substrate anticancer drugs colchicine, paclitaxel, and vincristine. This effect of PD173074 is specific to ABCB1, as no significant interaction was detected with other ABC transporters such as ABCC1 and ABCG2. The observed reversal effect seems to be primarily due to the decreased active efflux of [³H]-paclitaxel in ABCB1 overexpressing cells observed in efflux assay. In addition, no significant change in the ABCB1 expression was observed when ABCB1 overexpressing cells were exposed to 5 µM PD173074 for up to 3 days, thereby further suggesting its role in modulating the function of the transporter. In addition, PD173074 stimulated the ATPase activity of ABCB1 in a concentration-dependent manner, indicating a direct interaction with the transporter. Interestingly, PD173074 did not inhibit photolabeling of ABCB1 with [¹²5I]-iodoarylazidoprazosin (IAAP), showing that it binds at a site different from that of IAAP in the drug-binding pocket. CONCLUSIONS: Here, we report for the first time, PD173074, an inhibitor of the FGFR, to selectively reverse ABCB1 transporter-mediated MDR by directly blocking the efflux function of the transporter.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Antineoplásicos/agonistas , Resistencia a Múltiples Medicamentos/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Neoplasias/tratamiento farmacológico , Pirimidinas/farmacología , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Subfamilia B de Transportador de Casetes de Unión a ATP , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/agonistas , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Marcadores de Afinidad/farmacología , Regulación Alostérica , Antineoplásicos/metabolismo , Antineoplásicos/farmacología , Transporte Biológico/efectos de los fármacos , Línea Celular Tumoral , Colchicina/agonistas , Colchicina/farmacología , Células HEK293 , Humanos , Hidrólisis/efectos de los fármacos , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/metabolismo , Neoplasias/enzimología , Neoplasias/metabolismo , Paclitaxel/agonistas , Paclitaxel/metabolismo , Paclitaxel/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Recombinantes/agonistas , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/metabolismo , Moduladores de Tubulina/agonistas , Moduladores de Tubulina/metabolismo , Moduladores de Tubulina/farmacología , Vincristina/agonistas , Vincristina/farmacología
14.
Drug Metab Dispos ; 41(4): 916-22, 2013 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-23388705

RESUMEN

The high density of A1 adenosine receptors in the brain results in significant potential for central nervous system (CNS)-related adverse effects with A1 agonists. Tecadenoson is a selective A1 adenosine receptor agonist with close similarity to adenosine. We studied the binding and transmembrane transport of tecadenoson by recombinant human equilibrative nucleoside transporters (hENTs) hENT1 and hENT2, and human concentrative nucleoside transporters (hCNTs) hCNT1, hCNT2, and hCNT3 in vitro and by mouse mENT1 in vivo. Binding affinities of the five recombinant human nucleoside transporters for tecadenoson differed (hENT1 > hCNT1 > hCNT3 > hENT2 > hCNT2), and tecadenoson was transported largely by hENT1. Pretreatment of mice with a phosphorylated prodrug of nitrobenzylmercaptopurine riboside, an inhibitor of mENT1, significantly decreased brain exposure to tecadenoson compared with that of the untreated (control) group, suggesting involvement of mENT1 in transport of tecadenoson across the blood-brain barrier (BBB). In summary, ENT1 was shown to mediate the transport of tecadenoson in vitro with recombinant and native human protein and in vivo with mice. The micromolar apparent Km value of tecadenoson for transport by native hENT1 in cultured cells suggests that hENT1 will not be saturated at clinically relevant (i.e., nanomolar) concentrations of tecadenoson, and that hENT1-mediated passage across the BBB may contribute to the adverse CNS effects observed in clinical trials. In contrast, in cases in which a CNS effect is desired, the present results illustrate that synthetic A1 agonists that are transported by hENT1 could be used to target CNS disorders because of enhanced delivery to the brain.


Asunto(s)
Agonistas del Receptor de Adenosina A1/farmacocinética , Adenosina/análogos & derivados , Barrera Hematoencefálica/metabolismo , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Furanos/farmacocinética , Proteínas de Transporte de Nucleósidos/metabolismo , Profármacos/farmacología , Tioinosina/análogos & derivados , Adenosina/farmacocinética , Marcadores de Afinidad/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Encéfalo/metabolismo , Células Cultivadas , Tranportador Equilibrativo 1 de Nucleósido/antagonistas & inhibidores , Humanos , Moduladores del Transporte de Membrana/farmacología , Ratones , Tioinosina/farmacología
15.
Neurosci Lett ; 530(1): 85-90, 2012 Nov 14.
Artículo en Inglés | MEDLINE | ID: mdl-22981978

RESUMEN

The role of the purinergic system in the modulation of pain mechanisms suggests that it might be promising target for treating neuropathic pain. In this study we evaluated the effects of two different dialdehydic compounds: a modified stable adenosine (2-[1-(6-amminopurin-9-il)-2-osso-etossi]prop-2-enale, named MED1101), and oxidized ATP (Ox-ATP), in two different neuropathic pain rat models: the sciatic spared nerve injury (SNI) and paclitaxel evoked painful peripheral neuropathy (pPPN). Neuropathic animals were divided in groups as follows: (a) treated with intraperitoneal (i.p.) MED1101 or Ox-ATP for 21 days; (b) receiving vehicle (VEH) and (c) control (CTR) rats. The allodynic and hyperalgesic behavior was investigated by Von Frey filament test and thermal Plantar test, respectively. We evaluated by immunocytochemistry the astrocytic (GFAP) and microglial (Iba1) response on lumbar spinal cord sections. In either experimental models and using either substances, treated animals showed reduced allodynia and thermal hyperalgesia paralleled by a significant reduction of glial reaction in the spinal cord. These data prompt to hypothesize a potential role of dialdehydes as analgesic agent in chronic neuropathic pain and a possible role as anti-gliotic molecules.


Asunto(s)
Adenosina Trifosfato/análogos & derivados , Adenosina/análogos & derivados , Aldehídos/farmacología , Analgésicos/farmacología , Gliosis/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Adenosina/farmacología , Adenosina Trifosfato/farmacología , Marcadores de Afinidad/farmacología , Animales , Astrocitos/efectos de los fármacos , Astrocitos/patología , Enfermedad Crónica , Modelos Animales de Enfermedad , Descubrimiento de Drogas/métodos , Gliosis/patología , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/patología , Vértebras Lumbares , Masculino , Microglía/efectos de los fármacos , Microglía/patología , Neuralgia/patología , Ratas , Ratas Sprague-Dawley , Médula Espinal/citología
16.
Biochim Biophys Acta ; 1820(12): 1980-6, 2012 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22982588

RESUMEN

BACKGROUND: Live and injured bacteria cannot be successfully discriminated using flow cytometric methods (FCM) with commercial live/dead staining agents because injured cells have intact cell membranes and are counted as live cells. We previously reported that photoactivated ethidium monoazide (EMA) directly cleaves bacterial DNA both in vivo and in vitro (Microbiol. Immunol. 51:763-775, 2007). METHODS: We report that EMA cleaves the chromosomal DNA of antibiotic-injured, but not live, Listeria monocytogenes. The combination of FCM and EMA treatment was evaluated as a rapid method to discriminate between live and antibiotic-injured L. monocytogenes. Additionally, we evaluated our methodology using blood from pediatric patients infected with other gram-negative and gram-positive bacteria. RESULTS: For antibiotic-injured, but not live, L. monocytogenes in blood, photoactivated EMA suppressed SYTO9 staining, as the SYTO9 staining of the antibiotic-injured L. monocytogenes was weak compared with that of live cells. Similarly, the rapid and clear discrimination between live and injured bacteria (gram-negative and gram-positive) was performed using the blood of pediatric patients administered antibiotics. CONCLUSIONS: The combination of FCM with EMA treatment is a rapid method for evaluating the susceptibility of live pathogens in infants with bacteremia without the need for bacterial culture. GENERAL SIGNIFICANCE: This assay is more rapid than other currently available techniques due to the elimination of the time-consuming culture step and could be used in clinical settings to rapidly determine the success of antibiotic treatment in pediatric bacteremia through the discrimination of injured (i.e., susceptible to the administered antibiotics) and live pathogens.


Asunto(s)
Antibacterianos/farmacología , Bacteriemia/tratamiento farmacológico , ADN Bacteriano/metabolismo , Citometría de Flujo/métodos , Listeria monocytogenes/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Marcadores de Afinidad/farmacología , Azidas/farmacología , Bacteriemia/metabolismo , Bacteriemia/microbiología , ADN Bacteriano/genética , Eritrocitos/efectos de los fármacos , Eritrocitos/metabolismo , Eritrocitos/microbiología , Granulocitos/efectos de los fármacos , Granulocitos/metabolismo , Granulocitos/microbiología , Humanos , Lactante , Luz , Listeria monocytogenes/crecimiento & desarrollo , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/microbiología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/microbiología , Etiquetas de Fotoafinidad
17.
J Proteomics ; 75(10): 2924-33, 2012 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-22202183

RESUMEN

Functionalized quantum dots with dopamine dithiocarbamate (QDs-DDTC) were utilized for the first time as an efficient material for the quantification of efavirenz in human plasma of HIV infected patients and rapid identification of microwave tryptic digest proteins (cytochrome c, lysozyme and BSA) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The synthesized QDs-DDTC was characterized by using spectroscopic (UV-visible, FT-IR and (1)H NMR) and microscopic (SEM and TEM) techniques. Functionalized QDs-DDTC exhibited a high desorption/ionization efficiency for the rapid quantification of small molecules (efavirenz, tobramycin and aspartame) at low-mass region. QDs-DDTC has well ability to trap target species, and capable to transfer laser energy for efficient desorption/ionization of analytes with background-free detection. The use of QDs-DDTC as a matrix provided good linearity for the quantification of small molecules (R(2)=~0.9983), with good reproducibility (RSD<10%), in the analysis of efavirenz in the plasma of HIV infected patients by the standard addition method. We also demonstrated that the use of functionalized QDs-DDTC as affinity probes for the rapid identification of microwave tryptic digested proteins (cytochrome c, lysozyme and BSA) by MALDI-TOF-MS. QDs-DDTC-based MALDI-TOF-MS approach provides simplicity, rapidity, accuracy, and precision for the determination of efavirenz in human plasma of HIV infected patients and rapid identification of microwave tryptic digested proteins. This new material presents a marked advance in the development of matrix-free mass spectrometric methods for the rapid and precise quantitative determination of a variety of molecules. This article is part of a Special Issue entitled: Proteomics: The clinical link.


Asunto(s)
Marcadores de Afinidad , Benzoxazinas/análisis , Dopamina/análogos & derivados , Proteínas/análisis , Puntos Cuánticos , Tiocarbamatos/química , Marcadores de Afinidad/química , Marcadores de Afinidad/farmacología , Alquinos , Fármacos Anti-VIH/análisis , Fármacos Anti-VIH/sangre , Benzoxazinas/sangre , Ciclopropanos , Dopamina/química , Humanos , Microondas , Modelos Biológicos , Proteínas/metabolismo , Proteínas/efectos de la radiación , Proteolisis/efectos de la radiación , Proteómica/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factores de Tiempo , Tripsina/metabolismo
18.
J Med Chem ; 54(14): 5185-94, 2011 Jul 28.
Artículo en Inglés | MEDLINE | ID: mdl-21662977

RESUMEN

Multimodal imaging-therapeutic nanoprobe TiO(2)@RhdGd was prepared and successfully used for in vitro and in vivo cell tracking as well as for killing of cancer cells in vitro. TiO(2) nanoparticles were used as a core for phosphonic acid modified functionalities, responsible for contrast in MRI and optical imaging. The probe shows high (1)H relaxivity and relaxivity density values. Presence of fluorescent dye allows for visualization by means of fluorescence microscopy. The applicability of the probe was studied, using mesenchymal stem cells, cancer HeLa cells, and T-lymphocytes. The probe did not exhibit toxicity in any of these systems. Labeled cells were successfully visualized in vitro by means of fluorescence microscopy and MRI. Furthermore, it was shown that the probe TiO(2)@RhdGd can be changed into a cancer cell killer upon UV light irradiation. The above stated results represent a valuable proof of a principle showing applicability of the probe design for diagnosis and therapy.


Asunto(s)
Marcadores de Afinidad/síntesis química , Nanopartículas , Organofosfonatos/síntesis química , Titanio/química , Marcadores de Afinidad/química , Marcadores de Afinidad/farmacología , Animales , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Supervivencia Celular/efectos de los fármacos , Complejos de Coordinación/síntesis química , Complejos de Coordinación/química , Complejos de Coordinación/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Colorantes Fluorescentes/farmacología , Gadolinio , Células HeLa , Humanos , Imagen por Resonancia Magnética , Células Madre Mesenquimatosas/metabolismo , Ratones , Microscopía Fluorescente , Organofosfonatos/química , Organofosfonatos/farmacología , Relación Estructura-Actividad , Linfocitos T/metabolismo , Titanio/farmacología , Rayos Ultravioleta
19.
Biochemistry ; 50(18): 3724-35, 2011 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-21452853

RESUMEN

5'-Fluorosulfonylbenzonyl 5'-adenosine (FSBA) is an ATP analogue that covalently modifies several residues in the nucleotide-binding domains (NBDs) of several ATPases, kinases, and other proteins. P-glycoprotein (P-gp, ABCB1) is a member of the ATP-binding cassette (ABC) transporter superfamily that utilizes energy from ATP hydrolysis for the efflux of amphipathic anticancer agents from cancer cells. We investigated the interactions of FSBA with P-gp to study the catalytic cycle of ATP hydrolysis. Incubation of P-gp with FSBA inhibited ATP hydrolysis (IC(50 )= 0.21 mM) and the binding of 8-azido[α-(32)P]ATP (IC(50) = 0.68 mM). In addition, (14)C-FSBA cross-links to P-gp, suggesting that FSBA-mediated inhibition of ATP hydrolysis is irreversible due to covalent modification of P-gp. However, when the NBDs were occupied with a saturating concentration of ATP prior to treatment, FSBA stimulated ATP hydrolysis by P-gp. Furthermore, FSBA inhibited the photo-cross-linking of P-gp with [(125)I]iodoarylazidoprazosin (IAAP; IC(50) = 0.17 mM). As IAAP is a transport substrate for P-gp, this suggests that FSBA affects not only the NBDs but also the transport-substrate site in the transmembrane domains. Consistent with these results, FSBA blocked efflux of rhodamine 123 from P-gp-expressing cells. Additionally, mass spectrometric analysis identified FSBA cross-links to residues within or nearby the NBDs but not in the transmembrane domains, and docking of FSBA in a homology model of human P-gp NBDs supports the biochemical studies. Thus, FSBA is an ATP analogue that interacts with both the drug-binding and ATP-binding sites of P-gp, but fluorosulfonyl-mediated cross-linking is observed only at the NBDs.


Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/química , Adenosina Trifosfato/química , Adenosina/análogos & derivados , Marcadores de Afinidad/farmacología , Subfamilia B de Transportador de Casetes de Unión a ATP , Adenosina/farmacología , Sitios de Unión , Reactivos de Enlaces Cruzados/química , Reactivos de Enlaces Cruzados/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Concentración 50 Inhibidora , Cinética , Espectrometría de Masas/métodos , Nucleótidos/química , Unión Proteica , Transporte de Proteínas
20.
Mol Cell Endocrinol ; 337(1-2): 96-100, 2011 Apr 30.
Artículo en Inglés | MEDLINE | ID: mdl-21315799

RESUMEN

Thyroid hormone (TH) transporter proteins mediate transport of TH across the plasma membrane, thereby facilitating its intracellular bioavailability. As only a few transporters have been identified which are relatively specific for TH, including monocarboxylate transporter (MCT) 8 and MCT10, the need for identification of novel specific TH transporters is obvious. A possible strategy to identify TH transporters is their modification with a ligand-derived affinity-label and subsequent identification by mass spectrometry. Previously, N-bromoacetyl (BrAc)-iodothyronines have been reported as useful affinity-labels for human (h) MCT8. In the present study we reinvestigated possible BrAc[(125)I]T3-labeling of hMCT8 and hMCT10. The present study demonstrates that hMCT8 and hMCT10 both facilitate BrAc[(125)I]T3 transport, but are not labeled by BrAc[(125)I]T3. We provide evidence that human protein disulfide isomerase, which molecular mass is similar to hMCT8, is labeled by BrAc[(125)I]T3. In addition, differential inhibitory effects were observed of iodothyronines derivatives with different side chains on T3 transport by hMCT8 and hMCT10. In conclusion, we demonstrated that not hMCT8 and hMCT10, but human protein disulfide isomerase, is labeled by BrAc[(125)I]T3. The usefulness of BrAc[(125)I]T3 as a tool for the identification of novel TH transporters remains to be explored.


Asunto(s)
Marcadores de Afinidad/farmacología , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas Recombinantes/metabolismo , Triyodotironina/análogos & derivados , Marcadores de Afinidad/farmacocinética , Animales , Células COS , Chlorocebus aethiops , Humanos , Yoduro Peroxidasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Ratas , Simportadores , Triyodotironina/farmacocinética , Triyodotironina/farmacología
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