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1.
Chembiochem ; 19(6): 613-621, 2018 03 16.
Artículo en Inglés | MEDLINE | ID: mdl-29314498

RESUMEN

We describe the design, synthesis, and structure-activity relationships (SARs) of a series of 2-aminobenzothiazole inhibitors of Rho kinases (ROCKs) 1 and 2, which were optimized to low nanomolar potencies by use of protein kinase A (PKA) as a structure surrogate to guide compound design. A subset of these molecules also showed robust activity in a cell-based myosin phosphatase assay and in a mechanical hyperalgesia in vivo pain model.


Asunto(s)
Benzotiazoles/farmacología , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Quinasas Asociadas a rho/antagonistas & inhibidores , Benzotiazoles/síntesis química , Benzotiazoles/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Humanos , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-Actividad , Quinasas Asociadas a rho/metabolismo
2.
J Neurosci Methods ; 192(2): 249-53, 2010 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-20692294

RESUMEN

Cerebrospinal fluid (CSF) is commonly used for assessing biomarkers of drug efficacy or disease progression in the central nervous system. Studies of CSF from pre-clinical species can characterize biomarkers for use in clinical trials. However, obtaining CSF from pre-clinical species, particularly rodents, can be challenging due to small body sizes, and consequently, low volumes of CSF. Surgical cannulation of rats is commonly used to allow for CSF withdrawal from the cisterna magna. However, cannulae do not remain patent over multiple days, making chronic studies on the same rats difficult. Moreover, CSF biomarkers may be affected by cannulation. Thus cannulation may contribute confounding factors to the understanding of CSF biomarkers. To determine the potential impact on biomarkers, CSF was analyzed from cannulated rats, surgically implanted with catheters as well as from non-cannulated rats. Brain protein biomarkers (αII-spectrin SBDP150 and total tau) and albumin, were measured in the CSF using ELISA assays. Overall, cannulated rat CSF had elevated levels of the biomarkers examined compared to non-cannulated rat CSF. Additionally, the variation in biomarker levels observed among CSF from cannulated rats was greater than that observed for non-cannulated rat CSF. These results demonstrate that in some cases, biomarker assessment using CSF from cannulated rats may differ from that of non-cannulated animals and may contribute confounding factors to biomarker measurements and assay development for clinical use.


Asunto(s)
Biomarcadores/líquido cefalorraquídeo , Catéteres de Permanencia , Cisterna Magna/metabolismo , Albúminas/líquido cefalorraquídeo , Animales , Barrera Hematoencefálica , Cisterna Magna/cirugía , Ensayo de Inmunoadsorción Enzimática , Masculino , Ratas , Ratas Sprague-Dawley , Espectrina/líquido cefalorraquídeo , Proteínas tau/líquido cefalorraquídeo
3.
J Neurosci ; 28(19): 5063-71, 2008 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-18463259

RESUMEN

TRPA1 is an excitatory, nonselective cation channel implicated in somatosensory function, pain, and neurogenic inflammation. Through covalent modification of cysteine and lysine residues, TRPA1 can be activated by electrophilic compounds, including active ingredients of pungent natural products (e.g., allyl isothiocyanate), environmental irritants (e.g., acrolein), and endogenous ligands (4-hydroxynonenal). However, how covalent modification leads to channel opening is not understood. Here, we report that electrophilic, thioaminal-containing compounds [e.g., CMP1 (4-methyl-N-[2,2,2-trichloro-1-(4-nitro-phenylsulfanyl)-ethyl]-benzamide)] covalently modify cysteine residues but produce striking species-specific effects [i.e., activation of rat TRPA1 (rTRPA1) and blockade of human TRPA1 (hTRPA1) activation by reactive and nonreactive agonists]. Through characterizing rTRPA1 and hTRPA1 chimeric channels and point mutations, we identified several residues in the upper portion of the S6 transmembrane domains as critical determinants of the opposite channel gating: Ala-946 and Met-949 of rTRPA1 determine channel activation, whereas equivalent residues of hTRPA1 (Ser-943 and Ile-946) determine channel block. Furthermore, side-chain replacements at these critical residues profoundly affect channel function. Therefore, our findings reveal a molecular basis of species-specific channel gating and provide novel insights into how TRPA1 respond to stimuli.


Asunto(s)
Benzamidas/farmacología , Canales de Calcio/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Proteínas del Tejido Nervioso/metabolismo , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Canales de Potencial de Receptor Transitorio/metabolismo , Animales , Ancirinas , Canales de Calcio/química , Canales de Calcio/genética , Línea Celular , Humanos , Activación del Canal Iónico/fisiología , Mutación , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Estructura Terciaria de Proteína , Ratas , Especificidad de la Especie , Canal Catiónico TRPA1 , Canales Catiónicos TRPC , Canales de Potencial de Receptor Transitorio/química , Canales de Potencial de Receptor Transitorio/genética
4.
J Biomol Screen ; 12(1): 61-9, 2007 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-17099245

RESUMEN

Despite increasing use of cell-based assays in high-throughput screening (HTS) and lead optimization, one challenge is the adequate supply of high-quality cells expressing the target of interest. To this end, cell lines stably expressing targets are often established, maintained, and scaled up by cell culture. These steps require large investments of time and resources. Moreover, significant variability invariably occurs in cell yield, viability, expression levels, and target activities. In particular, stable expression of targets such as transient receptor potential A1 (TRPA1) causes toxicity, cell line degeneration, and loss of functional activity. Therefore, in an effort to identify TRPA1 antagonists, the authors used large-scale transiently transfected (LSTT) cells, enabling rapid establishment of assays suitable for HTS. LSTT cells, which could- be stored frozen for a long period of time (e.g., at least 42 weeks), retained TRPA1 protein expression and could be easily revived to produce robust and consistent signals in calcium influx and electrophysiological assays. Using cells from a single transfection, a chemical library of 700,000 compounds was screened, and TRPA1 antagonists were identified. The use of LSTT circumvented issues associated with stable TRPA1 expression, increased flexibility and consistency, and greatly reduced labor and cost. This approach will also be applicable to other pharmaceutical targets.


Asunto(s)
Moduladores del Transporte de Membrana/análisis , Moduladores del Transporte de Membrana/farmacología , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Transfección , Canales de Potencial de Receptor Transitorio/agonistas , Calcio/metabolismo , Canales de Calcio/metabolismo , Células Clonales , Electrofisiología , Fluorescencia , Congelación , Humanos , Proteínas del Tejido Nervioso/metabolismo , Canal Catiónico TRPA1 , Canales de Potencial de Receptor Transitorio/metabolismo
5.
Biochem Biophys Res Commun ; 326(4): 887-93, 2005 Jan 28.
Artículo en Inglés | MEDLINE | ID: mdl-15607752

RESUMEN

Kcv, the first identified viral potassium channel encoded by the green algae Paramecium bursaria chlorella virus (PBCV-1), conducted K(+) selective currents when expressed in heterologous systems. This K(+) channel was proposed to be important for PBCV-1 infection and replication. In the present study, we identified and functionally characterized a novel K(+) channel Kesv, encoded by Ectocarpus siliculosus virus that infects filamentous marine brown algae. Kesv encodes a protein of 124 amino acids and is 21.8% identical and 37.1% homologous to Kcv. Membrane topology programs predicted that Kesv consists of three transmembrane domains. When expressed in Xenopus oocytes, Kesv induced largely instantaneous, K(+) selective currents that were sensitive to block by Ba(2+) and amantadine. Thus, Kesv along with Kcv, constitutes an emerging family of viral potassium channels, which may play important roles in the life cycle of viruses.


Asunto(s)
Oocitos/fisiología , Phycodnaviridae/genética , Phycodnaviridae/metabolismo , Canales de Potasio/química , Canales de Potasio/fisiología , Potasio/metabolismo , Animales , Células Cultivadas , Regulación Viral de la Expresión Génica/fisiología , Potenciales de la Membrana/fisiología , Phycodnaviridae/clasificación , Canales de Potasio/clasificación , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidad de la Especie , Relación Estructura-Actividad , Proteínas Virales/química , Proteínas Virales/metabolismo , Xenopus laevis
6.
Mol Vis ; 10: 808-13, 2004 Oct 29.
Artículo en Inglés | MEDLINE | ID: mdl-15534583

RESUMEN

PURPOSE: The cyclic nucleotide gated cation channel subunit, CNGA3, (also known as the cone alpha CNG subunit), plays a vital role in signal transduction of human cone cells. Owing to its well established role in vision, the human CNGA3 isoform studied thus far was cloned from retinal tissue. However, non-human homologs of CNGA3 have been cloned from a variety of other tissues including kidney, heart, pineal gland, adrenal gland and testes. Among these, alternative splice forms of CNGA3 have been identified. The objective of this study was to explore alternative splice forms of human CNGA3 and determine their distribution among various human tissues. METHODS: RT-PCR was used to amplify full length open reading frames of CNGA3 from human testes RNA and to detect and distinguish among splice forms in 23 tissues. DNA sequencing was used to characterize full length splice forms and to confirm the identity of RT-PCR products from a number of tissues. RESULTS: Two new full length alternatively spliced forms of hCNGA3 (referred to as Variant 2 and 3) were isolated. These splice variants are different from the cone hCNGA3 (Variant 1) in that they lack exon 5. In addition, they differ from each other in that Variant 3 contains an extra exon that originates from the intron preceding exon 4. We demonstrate that CNGA3 transcripts are detectable in all 23 human tissues examined. In all tissues, except retina, Variant 2 specific PCR products had the brightest band intensity. In retina, the band from Variant 1 (containing exon 5) was most intense. In fact, among all tissues examined, Variant 1 can only be detected strongly in the retina. CONCLUSIONS: The extensive distribution of CNGA3 and the tissue specific expression of alternative splice forms indicate widespread and diverse roles for CNGA3. The unique expression of Variant 1 in the retina implies a significance to the amino acids encoded by exon 5 that may be necessary for the function of CNGA3 in human cone cells.


Asunto(s)
Empalme Alternativo/genética , Proteínas del Ojo/genética , Expresión Génica , Canales Iónicos/genética , Células Fotorreceptoras Retinianas Conos/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Canales Catiónicos Regulados por Nucleótidos Cíclicos , Proteínas del Ojo/metabolismo , Femenino , Humanos , Canales Iónicos/metabolismo , Masculino , Datos de Secuencia Molecular , Sistemas de Lectura Abierta/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ADN , Testículo/metabolismo , Distribución Tisular
7.
Clin Pharmacol Ther ; 75(6): 516-28, 2004 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15179406

RESUMEN

BACKGROUND: The metabolizing enzyme cytochrome P450 (CYP) 3A5 is polymorphically expressed as a result of genetic variants that do not encode functional protein. Because of overlapping substrate specificity with CYP3A4 and the multidrug efflux pump P-glycoprotein, the importance of CYP3A5 genetic polymorphism for pharmacokinetics is controversial. OBJECTIVE: Our objective was to determine whether genetic polymorphisms in CYP3A5 or MDR-1 (which encodes P-glycoprotein) influence the drug levels of ABT-773, a ketolide antibiotic that is a substrate for both CYP3A and P-glycoprotein. METHODS: Healthy volunteers given 3 different oral dose levels of ABT-773 were genotyped at 2 common CYP3A5 and 7 common MDR-1 polymorphisms. Individuals were categorized as CYP3A5-positive if they carried at least 1 functional CYP3A5*1 allele and as CYP3A5-negative if they did not. Area under the plasma concentration-time curves (AUCs) from 0 to 6 hours (AUC(t)) and maximum postdose plasma concentration (C(max)) after a single dose and on day 5 of a twice-daily regimen were calculated and correlated with genotypes. RESULTS: ABT-773 AUC(t) and C(max) were, on average, higher in CYP3A5-negative subjects given 450 mg ABT-773 (n = 9) than in CYP3A5-positive subjects with identical doses (n = 8). The relationship for AUC(t) was statistically significant both after a single dose (geometric mean and 95% confidence interval [CI], 5.0 microg.h/mL [3.9-6.4 microg.h/mL] versus 2.8 microg.h/mL [1.8-4.3 microg.h/mL]; P =.03) and on the fifth day of twice-daily dosing (12.4 microg.h/mL [8.7-17.6 microg.h/mL] versus 7.4 microg.h/mL [5.5-9.8 microg.h/mL], P =.04). The relationship for C(max) was statistically significant after a single dose (1220 microg/mL [867-1167 microg/mL] versus 727 microg/mL [506-1044 microg/mL], P =.04) and showed a trend in the same direction on the fifth day of twice-daily dosing (2566 microg/mL [1813-3631 microg/mL] versus 1621 microg/mL [1122-2343 microg/mL], P =.07). In contrast, AUC(t) and C(max) were not significantly different between CYP3A5-positive and CYP3A5-negative individuals given 150 mg or 300 mg ABT-773. ABT-773 plasma levels did not trend with MDR-1 genotypes. CONCLUSIONS: These results suggest that CYP3A5 genotype may be an important determinant of in vivo drug disposition and that this effect may be dose-dependent.


Asunto(s)
Sistema Enzimático del Citocromo P-450/genética , Eritromicina/sangre , Cetólidos , Adolescente , Adulto , Anciano , Área Bajo la Curva , Intervalos de Confianza , Estudios Cruzados , Citocromo P-450 CYP3A , Relación Dosis-Respuesta a Droga , Método Doble Ciego , Eritromicina/administración & dosificación , Eritromicina/análogos & derivados , Eritromicina/química , Femenino , Genes MDR/genética , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Estadísticas no Paramétricas
8.
J Biomol Screen ; 7(5): 466-75, 2002 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-14599363

RESUMEN

The cloned vanilloid receptor 1 (VR1) is a ligand-gated calcium channel that is believed to be the capsaicin-activated vanilloid receptor found in native tissues, based on similarities regarding molecular mass, tissue distribution, and electrophysiological properties. Using a Fluorescent Imaging Plate Reader (FLIPR), along with Fluo-3 to signal intracellular calcium levels ([Ca(++)](i)), rat VR1 (rVR1) and a human orthologue (hVR1) were pharmacologically characterized with various VR1 ligands. HEK-293 cells, stably expressing rVR1 or hVR1, exhibited dose-dependent increases in [Ca(++)](i) when challenged with capsaicin (EC(50)s congruent with 10 nM). Responses to capsaicin were blocked by the VR1 antagonist capsazepine and were dependent on VR1 expression. Potencies for 10 structurally diverse VR1 agonists revealed rVR1 potencies highly correlated to that of hVR1 (R(2) = 0.973). However, a subset of agonists (tinyatoxin, gingerol, and zingerone) was approximately 10-fold more potent for rVR1 compared to hVR1. Schild analysis for blockade of capsaicin-induced responses by capsazepine was consistent with competitive antagonism, whereas ruthenium red displayed noncompetitive antagonism. Compared to rVR1, hVR1 was more sensitive to blockade by both antagonists. For both rVR1 and hVR1, time-response waveforms elicited by resiniferatoxin increased more gradually compared to other agonists. Tinyatoxin also displayed slow responses with hVR1 but showed rapid responses with rVR1. Thus, FLIPR technology can be used to readily reveal differences between rVR1 and hVR1 pharmacology with respect to potencies, efficacies, and kinetics for several VR1 ligands.


Asunto(s)
Bioensayo/métodos , Calcio/análisis , Capsaicina/análogos & derivados , Receptores de Droga/efectos de los fármacos , Receptores de Droga/metabolismo , Secuencia de Aminoácidos , Animales , Bioensayo/instrumentación , Calcio/metabolismo , Capsaicina/farmacología , Células Cultivadas , Diterpenos/metabolismo , Diterpenos/farmacología , Fluorescencia , Humanos , Cinética , Ligandos , Datos de Secuencia Molecular , Ratas , Receptores de Droga/genética , Rojo de Rutenio/farmacología , Homología de Secuencia de Aminoácido , Especificidad de la Especie
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