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Microbial Cell Factories Feb 2015The hERG potassium channel is essential for repolarization of the cardiac action potential. Due to this vital function, absence of unintended and potentially...
The hERG potassium channel is essential for repolarization of the cardiac action potential. Due to this vital function, absence of unintended and potentially life-threatening interactions with hERG is required for approval of new drugs. The structure of hERG is therefore one of the most sought-after. To provide purified hERG for structural studies and new hERG biomimetic platforms for detection of undesirable interactions, we have developed a hERG expression platform generating unprecedented amounts of purified and functional hERG channels. Full-length hERG, with or without a C-terminally fused green fluorescent protein (GFP) His 8-tag was produced from a codon-optimized hERG cDNA in Saccharomyces cerevisiae. Both constructs complemented the high potassium requirement of a knock-out Saccharomyces cerevisiae strain, indicating correct tetramer assembly in vivo. Functionality was further demonstrated by Astemizole binding to membrane embedded hERG-GFP-His 8 with a stoichiometry corresponding to tetramer assembly. The 156 kDa hERG-GFP protein accumulated to a membrane density of 1.6%. Fluorescence size exclusion chromatography of hERG-GFP-His 8 solubilized in Fos-Choline-12 supplemented with cholesteryl-hemisuccinate and Astemizole resulted in a monodisperse elution profile demonstrating a high quality of the hERG channels. hERG-GFP-His 8 purified by Ni-affinity chromatography maintained the ability to bind Astemizole with the correct stoichiometry indicating that the native, tetrameric structure was preserved. To our knowledge this is the first reported high-yield production and purification of full length, tetrameric and functional hERG. This significant breakthrough will be paramount in obtaining hERG crystal structures, and in establishment of new high-throughput hERG drug safety screening assays.
Topics: Astemizole; Biomass; Cell Membrane; Chromatography, Affinity; DNA, Complementary; ERG1 Potassium Channel; Ether-A-Go-Go Potassium Channels; Green Fluorescent Proteins; Histamine H1 Antagonists, Non-Sedating; Humans; Microscopy, Fluorescence; Protein Binding; Protein Multimerization; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Temperature; Time Factors
PubMed: 25656388
DOI: 10.1186/s12934-015-0193-9 -
Journal of Biomolecular Screening Mar 2015The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems...
The emergence of human stem cell-derived cardiomyocyte (hSCCM)-based assays in the cardiovascular (CV) drug discovery sphere requires the development of improved systems for interrogating the rich information that these cell models have the potential to yield. We developed a new analytical framework termed SALVO (synchronization, amplitude, length, and variability of oscillation) to profile the amplitude and temporal patterning of intra- and intercellular calcium signals in hSCCM. SALVO quantified drug-induced perturbations in the calcium signaling "fingerprint" in spontaneously contractile hSCCM. Multiparametric SALVO outputs were integrated into a single index of in vitro cytotoxicity that confirmed the rank order of perturbation as astemizole > thioridazine > cisapride > flecainide > valdecoxib > sotalol > nadolol ≈ control. This rank order of drug-induced Ca(2+) signal disruption is in close agreement with the known arrhythmogenic liabilities of these compounds in humans. Validation of the system using a second set of compounds and hierarchical cluster analysis demonstrated the utility of SALVO to discriminate drugs based on their mechanisms of action. We discuss the utility of this new mechanistically agnostic system for the evaluation of in vitro drug cytotoxicity in hSCCM syncytia and the potential placement of SALVO in the early stage drug screening framework.
Topics: Anti-Arrhythmia Agents; Calcium Signaling; Cell Line; Cells, Cultured; Cluster Analysis; Drug Discovery; Drug Evaluation, Preclinical; Embryonic Stem Cells; Humans; Membrane Potentials; Myocytes, Cardiac; Troponin T
PubMed: 25367900
DOI: 10.1177/1087057114557232 -
BMC Cancer Oct 2014The oncogenic ether-à-go-go-1 potassium channel (EAG1) activity and expression are necessary for cell cycle progression and tumorigenesis. The active vitamin D...
BACKGROUND
The oncogenic ether-à-go-go-1 potassium channel (EAG1) activity and expression are necessary for cell cycle progression and tumorigenesis. The active vitamin D metabolite, calcitriol, and astemizole, a promising antineoplastic drug, target EAG1 by inhibiting its expression and blocking ion currents, respectively. We have previously shown a synergistic antiproliferative effect of calcitriol and astemizole in breast cancer cells in vitro, but the effect of this dual therapy in vivo has not been studied.
METHODS
In the present study, we explored the combined antineoplastic effect of both drugs in vivo using mice xenografted with the human breast cancer cell line T-47D and a primary breast cancer-derived cell culture (MBCDF). Tumor-bearing athymic female mice were treated with oral astemizole (50 mg/kg/day) and/or intraperitoneal injections of calcitriol (0.03 μg/g body weight twice a week) during 3 weeks. Tumor sizes were measured thrice weekly. For mechanistic insights, we studied EAG1 expression by qPCR and Western blot. The expression of Ki-67 and the relative tumor volume were used as indicators of therapeutic efficacy.
RESULTS
Compared to untreated controls, astemizole and calcitriol significantly reduced, while the coadministration of both drugs further suppressed, tumor growth (P < 0.05). In addition, the combined therapy significantly downregulated tumoral EAG1 and Ki-67 expression.
CONCLUSIONS
The concomitant administration of calcitriol and astemizole inhibited tumor growth more efficiently than each drug alone, which may be explained by the blocking of EAG1. These results provide the bases for further studies aimed at testing EAG1-dual targeting in breast cancer tumors expressing both EAG1 and the vitamin D receptor.
Topics: Animals; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Astemizole; Breast Neoplasms; Calcitriol; Cell Line, Tumor; Drug Synergism; Ether-A-Go-Go Potassium Channels; Female; Humans; Mice; Mice, Nude; Neoplasm Transplantation
PubMed: 25280486
DOI: 10.1186/1471-2407-14-745 -
Xenobiotica; the Fate of Foreign... Feb 20151. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys,...
1. An increasing number of studies have indicated the roles of CYP4 proteins in drug metabolism; however, CYP4 expression has not been measured in cynomolgus monkeys, an important animal species for drug metabolism studies. 2. In this study, cynomolgus CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, along with CYP2J2, were immunoquantified using selective antibodies in 28 livers and 35 small intestines, and their content was compared with CYP1A, CYP2A, CYP2B6, CYP2C9/19, CYP2D, CYP2E1, CYP3A4 and CYP3A5, previously quantified. 3. In livers, CYP2J2, CYP4A11, CYP4F2/3, CYP4F11 and CYP4F12, varied 1.3- to 4.3-fold, represented 11.2, 14.4, 8.0, 2.7 and 0.3% of total immunoquantified CYP1-4 proteins, respectively. 4. In small intestines, CYP2J2, CYP4F2/3, CYP4F11 and CYP4F12, varied 2.4- to 9.7-fold, represented 6.9, 36.4, 2.4 and 9.3% of total immunoquantified CYP1-4 proteins, respectively, making CYP4F the most abundant P450 subfamily in small intestines. CYP4A11 was under the detection limit in all of the samples analyzed. 5. Significant correlations were found in liver for CYP4A11 with lauric acid 11-/12-hydroxylation and for CYP4F2/3 and CYP4F11 with astemizole hydroxylation. 6. This study revealed the relatively abundant contents of cynomolgus CYP2J2, CYP4A11 and CYP4Fs in liver and/or small intestine, suggesting their potential roles for the metabolism of xenobitotics and endogenous substrates.
Topics: Animals; Cytochrome P-450 Enzyme System; Female; Intestine, Small; Liver; Macaca fascicularis; Male; Microsomes; Microsomes, Liver
PubMed: 25138712
DOI: 10.3109/00498254.2014.952800