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| 2. |
- Engen, Karin, et al.
(författare)
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Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening
- 2016
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Ingår i: Assay and drug development technologies. - : Mary Ann Liebert Inc. - 1540-658X .- 1557-8127. ; 14:3, s. 180-193
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Tidskriftsartikel (refereegranskat)abstract
- Intracerebroventricular injection of angiotensin IV, a ligand of insulin-regulated aminopeptidase (IRAP), has been shown to improve cognitive functions in several animal models. Consequently, IRAP is considered a potential target for treatment of cognitive disorders. To identify nonpeptidic IRAP inhibitors, we adapted an established enzymatic assay based on membrane preparations from Chinese hamster ovary cells and a synthetic peptide-like substrate for high-throughput screening purposes. The 384-well microplate-based absorbance assay was used to screen a diverse set of 10,500 compounds for their inhibitory capacity of IRAP. The assay performance was robust with Z-values ranging from 0.81 to 0.91, and the screen resulted in 23 compounds that displayed greater than 60% inhibition at a compound concentration of 10M. After hit confirmation experiments, purity analysis, and promiscuity investigations, three structurally different compounds were considered particularly interesting as starting points for the development of small-molecule-based IRAP inhibitors. After resynthesis, all three compounds confirmed low M activity and were shown to be rapidly reversible. Additional characterization included activity in a fluorescence-based orthogonal assay and in the presence of a nonionic detergent and a reducing agent, respectively. Importantly, the characterized compounds also showed inhibition of the human ortholog, prompting our further interest in these novel IRAP inhibitors.
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| 3. |
- Eriksson, Solveig, et al.
(författare)
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Cytochrome P450 genotyping by multiplexed real-time DNA sequencing with Pyrosequencing TM technology
- 2002
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Ingår i: Assay and drug development technologies. - 1540-658X .- 1557-8127. ; 1:1, s. 49-59
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Tidskriftsartikel (refereegranskat)abstract
- Individual differences in xenobiotic metabolism influence the therapeutic value of many drugs and are of major concern during the development of new drug candidates. A number of polymorphic cytochrome p450 enzymes account for a significant part of this variation. A better understanding of these genetic factors would be of value for drug development, as well as clinical practice. To fulfill the goal of a personalized medicine, methods for simple and accurate assessment of cytochrome p450 genes are required. We report on the development of multiplex assays for genotyping of the cytochrome p450 drug-metabolizing enzymes CYP2D6, CYP2C9, and CYP2C19 with Pyrosequencing technology. Eleven variable positions, representing 12 of the most frequent alleles, were scored: CYP2D6 alleles *2, *3, *4, *6, *7, *8, and *14, CYP2C19 alleles *2, *3, and *4, and CYP2C9 alleles *2 and *3. Four multiplex Pyrosequencing reactions per patient sample were performed to cover these positions, using either simplex or multiplex PCR for amplification of target DNA sequences. Unequivocal genotypes were obtained for all patient samples, and the results were validated by comparing with results obtained using PCR-RFLP. For positions addressed with both methods, the results were in complete agreement. Pyrosequencing technology offers a highly automated, rapid, and accurate method for identification of cytochrome p450 alleles, which is suitable for pharmacogenomic research, as well as for routine assessment of patient genotypes.
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| 5. |
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| 6. |
- Hansson, Pia, et al.
(författare)
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A Comparative Study of Fluorescence Assays in Screening for BRD4
- 2018
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Ingår i: Assay and drug development technologies. - : Mary Ann Liebert Inc. - 1540-658X .- 1557-8127. ; 16:7, s. 372-383
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Tidskriftsartikel (refereegranskat)abstract
- Fluorescence assay technologies are commonly used in high-throughput screening because of their sensitivity and ease of use. Different technologies have their characteristics and the rationale for choosing one over the other can differ between projects because of factors such as availability of reagents, assay performance, and cost. Another important factor to consider is the assay susceptibility to artifacts, which is almost as important as the ability of the assay to pick up active compounds. Spending time and money on false positives or missing the opportunity to build chemistry around false negatives is something that every drug project tries to avoid. We used a BET family Bromodomain, BRD4(1), to explore the outcome of a screening campaign using three fluorescent assay technologies as primary assays. A diverse 7,038 compound set was screened in fluorescence lifetime, fluorescence polarization, and homogeneous time-resolved fluorescence to look at primary hit rates, compound overlap, and hit confirmation rates. The results show a difference between the fluorescence assay technologies with three separate hit lists and some overlap. The confirmed hits from each assay were further evaluated for translation into cells (NanoBRET (TM)). Most of the actives confirmed in cells originated from compounds that overlapped between the assays. In addition, a well-annotated set of compounds with undesirable mechanism of inhibition was screened against BRD4(1) to compare the ability to discriminate true hits from artifact compounds. The results indicate a difference between the assays in their ability to generate false positives and negatives.
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| 7. |
- Jacobson, Lykke, et al.
(författare)
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An optimized automated assay for determination of metabolic stability using hepatocytes: assay validation, variance component analysis, and in vivo relevance.
- 2007
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Ingår i: Assay and drug development technologies. - : Mary Ann Liebert Inc. - 1540-658X .- 1557-8127. ; 5:3, s. 403-15
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Screening of new chemical entities for metabolic stability using hepatocytes is routinely used in drug discovery. To enhance compound throughput, an optimized automated microassay for determination of intrinsic clearance was developed. Dulbecco's modified Eagle's medium, Hanks' balanced salt solution, and Leibovitz L-15 medium (L-15) were tested for their ability to maintain cell viability during incubation in 96-well plates. L-15 was found to keep pH within 0.1 units and maintain high viability during several hours of incubation. Moreover, two different thawing protocols for cryopreserved hepatocytes were compared. Protocol 2 resulted in a nearly 100% increase in post-thaw yield, whereas no difference was observed in cell viability. The microassay was validated using human cryopreserved hepatocytes and 19 reference compounds covering the most important phase I and II liver metabolizing enzymes ranging from low to medium and high clearance compounds. The day-to-day variation was determined, revealing an overall good precision of the assay. In vitro-in vivo correlations, for both fresh rat and cryopreserved human hepatocytes, were calculated. For 86% (human) and 77% (rat) of the compounds, calculated hepatic clearance was within twofold observed clearance in vivo. Using the validation data, variance component analysis was applied to determine within and between-experiment variability, enabling estimation of variation and detection limit for any combination of repeated experiments and replicate samples. Based on the precision desired, this provides a tool to select the most optimal and cost-effective assay approach for different compounds considering the actual phase in the drug discovery program.
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| 8. |
- Lefler, D, et al.
(författare)
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Jak2 and Ca2+/calmodulin are key intermediates for bradykinin B-2 receptor-mediated activation of Na+/H+ exchange in KNRK and CHO cells
- 2003
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Ingår i: Assay and Drug Development Technologies. - : Mary Ann Liebert Inc. - 1540-658X .- 1557-8127. ; 1:2, s. 281-289
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Tidskriftsartikel (refereegranskat)abstract
- Na+/H+ exchangers are ubiquitous in mammalian cells, carrying out key functions, such as cell volume defense, acid-base homeostasis, and regulation of the cytoskeleton. We used two screening technologies (FLIPR and microphysiometry) to characterize the signal transduction pathway used by the bradykinin beta(2) receptor to activate Na+/H+ exchange in two cell lines, KNRK and CHO. In both cell types, beta(2) receptor activation resulted in rapid increases in the rate of proton extrusion that were sodium-dependent and could be blocked by the Na+/H+ exchange inhibitors EIPA and MIA or by replacing extracellular sodium with TMA. Activation of Na+/H+ exchange by bradykinin was concentration-dependent and could be blocked by the selective beta(2) receptor antagonist HOE140, but not by the beta(1) receptor antagonist des-Arg(10)-HOE140. Inhibitors of Jak2 tyrosine kinase (genistein and AG490) and of CAM (W-7 and calmidazolium) attenuated bradykinin-induced activation of Na+/H+ exchange. Bradykinin induced formation of a complex between CAM and Jak2, supporting a regulatory role for Jak2 and CAM in the activation of Na+/H+ exchange in KNRK and CHO cells. We propose that this pathway (beta(2) receptor --> Jak2 --> CAM --> Na+/H+ exchanger) is a fundamental regulator of Na+/H+ exchange activity.
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| 9. |
- Lundin, Arne, et al.
(författare)
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A real-time bioluminescent HTS method for measuring protein kinase activity influenced neither by ATP concentration nor by luciferase inhibition
- 2008
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Ingår i: Assay and drug development technologies. - : Mary Ann Liebert. - 1540-658X .- 1557-8127. ; 6:4, s. 531-541
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Tidskriftsartikel (refereegranskat)abstract
- The firefly luciferin-luciferase reaction has been used to set up an assay for protein kinase based on measuring ATP consumption rate as the first-order rate constant for the kinase reaction. The assay obviates the problems encountered with previous bioluminescent protein kinase assays such as interference with the luciferase reaction from library compounds, nonlinear standard curves, and limited dynamic ranges. In the assay described in the present paper luciferase and luciferin are present during the entire kinase reaction, and the light emission can be measured continuously. In an HTS situation the light emission is measured only twice, i.e., initially and after a predetermined time. After a fivefold reduction of the ATP concentration a Z' value of 0.96 was obtained. Light emission data from samples with kinase are normalized with light emission data from blanks without kinase. First-order rate constants for the kinase reaction calculated from normalized light emission are not affected by a moderate degree of inactivation of luciferase and luciferin during the measuring time. The constants have the same value at all ATP concentrations much lower than the K(m) of the luciferase and the kinase. These factors make the assay very robust and influenced neither by ATP concentration nor by luciferase inhibition. The measuring time depends on the kinase activity and can be varied from minutes to more than 8 h provided the kinase is stable and the evaporation of water from the wells is acceptable. The assay is linear with respect to kinase activity over three orders of magnitude. The new reagents also allowed us to determine K(m) values for ATP and for Kemptide.
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| 10. |
- Napper, A, et al.
(författare)
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Interview with Thomas Lundbäck, PhD
- 2016
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Ingår i: Assay and drug development technologies. - : Mary Ann Liebert Inc. - 1557-8127 .- 1540-658X. ; 14:4, s. 221-225
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)
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