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J Biomol Screen [journal]
- High-Throughput Screening of Human Leukemia Xenografts to Identify Dexamethasone Sensitizers. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Aug 7.
Acute lymphoblastic leukemia (ALL) is the most common pediatric malignancy. Glucocorticoids (e.g., dexamethasone) form a critical component of chemotherapy regimens for pediatric ALL, and the initial response to glucocorticoid therapy is a major prognostic factor, where resistance is predictive of poor outcome. We have previously established a clinically relevant ALL xenograft model, consisting of primary pediatric ALL biopsies engrafted into immune-deficient mice, in which in vitro and in vivo dexamethasone sensitivity significantly correlated with patient outcome. In this study, we used high-throughput screening (HTS) to identify novel compounds that reverse dexamethasone resistance in a xenograft (ALL-19) derived from a chemoresistant pediatric ALL patient that is representative of the most common pediatric ALL subtype (B-cell precursor [BCP-ALL]). The compound 2-(4-chlorophenoxy)-2-methyl-N-(2-(piperidin-1-yl)phenyl)propanamide showed little cytotoxic activity alone (IC50 = 31 µM), but when combined with dexamethasone, it caused a marked decrease in cell viability. Fixed-ratio combination assays were performed against a broad panel of dexamethasone-resistant and -sensitive xenografts representative of BCP-ALL, T-cell ALL, and Mixed Lineage Leukemia-rearranged ALL, and synergy was observed in six of seven xenografts. We describe here the development of a novel 384-well cell-based high-throughput screening assay for identifying potential dexamethasone sensitizers using a clinically relevant ALL xenograft model.
- Corrigendum. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Aug 5; 19(8):1231.
Ouimet, T;, Duquesnoy, S.; Poras, H.; Fournié-Zaluski, M.-C.; et al. Comparison of Fluorigenic Peptide Substrates PL50, SNAPtide, and BoTest A/E for BoNT/A Detection and Quantification: Exosite Binding Confers High-Assay Sensitivity. J. Biomol. Screen. 2013: , 18(6), 726-735. (Original doi: 10.1177/1087057113476089).
- Microplates, Assay Reagents, Screening Consumables, and Kits. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Aug 5; 19(8):1227-1230.
- Detection of PI3K Inhibition in Human Neuroblastoma Using Multiplex Luminex Bead Immunoassay: A Targeted Approach for Pathway Analysis. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Aug 4.
Neuroblastoma (NB) is a common solid tumor in children. Outcomes for advanced stage NB have not improved, at least in part because of multimodality therapy resistance. Better comprehension of novel molecular targets will likely lead to improved therapies with specific cytotoxic agents. For instance, the role of deregulated IGF-1R/AKT/PI3K/mTOR (PI3K) pathway activity has attracted much attention across several tumors, including NB. Thus, modulating this pathway via anti-PI3K drugs has taken center stage in many cancer clinical trials. However, varied clinical effects have hampered the precise application of these agents. Tumor PI3K pathway profiling may reveal a method to enhance the efficacy of these inhibitors. To this end, solid-phase antibody-based array platforms have emerged as a direct, rapid means of profiling intracellular signaling pathways. We tested the efficacy of four PI3K inhibitors against a panel of human NB cell lines using Luminex xMAP bead array technology to establish PI3K phosphoprotein profiles. We demonstrate the utility of the xMAP approach in following intracellular signaling signatures specific for PI3K targeted therapy. Further validation is required before xMAP is used routinely for clinical PI3K pathway evaluation, but this method may eventually be personalized by taking into account each child's basal NB pathway status.
- Software, Database, and Information Services. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Aug 4.
- Echinomycin, a Potential Binder of FKBP12, Shows Minor Effect on Calcineurin Activity. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Aug 1.
Echinomycin, a member of the quinoxaline family of antibiotics, is known to be a small-molecule inhibitor of hypoxia inducible factor-1 (HIF-1) DNA binding activity. Recently, it has been shown to suppress mammalian target of rapamycin (mTOR) signaling and growth in leukemia cell lines. In this study, we investigated whether echinomycin interacts with the FKBP12 protein. Molecular docking was used, and the predicted binding energy was -10.61 kcal/mol. Moreover, surface plasmon resonance imaging and fluorescence quenching techniques were used to validate this interaction. Echinomycin binds to FKBP12 with a strong binding affinity comparable with rapamycin. Furthermore, the echinomycin-FKBP12 complex has been shown to affect calcineurin activity when tested in a calcineurin phosphatase inhibition assay. All of these studies have shown that echinomycin may have a double impact on HIF signaling by direct inhibition and through mTOR.
- Identification of Selective Agonists and Positive Allosteric Modulators for µ- and δ-Opioid Receptors from a Single High-Throughput Screen. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jul 21.
Hetero-oligomeric complexes of G protein-coupled receptors (GPCRs) may represent novel therapeutic targets exhibiting different pharmacology and tissue- or cell-specific site of action compared with receptor monomers or homo-oligomers. An ideal tool for validating this concept pharmacologically would be a hetero-oligomer selective ligand. We set out to develop and execute a 1536-well high-throughput screen of over 1 million compounds to detect potential hetero-oligomer selective ligands using a β-arrestin recruitment assay in U2OS cells coexpressing recombinant µ- and δ-opioid receptors. Hetero-oligomer selective ligands may bind to orthosteric or allosteric sites, and we might anticipate that the formation of hetero-oligomers may provide novel allosteric binding pockets for ligand binding. Therefore, our goal was to execute the screen in such a way as to identify positive allosteric modulators (PAMs) as well as agonists for µ, δ, and hetero-oligomeric receptors. While no hetero-oligomer selective ligands were identified (based on our selection criteria), this single screen did identify numerous µ- and δ-selective agonists and PAMs as well as nonselective agonists and PAMs. To our knowledge, these are the first µ- and δ-opioid receptor PAMs described in the literature.
- Screening Robotics and Automation. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jul 18; 19(7):1131-1132.
- High-Throughput Screening and Stability Optimization of Anti-Streptavidin IgG1 and IgG2 Formulations. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jul 14.
Selection of a suitable formulation that provides adequate product stability is an important aspect of the development of biopharmaceutical products. Stability of proteins includes not only resistance to chemical modifications but also conformational and colloidal stabilities. While chemical degradation of antibodies is relatively easy to detect and control, propensity for conformational changes and/or aggregation during manufacturing or long-term storage is difficult to predict. In many cases, the formulation factors that increase one type of stability may significantly decrease another type under the same or different conditions. Often compromise is necessary to minimize the adverse effects of an antibody formulation by careful optimization of multiple factors responsible for overall stability. In this study, high-throughput stress and characterization techniques were applied to 96 formulations of anti-streptavidin antibodies (an IgG1 and an IgG2) to choose optimal formulations. Stress and analytical methods applied in this study were 96-well plate based using an automated liquid handling system to prepare the different formulations and sample plates. Aggregation and clipping propensity were evaluated by temperature and mechanical stresses. Multivariate regression analysis of high-throughput data was performed to find statistically significant formulation factors that alter measured parameters such as monomer percentage or unfolding temperature. The results of the regression models were used to maximize the stabilities of antibodies under different formulations and to find the optimal formulation space for each molecule. Comparison of the IgG1 and IgG2 data indicated an overall greater stability of the IgG1 molecule under the conditions studied. The described method can easily be applied to both initial preformulation screening and late-stage formulation development of biopharmaceutical products.
- Usefulness of Monitoring γ-H2AX and Cell Cycle Arrest in HepG2 Cells for Estimating Genotoxicity Using a High-Content Analysis System. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jun 30.
Formation of the phosphorylated protein γ-H2AX is a well-established marker of DNA strand breakage induced by DNA-damaging compounds. Many of these genotoxic compounds also inhibit cell division, leading to arrest at specific points in the cell cycle. Detection of γ-H2AX in combination with cell cycle arrest may therefore be useful for estimating the genotoxicity of experimental compounds. In this study, we examined γ-H2AX formation and cell cycle arrest using high-content screening (HCS) as a method for determining genotoxicity. HepG2 cells were treated with a panel of compounds and then stained with Hoechst 33342 and anti-γ-H2AX, anti-phospho-histone H3, and anti-tubulin antibodies. In total, 19 genotoxic and 7 nongenotoxic compounds were tested in this study. γ-H2AX production was observed within 1 h posttreatment for the majority of Ames-positive compounds, topoisomerase inhibitors, and DNA polymerase inhibitors. Cell cycle arrest in either the S or G2 phase was detected for all DNA-damaging compounds 24 h posttreatment, whereas tubulin-targeting compounds were shown to induce cell cycle arrest in the mitotic phase. Together, these results show that HCS is a simple, rapid, and effective tool for estimating the genotoxicity of compounds through detection of γ-H2AX production and cell cycle arrest.