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J Biomol Screen [journal]
- 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.
- Characterization of Human Hippocampal Neural Stem/Progenitor Cells and Their Application to Physiologically Relevant Assays for Multiple Ionotropic Glutamate Receptors. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jun 30.
The hippocampus is an important brain region that is involved in neurological disorders such as Alzheimer disease, schizophrenia, and epilepsy. Ionotropic glutamate receptors-namely, N-methyl-D-aspartate (NMDA) receptors (NMDARs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors (AMPARs), and kainic acid (KA) receptors (KARs)-are well known to be involved in these diseases by mediating long-term potentiation, excitotoxicity, or both. To predict the therapeutic efficacy and neuronal toxicity of drug candidates acting on these receptors, physiologically relevant systems for assaying brain region-specific human neural cells are necessary. Here, we characterized the functional differentiation of human fetal hippocampus-derived neural stem/progenitor cells-namely, HIP-009 cells. Calcium rise assay demonstrated that, after a 4-week differentiation, the cells responded to NMDA (EC50 = 7.5 ± 0.4 µM; n = 4), AMPA (EC50 = 2.5 ± 0.1 µM; n = 3), or KA (EC50 = 33.5 ± 1.1 µM; n = 3) in a concentration-dependent manner. An AMPA-evoked calcium rise was observed in the absence of the desensitization inhibitor cyclothiazide. In addition, the calcium rise induced by these agonists was inhibited by antagonists for each receptor-namely, MK-801 for NMDA stimulation (IC50 = 0.6 ± 0.1 µM; n = 4) and NBQX for AMPA and KA stimulation (IC50 = 0.7 ± 0.1 and 0.7 ± 0.03 µM, respectively; n = 3). The gene expression profile of differentiated HIP-009 cells was distinct from that of undifferentiated cells and closely resembled that of the human adult hippocampus. Our results show that HIP-009 cells are a unique tool for obtaining human hippocampal neural cells and are applicable to systems for assay of ionotropic glutamate receptors as a physiologically relevant in vitro model.
- Identification and Characterization of Small-Molecule Inhibitors of the R132H/R132H Mutant Isocitrate Dehydrogenase 1 Homodimer and R132H/Wild-Type Heterodimer. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jun 30.
Recurrent genetic mutations in isocitrate dehydrogenase 1 and 2 (IDH1 and IDH2) have been identified in multiple tumor types. The most frequent mutation, IDH1 R132H, is a gain-of-function mutation resulting in an enzyme-catalyzing conversion of α-ketoglutarate (α-KG) to 2-hydroxyglutarate (2-HG). A high-throughput assay quantifying consumption of NADPH by IDH1 R132H has been optimized and implemented to screen 3 million compounds in 1536-well formats. The primary high-throughput screening hits were further characterized by RapidFire-mass spectrometry measuring 2-HG directly. Multiple distinct chemotypes were identified with nanomolar potencies (6-300 nM). All inhibitors were found to be inactive against the wild-type IDH1 homodimers. An IDH1 heterodimer between wild-type and R132H mutant is capable of catalyzing conversion of α-KG to 2-HG and isocitrate to α-KG. Interestingly, one of the inhibitors, EXEL-9324, was found to inhibit both conversions by the IDH1 heterodimer. This indicates the R132H/WT heterodimer may adopt conformations distinct from that of the R132H/R132H homodimer. Further enzymatic studies support this conclusion as the heterodimer exhibited a significantly lower apparent Michaelis-Menten constant for α-KG (Km =110 µM) compared with the R132H homodimer (Km = 1200 µM). The enhanced apparent affinity for α-KG suggests R132H/WT heterodimeric IDH1 can produce 2-HG more efficiently at normal intracellular levels of α-KG (approximately 100 µM).
- Compound Precipitation in High-Concentration DMSO Solutions. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jun 30.
Screening compounds precipitate out of high-concentration DMSO solutions and cause issues in the liquid-handling equipment used to process the samples as well as false-positives and false-negatives in the biological data. This report examines the extent of the precipitation issue, correlation with physico-chemical properties of compounds, and measured concentrations. Possible ways to allow recovery of the precipitated solids are also described. This study also investigated whether particulates were present from the time of initial solubilization in DMSO or whether precipitation occurs during freeze-thaw cycling.
- Corrigendum. [JOURNAL ARTICLE]
- J Biomol Screen 2014 Jun 24; 19(6):982-986.
Harris, D.; Olechno, J.; Datwani, S.; Ellson, R. Gradient, Contact-Free Volume Transfers Minimize Compound Loss in Dose-Response Experiments. J. Biomol. Screen. 2010: , 15, 86-94. (Original doi: 10.1177/1087057113500121).