| 1. |
- Bergbrede, T., et al.
(author)
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An Adaptable High-Throughput Technology Enabling the Identification of Specific Transcription Modulators
- 2017
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In: Slas Discovery. - : Elsevier BV. - 2472-5552. ; 22:4, s. 378-386
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Journal article (peer-reviewed)abstract
- Mitochondria harbor the oxidative phosphorylation (OXPHOS) system, which under aerobic conditions produces the bulk of cellular adenosine triphosphate (ATP). The mitochondrial genome encodes key components of the OXPHOS system, and it is transcribed by the mitochondrial RNA polymerase, POLRMT. The levels of mitochondrial transcription correlate with the respiratory activity of the cell. Therefore, compounds that can increase or decrease mitochondrial gene transcription may be useful for fine-tuning metabolism and could be used to treat metabolic diseases or certain forms of cancer. We here report the establishment of a novel high-throughput assay technology that has allowed us to screen a library of 430,000 diverse compounds for effects on mitochondrial transcription in vitro. Following secondary screens facilitated by the same assay principle, we identified 55 compounds that efficiently and selectively inhibit mitochondrial transcription and that are active also in cell culture. Our method is easily adaptable to other RNA or DNA polymerases and varying spectroscopic detection technologies.
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| 2. |
- Ekblad, Torun, et al.
(author)
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Identification of Poly(ADP-Ribose) Polymerase Macrodomain Inhibitors Using an AlphaScreen Protocol
- 2018
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In: SLAS Discovery. - : Sage Publications. - 2472-5560 .- 2472-5552. ; 23:4, s. 353-362
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Journal article (peer-reviewed)abstract
- Macrodomains recognize intracellular adenosine diphosphate (ADP)-ribosylation resulting in either removal of the modification or a protein interaction event. Research into compounds that modulate macrodomain functions could make important contributions. We investigated the interactions of all seven individual macrodomains of the human poly(ADP-ribose) polymerase (PARP) family members PARP9, PARP14, and PARP15 with five mono-ADP-ribosylated (automodified) ADP-ribosyltransferase domains using an AlphaScreen assay. Several mono-ADP-ribosylation-dependent interactions were identified, and they were found to be in the micromolar affinity range using surface plasmon resonance (SPR). We then focused on the interaction between PARP14 macrodomain-2 and the mono-ADP-ribosylated PARP10 catalytic domain, and probed a similar to 1500-compound diverse library for inhibitors of this interaction using AlphaScreen. Initial hit compounds were verified by concentration-response experiments using AlphaScreen and SPR, and they were tested against PARP14 macrodomain-2 and -3. Two initial hit compounds and one chemical analog each were further characterized using SPR and microscale thermophoresis. In conclusion, our results reveal novel macrodomain interactions and establish protocols for identification of inhibitors of such interactions.
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| 3. |
- Jagadeesan, Kishore, et al.
(author)
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Filter Plate–Based Screening of MIP SPE Materials for Capture of the Biomarker Pro-Gastrin-Releasing Peptide
- 2017
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In: SLAS Discovery. - : Sage Publications. - 2472-5560 .- 2472-5552. ; 22:10, s. 1253-1261
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Journal article (peer-reviewed)abstract
- Affinity-based solid-phase extraction (SPE) is an attractive low-cost sample preparation strategy for biomarker analysis. Molecularly imprinted polymers (MIPs) as affinity sorbents offer unique opportunities for affinity SPE, due to their low manufacturing cost and high robustness. A limitation is the prediction of their affinity; therefore, screening of analyte recovery and specificity within a large range of SPE conditions is important in order to ensure high-sensitivity detection and assay reproducibility. Here, a µ-SPE method for screening of the MIP-SPE materials using a commercial 384-well filter plate is presented. The method allows for rapid and automated screening using 10?30 µL of packed SPE sorbent per well and sample volumes in the range of 10?70 µL. This enables screening of many different SPE sorbents while simultaneously identifying optimal SPE conditions. In addition, the 384-well format also facilitates detection with a multitude of analytical platforms. Performance of the µ-MIP-SP
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| 4. |
- Kensert, Alexander, et al.
(author)
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Transfer Learning with Deep Convolutional Neural Networks for Classifying Cellular Morphological Changes
- 2019
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In: SLAS Discovery. - : Elsevier BV. - 2472-5560 .- 2472-5552. ; 24:4, s. 466-475
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Journal article (peer-reviewed)abstract
- The quantification and identification of cellular phenotypes from high-content microscopy images has proven to be very useful for understanding biological activity in response to different drug treatments. The traditional approach has been to use classical image analysis to quantify changes in cell morphology, which requires several nontrivial and independent analysis steps. Recently, convolutional neural networks have emerged as a compelling alternative, offering good predictive performance and the possibility to replace traditional workflows with a single network architecture. In this study, we applied the pretrained deep convolutional neural networks ResNet50, InceptionV3, and InceptionResnetV2 to predict cell mechanisms of action in response to chemical perturbations for two cell profiling datasets from the Broad Bioimage Benchmark Collection. These networks were pretrained on ImageNet, enabling much quicker model training. We obtain higher predictive accuracy than previously reported, between 95% and 97%. The ability to quickly and accurately distinguish between different cell morphologies from a scarce amount of labeled data illustrates the combined benefit of transfer learning and deep convolutional neural networks for interrogating cell-based images.
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| 5. |
- Matuszewski, Damian J., et al.
(author)
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Image-Based Detection of Patient-Specific Drug-Induced Cell-Cycle Effects in Glioblastoma
- 2018
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In: SLAS Discovery. - : Elsevier BV. - 2472-5560 .- 2472-5552. ; 23:10, s. 1030-1039
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Journal article (peer-reviewed)abstract
- Image-based analysis is an increasingly important tool to characterize the effect of drugs in large-scale chemical screens. Herein, we present image and data analysis methods to investigate population cell-cycle dynamics in patient-derived brain tumor cells. Images of glioblastoma cells grown in multiwell plates were used to extract per-cell descriptors, including nuclear DNA content. We reduced the DNA content data from per-cell descriptors to per-well frequency distributions, which were used to identify compounds affecting cell-cycle phase distribution. We analyzed cells from 15 patient cases representing multiple subtypes of glioblastoma and searched for clusters of cell-cycle phase distributions characterizing similarities in response to 249 compounds at 11 doses. We show that this approach applied in a blind analysis with unlabeled substances identified drugs that are commonly used for treating solid tumors as well as other compounds that are well known for inducing cell-cycle arrest. Redistribution of nuclear DNA content signals is thus a robust metric of cell-cycle arrest in patient-derived glioblastoma cells.
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| 6. |
- Siva, Kavitha, et al.
(author)
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A Phenotypic Screening Assay Identifies Modulators of Diamond Blackfan Anemia
- 2019
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In: SLAS Discovery. - : Elsevier BV. - 2472-5552. ; 24:3, s. 304-313
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Journal article (peer-reviewed)abstract
- Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome caused by mutations in ribosomal protein genes. Pathogenic mechanisms are poorly understood but involve severely reduced proliferation of erythroid precursors. Because current DBA therapies are ineffective and associated with severe side effects, disease-specific therapies are urgently needed. We hypothesized that druggable molecular pathways underlying the defect can be revealed through phenotypic small-molecule screens. Accordingly, a screening assay was developed using c-kit+ fetal liver erythroid progenitors from a doxycycline-inducible DBA mouse model. The addition of doxycycline to the culture medium induces the phenotype and reduces proliferation to <10% of normal, such that rescue of proliferation can be used as a simple readout for screening. Here, we describe the assay rationale and efforts toward validation of a microtiter plate-compatible assay and its application in a pilot screen of 3871 annotated compounds. Ten hits demonstrated concentration-dependent activity, and we report a brief follow-up of one of these compounds. In conclusion, we established a robust scalable assay for screening molecules that rescue erythropoiesis in DBA.
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| 7. |
- Zetterström, Caroline E., 1982-, et al.
(author)
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Screening for Inhibitors of Acetaldehyde Dehydrogenase (AdhE) from Enterohemorrhagic Escherichia coli (EHEC)
- 2018
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In: SLAS Discovery. - : Sage Publications. - 2472-5560 .- 2472-5552. ; 23:8, s. 815-822
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Journal article (peer-reviewed)abstract
- Acetaldehyde dehydrogenase (AdhE) is a bifunctional acetaldehyde-coenzyme A (CoA) dehydrogenase and alcohol dehydrogenase involved in anaerobic metabolism in gram-negative bacteria. This enzyme was recently found to be a key regulator of the type three secretion (T3S) system in Escherichia coli. AdhE inhibitors can be used as tools to study bacterial virulence and a starting point for discovery of novel antibacterial agents. We developed a robust enzymatic assay, based on the acetaldehyde-CoA dehydrogenase activity of AdhE using both absorption and fluorescence detection models (Z' > 0.7). This assay was used to screen similar to 11,000 small molecules in 384-well format that resulted in three hits that were confirmed by resynthesis and validation. All three compounds are noncompetitive with respect to acetaldehyde and display a clear dose-response effect with hill slopes of 1-2. These new inhibitors will be used as chemical tools to study the interplay between metabolism and virulence and the role of AdhE in T3S regulation in gram-negative bacteria, and as starting points for the development of novel antibacterial agents.
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| 8. |
- Aftab, Obaid, 1984-, et al.
(author)
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Detection of cell aggregation and altered cell viability by automated label-free video microscopy : A promising alternative to endpoint viability assays in high throughput screening
- 2015
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In: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 20:3, s. 372-381
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Journal article (peer-reviewed)abstract
- Automated phase-contrast video microscopy now makes it feasible to monitor a high-throughput (HT) screening experiment in a 384-well microtiter plate format by collecting one time-lapse video per well. Being a very cost-effective and label-free monitoring method, its potential as an alternative to cell viability assays was evaluated. Three simple morphology feature extraction and comparison algorithms were developed and implemented for analysis of differentially time-evolving morphologies (DTEMs) monitored in phase-contrast microscopy videos. The most promising layout, pixel histogram hierarchy comparison (PHHC), was able to detect several compounds that did not induce any significant change in cell viability, but made the cell population appear as spheroidal cell aggregates. According to recent reports, all these compounds seem to be involved in inhibition of platelet-derived growth factor receptor (PDGFR) signaling. Thus, automated quantification of DTEM (AQDTEM) holds strong promise as an alternative or complement to viability assays in HT in vitro screening of chemical compounds.
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| 9. |
- Fitzpatrick, Paul A., et al.
(author)
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Robotic Mammosphere Assay for High-Throughput Screening in Triple-Negative Breast Cancer
- 2017
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In: Slas Discovery. - : Elsevier BV. - 2472-5552. ; 22:7, s. 827-836
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Journal article (peer-reviewed)abstract
- In order to identify novel treatment principles specifically affecting cancer stem cells in triple-negative breast cancer, we have developed a high-throughput screening method based on the mammosphere and anoikis resistance assays allowing us to screen compounds using a functional readout. The assay was validated against manual protocols and through the use of positive controls, such as the response to hypoxia and treatment with the known cancer stem cell-targeting compound salinomycin. Manual and robotic procedures were compared and produced similar results in cell handling, cell cultures, and counting techniques, with no statistically significant difference produced from either method. The variance between samples processed manually versus robotically was no greater than 0.012, while Levene's test of significance was 0.2, indicating no significant difference between mammosphere data produced manually or robotically. Through the screening of 989 FDA-approved drugs and a follow-up screen assessing the antineoplastic subgroup, we have identified three therapeutic compounds with the ability to modulate the breast cancer stem cell fraction in the triple-negative breast cancer cell line MDA-MB-231, highlighting their potential usage as stem cell-specific adjuvant treatments.
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| 10. |
- Goodwin, Richard J. A., et al.
(author)
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Exemplifying the Screening Power of Mass Spectrometry Imaging over Label-Based Technologies for Simultaneous Monitoring of Drug and Metabolite Distributions in Tissue Sections
- 2016
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In: Journal of Biomolecular Screening. - : Elsevier BV. - 1087-0571 .- 1552-454X. ; 21:2, s. 187-193
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Journal article (peer-reviewed)abstract
- Mass spectrometry imaging (MSI) provides pharmaceutical researchers with a suite of technologies to screen and assess compound distributions and relative abundances directly from tissue sections and offer insight into drug discovery-applicable queries such as blood-brain barrier access, tumor penetration/retention, and compound toxicity related to drug retention in specific organs/cell types. Label-free MSI offers advantages over label-based assays, such as quantitative whole-body autoradiography (QWBA), in the ability to simultaneously differentiate and monitor both drug and drug metabolites. Such discrimination is not possible by label-based assays if a drug metabolite still contains the radiolabel. Here, we present data exemplifying the advantages of MSI analysis. Data of the distribution of AZD2820, a therapeutic cyclic peptide, are related to corresponding QWBA data. Distribution of AZD2820 and two metabolites is achieved by MSI, which [C-14] AZD2820 QWBA fails to differentiate. Furthermore, the high mass-resolving power of Fourier transform ion cyclotron resonance MS is used to separate closely associated ions.
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