O. Joseph Trask

High-Throughput Automated Confocal Microscopy Imaging Screen of a Kinase-Focused Library to Identify p38 Mitogen-Activated Protein Kinase Inhibitors Using the GE InCell 3000 Analyzer

The integration of fluorescent microscopy imaging technologies and image analysis into high-content screening (HCS) has been applied throughout the drug discovery pipeline to identify, evaluate, and advance compounds from early lead generation through preclinical candidate selection. In this chapter we describe the development, validation, and implementation of an HCS assay to screen compounds from a kinase-focused small-molecule library to identify inhibitors of the p38 pathway using the GE InCell 3000 automated imaging platform. The assay utilized a genetically modified HeLa cell line stably expressing mitogen-activated, protein-activating protein kinase-2 fused to enhanced green fluorescent protein (MK2-EGFP) and measured the subcellular distribution of the MK2-EGFP as a direct readout of p38 activation. The MK2-EGFP translocation assay performed in 384-well glass bottom microtiter plates exhibited a robust Z-factor of 0.46 and reproducible EC50 and IC50 determinations for activators and inhibitors, respectively. A total of 32,891 compounds were screened in singlicate at 50 microM and 156 were confirmed as inhibitors of p38-mediated MK2-EGFP translocation in follow-up IC50 concentration response curves. Thirty-one compounds exhibited IC50s less than 1 microM, and at least one novel structural class of p38 inhibitor was identified using this HCA/HCS chemical biology screening approach.

Composite Primary Neuronal High-Content Screening Assay for Huntington’s Disease Incorporating Non-Cell-Autonomous Interactions

Huntington’s disease (HD) is a fatal neurodegenerative disease characterized by progressive cognitive, behavioral, and motor deficits and caused by expansion of a polyglutamine repeat in the Huntingtin protein (Htt). Despite its monogenic nature, HD pathogenesis includes obligatory non-cell-autonomous pathways involving both the cortex and the striatum, and therefore effective recapitulation of relevant HD disease pathways in cell lines and primary neuronal monocultures is intrinsically limited. To address this, the authors developed an automated high-content imaging screen in high-density primary cultures of cortical and striatal neurons together with supporting glial cells. Cortical and striatal neurons are transfected separately with different fluorescent protein markers such that image-based high-content analysis can be used to assay these neuronal populations separately but still supporting their intercellular interactions, including abundant synaptic interconnectivity. This assay was reduced to practice using transfection of a mutant N-terminal Htt domain and validated via a screen of ~400 selected small molecules. Both expected as well as novel candidate targets for HD emerged from this screen; of particular interest were target classes with close relative proximity to clinical testing. These findings suggest that composite primary cultures incorporating increased levels of biological complexity can be used for high-content imaging and “high-context” screening to represent molecular targets that otherwise may be operant only in the complex tissue environment found in vivo during disease pathogenesis.

Assessing dose-dependent differences in DNA-damage, p53 response and genotoxicity for quercetin and curcumin

As part of a longer-term goal to create a quantitative mechanistic model of the p53-Mdm2 DNA-damage pathway, we are studying cellular responses to compounds causing DNA-damage by various modes-of action, including two natural polyphenols: quercetin (QUE) and curcumin (CUR). QUE and CUR are weak mutagens in some in vitro assays and possess both anti- or pro-oxidant effects depending on dose. This study examines the dose-response of DNA-damage pathway to these compounds in HT1080 cells (a human cell line with wild-type p53) at doses relevant to human exposure. CUR was more potent in causing reactive oxygen species, DNA damage (measured as phospho-H2AX) and p53 induction, with lowest observed effect levels (LOELs; 3-8 μM) approximately three-fold lower than QUE (20-30 μM). CUR showed a strong G2/M arrest and apoptosis at ≈ 10 μM. QUE caused S phase arrest at low doses (8 μM) and apoptosis was only induced at much higher doses (60 μM). At concentrations with similar levels of p-H2AX and p53 biomarkers, CUR caused greater micronuclei frequency. CUR induced clear increases micronuclei at 3-6 μM, while QUE had a weaker micronuclei response even at the highest doses. Thus, even with two compounds sharing common chemistries, DNA-damage response patterns differed significantly in terms of dose and cell fate.

Screening for Activators of the Wingless Type/Frizzled Pathway by Automated Fluorescent Microscopy

Development of means to screen primary human cells rather than established cell lines is important in improving the predictive value of cellular assays in drug discovery. We describe a method of using automated fluorescent microscopy to detect activators of the wingless type/Frizzled (Wnt/Fzd) pathway in primary human preosteoblasts. This technique relies on detection of endogenous beta-catenin translocation to the nucleus as an indicator of pathway activation, requires only a limited number of primary cells, and is robust enough for automation and high-content, high-throughput screening. Identification of activators of the Wnt/Fzd pathway in human preosteoblasts may be useful in providing lead compounds for the treatment of osteoporosis.

A cellular genetics approach identifies gene-drug interactions and pinpoints drug toxicity pathway nodes

New approaches to toxicity testing have incorporated high-throughput screening across a broad-range of in vitro assays to identify potential key events in response to chemical or drug treatment. To date, these approaches have primarily utilized repurposed drug discovery assays. In this study, we describe an approach that combines in vitro screening with genetic approaches for the experimental identification of genes and pathways involved in chemical or drug toxicity. Primary embryonic fibroblasts isolated from 32 genetically-characterized inbred mouse strains were treated in concentration-response format with 65 compounds, including pharmaceutical drugs, environmental chemicals, and compounds with known modes-of-action. Integrated cellular responses were measured at 24 and 72 h using high-content imaging and included cell loss, membrane permeability, mitochondrial function, and apoptosis. Genetic association analysis of cross-strain differences in the cellular responses resulted in a collection of candidate loci potentially underlying the variable strain response to each chemical. As a demonstration of the approach, one candidate gene involved in rotenone sensitivity, Cybb, was experimentally validated in vitro and in vivo. Pathway analysis on the combined list of candidate loci across all chemicals identified a number of over-connected nodes that may serve as core regulatory points in toxicity pathways.

Standardization of High Content Imaging and Informatics

Undoubtedly, the advancement of HCS technology with improved light sources, cameras and detectors, optics, and new reagents and optical microplates has helped generate better-quality images. This progress has reduced but not eliminated the dependence on flat-field illumination correction and background subtraction. As advancements in microscopy technology has occurred, the scientific community has embraced newer cell-based models such as 3D scaffolds, mixed culture systems, and new fluorescent probes that require an even greater emphasis on image quality to make accurate and quantitative measurements. This is most evident when processing z-stack images captured on HCS platforms. For these reasons, it is absolutely critical that microscope-based HCS platforms are properly calibrated and operated to reduce unnecessary noise, and to optimize axial alignment of x and y horizontal dimensions, and the vertical z position. The scientific community must embrace the urgent need for standardization from cells to HCS data to enhance reproducibility and reduce unnecessary economic loss. Together, the scientists and end users partnering with the manufacturing companies, colleagues, and government agencies can strive to build the necessary toolboxes and protocols to ensure the highest quality of all aspects of HCS data. While standards alone may not entirely solve the problem of irreproducibility, they most certainly will enhance the likelihood that published HCS data can be duplicated, and minimize the experimental variables that require adjustment, thus saving much time and money. In keeping with the mission of SBI2, we and the HCS community will embark on the development and implementation of the standards (Image File Format Standard, Data Integrity Standard, and the Reference Material Standard) discussed above. Moreover, there is a requirement for additional standards and guidelines for HCS best practices to assure both data quality and reproducibility. These include standardization of the nomenclature and terminology used in the “algorithms” used in image analysis processing, and of the parameters generated by these analyses, and even guidelines for data mining and the statistical analysis of HCS data. To get involved or if you would like to learn more about how SBI2 is working to bring standards forward in the community, please contact us at gro.2ibs@ofni.

Abstract 5560: A high-throughput cellular genetics approach to identifying genes associated with sorafenib response and toxicity

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Sorafenib is an oral multikinase inhibitor, approved for hepatocellular, renal and thyroid carcinomas, which decreases tumor angiogenesis and proliferation. The antitumor efficacy and toxicity profiles of sorafenib vary among patients. No predictive biomarkers of sorafenib activity exist to help guide clinicians. Novel pathways and targets of sorafenib activity remain to be identified. We aimed to identify novel genes associated with sorafenib activity by using an in vitro methodology based upon mouse genomics. Methods: We profiled primary mouse embryonic fibroblasts (MEFs) from 32 inbred strains for sorafenib cytotoxicity utilizing high content imaging and simultaneous evaluation of cell health parameters. The 32 strains have been genomically characterized previously (PMID: 21623374). MEF cells were treated with varying concentrations (0-300 µM) of sorafenib, incubated for 24 h or 72 h, and then fixed and stained. Nuclear staining was used to assess sorafenib cytotoxicity and establish our cell viability phenotype. Dose response curves were generated from data, and EC50 values for each strain were identified using a Brain-Cousens model. Genome-wide association mapping, using the SNPster algorithm, was performed on cell viability EC50 values to identify quantitative trait loci (QTLs) associated with sorafenib cytotoxicity. Approximately 277,000 single nucleotide polymorphisms were tested, and genomic loci with p-values < 3.5x10-5 were selected for additional analyses. Results: Interstrain EC50 variability among the 32 MEF strains was observed after 24 h (21-121 µM) and 72 h (17-32 µM) sorafenib incubations. We identified three total peaks associated with cell viability: two on chromosome 13 (23 Mb apart; p = 3.4x10-5 and = 1.6x10-5, respectively), and one on chromosome 4 (p = 2.2x10-5). From these three peaks, we have identified candidate genes that may underlie variability in sorafenib cytotoxicity. A total of 16 genes expressed in MEF cells at mRNA level are present in these QTLs. Of particular interest, we identified one locus that contains Nfyc, a gene that encodes the C subunit of the NF-Y transcription factor. This transcription factor complex is conserved between humans and mice. In humans, NF-Y regulates MYC signaling and DNA-dependent transcription of PDGFR-β (a primary target of sorafenib) (PMID: 12167641). Conclusions: Our innovative high-throughput cellular genetics approach has identified three regions with genetic loci potentially associated with sorafenib cytotoxicity. This approach is capable of identifying robust interstrain cellular differences in sorafenib activity. Functional validation of Nyfc and other promising candidates should be conducted. Citation Format: Daniel J. Crona, Oscar Suzuki, O. Joseph Trask, Amber Frick, Bethany Parks, Tim Wiltshire, Federico Innocenti. A high-throughput cellular genetics approach to identifying genes associated with sorafenib response and toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5560. doi:10.1158/1538-7445.AM2014-5560