While quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) is a standard tool for many laboratories studying gene regulation, it is not commonly used for small molecule screening. More commonly, high throughput screens (HTSs) designed to detect transcriptional changes use a gene reporter, such as green fluorescent protein (GFP), β-galactosidase, or luciferase. The downsides of this approach include the genetic manipulation required to make reporter lines, the artifacts introduced by this indirect measurement, and the limited number of genes that can be monitored. Here we describe a method for using qRT-PCR to assay the regulation of multiple genes in a 384-well format. We envision this technology being utilized in three main scenarios: screening against cell lines that are not amenable to genetic manipulation (such as lines derived from patients), screening for transcriptional regulators without well-defined functions, or as a secondary screen validating results obtained using a traditional reporter cell line or biochemical readout. Additionally, we provide useful guidelines and protocols for culturing and plating mouse embryonic stem (ES) cells for high throughput screening. While embryonic stem cells are of great interest for regenerative medicine and are a useful tool for studying the epigenetic regulation of cell identity, they are difficult to culture and require extra care and consideration. We provide a method for culturing and plating mouse ES cells in 384-well format.
The SWI/SNF-like adenosine triphosphate (ATP)-dependent chromatin remodeling complex, esBAF, is both necessary and, in some contexts, sufficient to induce the pluripotent state. Furthermore, mutations in various BAF subunits are associated with cancer. Little is known regarding the precise mechanism(s) by which this complex exerts its activities. Thus, it is unclear which protein interactions would be important to disrupt to isolate a relevant readout of mechanism. To address this, we developed a gene expression-based assay to identify inhibitors of the native esBAF complex. Specifically, a quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) assay was developed in mouse embryonic stem (ES) cells to monitor expression of Bmi1, a developmentally important gene repressed by the esBAF complex. The assay was miniaturized to a 384-well format and used to screen a diverse collection of compounds, including novel products of diversity-oriented synthesis (DOS). Confirmed hits were validated using a knock-in ES cell reporter line in which luciferase is inserted into the Bmi1 locus. Several of the validated hits regulate a panel of target genes in a manner similar to the BAF chromatin-remodeling complex. Together these data indicate that expression-based screening using qRT-PCR is a successful approach to identify compounds targeting the regulation of key developmental genes in ES cells.
Abstract In 2002, the National Cancer Institute created the Initiative for Chemical Genetics (ICG), to enable public research using small molecules to accelerate the discovery of cancer-relevant small-molecule probes. The ICG is a public-access research facility consisting of a tightly integrated team of synthetic and analytical chemists, assay developers, high-throughput screening and automation engineers, computational scientists, and software developers. The ICG seeks to facilitate the cross-fertilization of synthetic chemistry and cancer biology by creating a research environment in which new scientific collaborations are possible. To date, the ICG has interacted with 76 biology laboratories from 39 institutions and more than a dozen organic synthetic chemistry laboratories around the country and in Canada. All chemistry and screening data are deposited into the ChemBank web site (http://chembank.broad.harvard.edu/) and are available to the entire research community within a year of generation. ChemBank is both a data repository and a data analysis environment, facilitating the exploration of chemical and biological information across many different assays and small molecules. This report outlines how the ICG functions, how researchers can take advantage of its screening, chemistry and informatic capabilities, and provides a brief summary of some of the many important research findings. (Cancer Res 2006; 66(18): 8935-42)
ChemBank ( http://chembank.broad.harvard.edu/ ) is a public, web-based informatics environment developed through a collaboration between the Chemical Biology Program and Platform at the Broad Institute of Harvard and MIT. This knowledge environment includes freely available data derived from small molecules and small-molecule screens and resources for studying these data. ChemBank is unique among small-molecule databases in its dedication to the storage of raw screening data, its rigorous definition of screening experiments in terms of statistical hypothesis testing, and its metadata-based organization of screening experiments into projects involving collections of related assays. ChemBank stores an increasingly varied set of measurements derived from cells and other biological assay systems treated with small molecules. Analysis tools are available and are continuously being developed that allow the relationships between small molecules, cell measurements, and cell states to be studied. Currently, ChemBank stores information on hundreds of thousands of small molecules and hundreds of biomedically relevant assays that have been performed at the Broad Institute by collaborators from the worldwide research community. The goal of ChemBank is to provide life scientists unfettered access to biomedically relevant data and tools heretofore available primarily in the private sector.
<p>Supplementary Materials and Methods. Supplementary Figure S1: Cell viability in various concentrations of DNA-damaging agents and novel ATR pathway inhibitors. Supplementary Figure S2: Effects of novel and established ATR pathway inhibitors on Chk1 phosphorylation induced by hydroxyurea or cisplatin as assessed by flow cytometry.</p>
Abstract High cancer death rates indicate the need for new anti-cancer therapeutic agents. Approaches to discover new cancer drugs include target-based drug discovery and phenotypic screening. Here, we identify phosphodiesterase 3A modulators as cell-selective cancer cytotoxic compounds by phenotypic compound library screening and target deconvolution by predictive chemogenomics. We found that sensitivity to 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one, or DNMDP, across 766 cancer cell lines correlates with expression of the phosphodiesterase 3A gene, PDE3A. Like DNMDP, a subset of known PDE3A inhibitors kill selected cancer cells while others do not. Furthermore, PDE3A depletion leads to DNMDP resistance. We demonstrate that DNMDP binding to PDE3A promotes an interaction between PDE3A and Schlafen 12 (SLFN12), suggesting a neomorphic activity. Co-expression of SLFN12 with PDE3A correlates with DNMDP sensitivity, while depletion of SLFN12 results in decreased sensitivity to DNMDP. Our results implicate PDE3A modulators as candidate cancer therapeutic agents and demonstrate the power of predictive chemogenomics in small-molecule discovery. Citation Format: Lucian de Waal, Timothy A. Lewis, Matthew G. Rees, Aviad Tsherniak, Xiaoyun Wu, Peter S. Choi, Lara Gechijian, Christina Hartigan, Patrick W. Faloon, Mark J. Hickey, Nicola Tolliday, Steven A. Carr, Paul A. Clemons, Benito Munoz, Bridget K. Wagner, Alykhan F. Shamji, Angela N. Koehler, Monica Schenone, Alex B. Burgin, Stuart L. Schreiber, Heidi Greulich, Matthew Meyerson. Identification of selective cancer cytotoxic modulators of phosphodiesterase 3a by predictive chemogenomics. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C136.
