Identification of specific inhibitors of human RAD51 recombinase using high-throughput screening.

RAD51 is a key protein of homologous recombination that plays a critical role in the repair of DNA double-strand breaks (DSB) and interstrand cross-links (ICL). To better understand the cellular function(s) of human RAD51, we propose to develop specific RAD51 inhibitors. RAD51 inhibitors may also help to increase the potency of anticancer drugs that act by inducing DSBs or ICLs, e.g., cisplatin or ionizing radiation. In vitro, RAD51 promotes DNA strand exchange between homologous ss- and dsDNA. Here, we developed a DNA strand exchange assay based on fluorescence resonance energy transfer and used this assay to identify RAD51 inhibitors by high-throughput screening of the NIH Small Molecule Repository (>200,000 compounds). Seventeen RAD51 inhibitors were identified and analyzed for selectivity using additional nonfluorescent DNA-based assays. As a result, we identified a compound (B02) that specifically inhibited human RAD51 (IC(50) = 27.4 µM) but not its E. coli homologue RecA (IC(50) > 250 µM). Two other compounds (A03 and A10) were identified that inhibited both RAD51 and RecA but not the structurally unrelated RAD54 protein. The structure-activity relationship (SAR) analysis allowed us to identify the structural components of B02 that are critical for RAD51 inhibition. The described approach can be used for identification of specific inhibitors of other human proteins that play an important role in DNA repair, e.g., RAD54 or Bloom's syndrome helicase.

High-throughput screening of a small molecule library for promoters and inhibitors of mesenchymal stem cell osteogenic differentiation.

The use of high-throughput screening (HTS) techniques has long been employed by the pharmaceutical industry to increase discovery rates for new drugs that could be useful for disease treatment, yet this technology has only been minimally applied in other applications such as in tissue regeneration. In this work, an assay for the osteogenic differentiation of human mesenchymal stem cells (hMSCs) was developed and used to screen a library of small molecules for their potential as either promoters or inhibitors of osteogenesis, based on levels of alkaline phosphatase activity and cellular viability. From a library of 1,040 molecules, 36 promoters, and 20 inhibitors were identified as hits based on statistical criteria. Osteopromoters from this library were further investigated using standard culture techniques and a wider range of outcomes to verify that these compounds drive cellular differentiation. Several hits led to some improvement in the expression of alkaline phosphatase, osteogenic gene expression, and matrix mineralization by hMSCs when compared to the standard dexamethasone supplemented media and one molecule was investigated in combination with a recently identified biodegradable and osteoconductive polymer. This work illustrates the ability of HTS to more rapidly identify potential molecules to control stem cell differentiation.

Parallel High-Throughput Automated Assays to Measure Cell Growth and Beta-Galactosidase Reporter Gene Expression in the Yeast Saccharomyces cerevisiae.

Parallel high-throughput automated assays are described for the measurement of cell growth and ß-galactosidase reporter gene expression from a single culture of the yeast S. cerevisiae. The dual assay measures the effect of test compounds on expression of a specific gene of interest linked to the ß-galactosidase reporter gene, and simultaneously tests for compound toxicity and other effects on cell growth. Examples of assay development and validation results are used to illustrate how this protocol may be used to screen two yeast cell lines in parallel. Yeast cells are grown overnight in V-bottom polypropylene 384-well plates, after which portions of the cell suspension are transferred to clear and to white flat-bottom 384-well plates for measurement of cell growth and reporter gene expression, respectively. Cell growth is determined by measurement of absorbance at 595 nm, and ß-galactosidase expression is quantified by Beta-Glo, a commercially available luminescent ß-galactosidase substrate. Curr. Protoc. Chem. Biol. 3:1-14 © 2011 by John Wiley & Sons, Inc.

Discovery of chemical modulators of a conserved translational control pathway by parallel screening in yeast.

Eukaryotic initiation factor 2 (eIF2) B is a guanine nucleotide exchange factor that plays a central role in translation initiation and its control, especially in response to diverse cellular stresses. In addition, inherited mutations in human eIF2B subunits cause a fatal brain disorder commonly called childhood ataxia with central nervous system hypomyelination or leukoencephalopathy with vanishing white matter. In yeast, inhibiting activity of eIF2B up-regulates expression of the transcriptional activator general control nondepressible (GCN) 4. We report here evaluation of high-throughput screening (HTS) using a yeast-based reporter gene assay, in which strains containing either wild-type or a mutant eIF2B were screened in parallel to identify compounds modifying eIF2B-dependent responses. The goals of the HTS were twofold: first, to discover compounds that restore normal function to mutant eIF2B, which may have therapeutic utility for the fatal human disease; and second, to identify compounds that activate a GCN4 response, which might be useful experimental tools. The HTS assay measured cell growth by absorbance, and activation of gene expression via a beta-galactosidase reporter gene fusion. Because mutant eIF2B activates GCN4 in the absence of stress inducers, the mutant strain was screened for reduction in GCN4 activation. HTS revealed apparent mutant-selective inhibitors but did not reliably predict selectivity as these hits affected both wild-type and mutant strains equally on dose-response confirmation. Wild-type strain results from the HTS identified two GCN4 response activators, both of which were confirmed to be wild-type selective in dose-response testing, suggesting that these compounds may activate GCN4 by a mechanism that down-regulates eIF2B activity.

High-throughput screening for modulators of mesenchymal stem cell chondrogenesis.

Mesenchymal stem cells (MSCs) are an attractive cell source for regenerative medicine and the study of skeletal development. Despite considerable interest in MSC chondrogenesis, the signal transduction and molecular mechanisms underlying this process remain largely undefined. To explore the signaling topology regulating chondrogenic differentiation, as well as to discover novel modulators, we developed and validated a high-throughput screening (HTS) assay for MSC chondrogenesis. Adapting standard assay procedures to enable HTS, we successfully minimized cell number, handling, and culture duration. Using our optimized methodology with automation, we evaluated a comprehensive screen using four growth factors, TGF-beta3, BMP-2, IGF-1, and FGF-2, to demonstrate the feasibility of large combinatorial screens. We examined the chondrogenic effects of these growth factors in different combinations and doses (81 combinations total with 16 replicates per group) and found variable effects on GAG content with different combinations. In general, TGF-beta3 had a pro-chondrogenic effect while FGF-2 had a proliferative effect. BMP-2 was both proliferative and pro-chondrogenic while the effect of IGF-1 in our system was variable. We also carried out an HTS campaign of the National Institute of Neurological Disorders and Stroke (NINDS) chemical library of small molecules (1040 compounds) and identified 5 potential inducers and 24 potential inhibitors of chondrogenesis. Of these compounds, several were identified from the hypnotic, anti-neoplastic, or anti-protein synthesis classes of molecules. These studies demonstrate our ability to carry out high-throughput screening assays for modulators of chondrogenesis.

Evaluation of an orthogonal pooling strategy for rapid high-throughput screening of proteases.

Orthogonal pooling was evaluated as a strategy for the rapid screening of multiple cysteine and serine proteases against large compound libraries. To validate the method the human cysteine protease cathepsin B was screened against a library of 64,000 individual compounds and also against the same library mixed 10 compounds per well. The orthogonal pooling method used resulted in each compound being present in two wells, mixed with a different set of nine other compounds in each location. Thus hits were identified based on activity in both locations, avoiding the need for retesting of each component of active mixtures. Hits were tested in dose-response both in the dithiothreitol (DTT)-containing buffer used in the primary HTS and in buffer containing cysteine in place of DTT to rule out artifacts due to oxidative inactivation of the enzyme. Comparison of the confirmed actives from single-compound and mixture screening showed that mixture screening identified all of the actives from single-compound HTS. Based on these results the orthogonal pooling strategy has been used successfully to rapidly screen several cysteine and serine proteases.

A Saccharomyces cerevisiae cell-based quantitative beta-galactosidase assay compatible with robotic handling and high-throughput screening.

Reporter-gene assays that employ the Escherichia coli lacZ gene are ubiquitously employed in biological research. However, we were not able to readily identify a quantitative method that worked reliably with yeast (Saccharomyces cerevisiae) cells and that was compatible with high-throughput screening and robotic liquid handling tools. We have therefore adapted a commercially available assay employing a 6-O-beta-galactopyranosyl-luciferin substrate to provide the required sensitivity with minimal sample handling times. Our assay uses only one-tenth of the reagents suggested by the reagent manufacturer (Promega) for equivalent assays with mammalian cell cultures and produces rapid, sensitive and reproducible analysis with as little as 1 microl yeast cell culture and with < 100 cells. We demonstrate that the assay is compatible with yeast strains generated by the systematic yeast deletion project and functions equally well with genomically integrated or plasmid-encoded lacZ reporters and with cells grown in complex or defined media. The high-sensitivity, miniaturized format reduced sample handling required will make this assay useful for a wide range of applications.

Identification and characterization of 3-substituted pyrazolyl esters as alternate substrates for cathepsin B: the confounding effects of DTT and cysteine in biological assays.

Substituted pyrazole esters were identified as hits in a high throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR) to identify inhibitors of the enzyme cathepsin B. Members of this class, along with functional group analogs, were synthesized in an effort to define the structural requirements for activity. Analog characterization was hampered by the need to include a reducing agent such as dithiothreitol (DTT) or cysteine in the assay, highlighting the caution required in interpreting biological data gathered in the presence of such nucleophiles. Despite the confounding effects of DTT and cysteine, our studies demonstrate that the pyrazole 1 acts as alternate substrate for cathepsin B, rather than as an inhibitor.

Depolymerization of starch and pectin using superporous matrix supported enzymes.

Immobilized enzyme catalyzed biotransformations involving macromolecular substrates and/or products are greatly retarded due to slow diffusion of large substrate molecules in and out of the typical enzyme supports. Slow diffusion of macromolecules into the matrix pores can be speeded up by use of macroporous supports as enzyme carriers. Depolymerization reactions of polysaccharides like starch, pectin, and dextran to their respective low molecular weight products are some of the reactions that can benefit from use of such superporous matrices. In the present work, an indigenously prepared rigid cross-linked cellulose matrix (called CELBEADS) has been used as support for immobilizing alpha amylase (1,4-alpha-D-glucan glucanohydrolase, EC 3.2.1.1.) and pectinase (endo-PG: poly(1,4-alpha-galactouronide) glycanohydrolase, EC 3.2.1.15). The immobilized enzymes were used for starch and pectin hydrolysis respectively, in batch, packed bed and expanded bed modes. The macroporosity of CELBEADS was found to permit through-flow and easy diffusion of substrates pectin and starch to enzyme sites in the porous supports and gave reaction rates comparable to the rates obtained using soluble enzymes.