Journal of Biomolecular Screening RSS feed -- OnlineFirst Articles

Practical Aspects of the SAMPL Challenge: Providing an Extensive Experimental Data Set for the Modeling Community

Newman, J., Fazio, V. J., Caradoc-Davies, T. T., Branson, K., Peat, T. S. — Thu, 12 Nov 2009 08:51:43 PST

To provide an experimental basis for a comprehensive molecular modeling evaluation study, 500 fragments from the Maybridge fragment library were soaked into crystals of bovine pancreatic trypsin and the structures determined by X-ray crystallography. The soaking experiments were performed in both single and pooled aliquots to determine if combination of fragments is an appropriate strategy. A further set of data was obtained from co-crystallizing the pooled fragments with the protein. X-ray diffraction data were collected on approximately 1000 crystals at the Australian Synchrotron, and these data were subsequently processed, and the preliminary analysis was performed with a custom software application (Jigsaw), which combines available software packages for structure solution and analysis. (Journal of Biomolecular Screening XXXX:xx-xx)

Development of a High-Throughput Cell-Based Reporter Assay to Identify Stabilizers of Tumor Suppressor Pdcd4

Mon, 09 Nov 2009 11:59:21 PST

The novel tumor suppressor Pdcd4 affects tumorigenesis by inhibiting translation. Pdcd4 is phosphorylated and subsequently lost by proteasomal degradation in response to tumorpromoting conditions. Here, the authors describe the development of a reporter cell system to monitor the stability of Pdcd4. The phosphorylationdependent degradation domain ("target") or an adjacent ("off-target") region of Pdcd4 was cloned into a luciferase expression system. The target constructs were responsive to Pdcd4 degrading conditions (e.g., TPA, p70^S6K1 overactivation), whereas the off-target constructs remained stable. The system was optimized for and shown to be reliable in a high-throughput compatible 384-well format. Screening of 15,275 pure compounds resulted in a hit rate of 0.30% (>50% inhibition of TPA-induced loss of signal, confirmed by reassay). Among the hits were inhibitors of previously identified critical signaling events for TPA-induced Pdcd4 degradation. One compound was identified to be nonspecific using the off-target control cell line. Screening of 135,678 natural product extracts yielded 42 confirmed, specific hits. Z' averaged 0.58 across 446 plates. Further characterization of active natural products and synthetic compounds is expected to identify novel Pdcd4 stabilizers that may be useful in targeting translation to prevent or treat cancers.

Identification and Preliminary Characterization of Novel Small Molecules That Inhibit Growth of Human Lung Adenocarcinoma Cells

Somwar, R., Shum, D., Djaballah, H., Varmus, H. — Tue, 03 Nov 2009 09:37:34 PST

Drug treatment for human lung cancers remains unsatisfactory, despite the identification of many potential therapeutic targets (such as mutant KRAS protein) and the approval of agents that inhibit the tyrosine kinase activity of mutant epidermal growth factor receptor (EGFR). To seek new therapeutic strategies against lung tumors, the authors have screened 189,290 small molecules for their ability to retard growth of human lung adenocarcinoma cell lines, which harbor mutations in EGFR or KRAS. Four candidates that are structurally different from common tyrosine kinase inhibitors were selected for further study. The authors describe one small molecule (designated lung cancer screen–1 [LCS-1]) in detail here. Identification of the targets of LCS-1 and other growth inhibitors found in this screen may help to develop new agents for the treatment of lung adenocarcinomas, including those driven by mutant EGFR and KRAS. (Journal of Biomolecular Screening XXXX:xx-xx)

Generation of Site-Specific Retargeting Platform Cell Lines for Drug Discovery Using phiC31 and R4 Integrases

Mon, 02 Nov 2009 14:24:06 PST

One of the challenges in developing cell lines for high-throughput screening in drug discovery is the labor- and time-intensive process required to create stable clonal cell lines that express specific reporters or drug targets. The authors report here the generation of a site-specific retargeting platform in 3 different cell lines: adherent HEK293, suspension CHO-S, and a human embryonic cell line (BGO1V). These platform cell lines were generated by using a combination of 2 site-specific integrases to develop a system that allows one to efficiently target a gene of interest to a specific locus and generates rapid production of homogeneous cell pools that stably express the gene of interest. The phiC31 integrase was used to create a platform line by placing a target site for the R4 integrase into a pseudo attP site, and then the R4 integrase was used to place a gene of interest into specific R4 target site. The authors demonstrate the successful and rapid retargeting of a G-protein-coupled receptor (cholecystokinin receptor A, CCKAR), an ion channel (the transient receptor potential cation channel, subfamily M, member 8, TRPM8), and a GFP-c-Jun(1-79) fusion protein into the specific loci in these cell lines and show that these retargeted cell lines exhibit functional and pharmacological responses consistent with those reported in the literature. (Journal of Biomolecular Screening XXXX:xx-xx)

Successful Identification of Glycine Transporter Inhibitors Using an Adaptation of a Functional Cell-Based Assay

Mon, 19 Oct 2009 14:58:55 PDT

Glycine transporter (GlyT1) function is typically measured by radiolabeled glycine uptake using lysis methods or scintillation proximity assays (SPAs), which have limited throughput. This study shows the adaptation of the standard cell lysis method to a screening assay with improved throughput and assay characteristics. The assay takes advantage of the 384well format, standard laboratory automation, and cryopreserved CHOK1 cells stably overexpressing human GlyT1a transporter (CHOK1/hGlyT1a) that were validated and banked in advance of screening. The assay was evaluated for the time course of glycine uptake, K_m, V_max, Z' factor analysis, and IC₅₀ value determination with reference GlyT1 inhibitors. Screening of 118,000 compounds at 10 mM identified 4556 compounds (3.9%) as inhibitors. Positive compounds (>50% inhibition) were retested in the assay at 4 inhibitor concentrations. Compounds demonstrating greater than 40% inhibition at 10 mM were considered as confirmed positives, yielding a 68% confirmation rate from the original screen. To eliminate compounds that nonspecifically inhibited glycine uptake, IC₅₀values were determined in both GlyT1 and GlyT2 assays, and those compounds that inhibited GlyT2 were removed from consideration. The screening campaign identified 300 small molecules as selective GlyT1 inhibitors for lead optimization, demonstrating the utility of this costeffective method. (Journal of Biomolecular Screening XXXX:xx-xx)

Dose-Response Modeling of High-Throughput Screening Data

Parham, F., Austin, C., Southall, N., Huang, R., Tice, R., Portier, C. — Wed, 14 Oct 2009 09:34:04 PDT

The National Toxicology Program is developing a high-throughput screening (HTS) program to set testing priorities for compounds of interest, to identify mechanisms of action, and potentially to develop predictive models for human toxicity. This program will generate extensive data on the activity of large numbers of chemicals in a wide variety of biochemical- and cell-based assays. The first step in relating patterns of response among batteries of HTS assays to in vivo toxicity is to distinguish between positive and negative compounds in individual assays. Here, the authors report on a statistical approach developed to identify compounds positive or negative in an HTS cytotoxicity assay based on data collected from screening 1353 compounds for concentration-response effects in 9 human and 4 rodent cell types. In this approach, the authors develop methods to normalize the data (removing bias due to the location of the compound on the 1536-well plates used in the assay) and to analyze for concentration-response relationships. Various statistical tests for identifying significant concentration-response relationships and for addressing reproducibility are developed and presented. (Journal of Biomolecular Screening XXXX:xx-xx)

High-Throughput Affinity-Based Technologies for Small-Molecule Drug Discovery

Zhu, Z., Cuozzo, J. — Mon, 12 Oct 2009 14:41:55 PDT

High-throughput affinity-based technologies are rapidly growing in use as primary screening methods in drug discovery. In this review, their principles and applications are described and their impact on small-molecule drug discovery is evaluated. In general, these technologies can be divided into 2 groups: those that detect binding interactions by measuring changes to the protein target and those that detect bound compounds. Technologies detecting binding interactions by focusing on the protein have limited throughput but can reveal mechanistic information about the binding interaction; technologies detecting bound compounds have very high throughput, some even significantly higher than current high-throughput screening technologies, but offer limited information about the binding interaction. In addition, the appropriate use of affinity-based technologies is discussed. Finally, nanotechnology is predicted to generate a significant impact on the future of affinity-based technologies. (Journal of Biomolecular Screening XXXX:xx-xx)

Development of High-Throughput TR-FRET and AlphaScreen Assays for Identification of Potent Inhibitors of PDK1

Mon, 12 Oct 2009 14:41:55 PDT

The PI3K/Akt signaling pathway plays a key role in cancer cell growth, survival, and tumor angiogenesis. 3-Phosphoinositide dependent protein kinase 1 (PDK1) is a Ser/Thr protein kinase, which catalyzes the phosphorylation of a conserved residue in the activation loop of a number of AGC kinases, including proto-oncogenes Akt, p70S6K, and RSK kinases. To find new small-molecule inhibitors of this important regulator kinase, the authors have developed PDK1-specific high-throughput enzymatic assays in time-resolved fluorescence resonance energy transfer (TR-FRET) and AlphaScreen^® formats, monitoring phosphorylation of a biotinylated peptide substrate derived from the activation loop of Akt. Development of homogeneous assays enabled screening of a focused kinase library of ~21,500 compounds in 1536-well TR-FRET format in duplicate. Upon validation of hits in an alternative 384-well AlphaScreen^® assay, several classes of structurally diverse PDK1 inhibitors, including tetracyclics, tricyclics, azaindoles, indazoles, and indenylpyrazoles, were identified, thus confirming the utility and sensitivity of the developed assays. Further testing in PC3 prostate cancer cells confirmed that representatives of the tetracyclic series showed intracellular modulation of the PDK1 activity, as evident from decreased phosphorylation levels of AKT, RSK, and S6-ribosomal protein. (Journal of Biomolecular Screening XXXX:xx-xx)

Two G{alpha}i1 Rate-Modifying Mutations Act in Concert to Allow Receptor-Independent, Steady-State Measurements of RGS Protein Activity

Mon, 12 Oct 2009 14:41:54 PDT

RGS proteins are critical modulators of G-protein-coupled receptor (GPCR) signaling given their ability to deactivate G subunits via GTPase-accelerating protein (GAP) activity. Their selectivity for specific GPCRs makes them attractive therapeutic targets. However, measuring GAP activity is complicated by slow guanosine diphosphate (GDP) release from G and lack of solution phase assays for detecting free GDP in the presence of excess guanosine triphosphate (GTP). To overcome these hurdles, the authors developed a G_i1mutant with increased GDP dissociation and decreased GTP hydrolysis rates, enabling detection of GAP activity using steady-state GTP hydrolysis. G_i1(R178M/A326S) GTPase activity was stimulated 6- to 12-fold by RGS proteins known to act on G_isubunits and not affected by those unable to act on G_i, demonstrating that the G/RGS domain interaction selectivity was not altered by mutation. The selectivity and affinity of G_i1(R178M/A326S) interaction with RGS proteins was confirmed by molecular binding studies. To enable nonradioactive, homogeneous detection of RGS protein effects on G_i1(R178M/A326S), the authors developed a Transcreener^®fluorescence polarization immunoassay based on a monoclonal antibody that recognizes GDP with greater than 100-fold selectivity over GTP. Combining (R178M/A326S) with a homogeneous, fluorescence-based GDP detection assay provides a facile means to explore the targeting of RGS proteins as a new approach for selective modulation of GPCR signaling. (Journal of Biomolecular Screening XXXX:xxx-xxx)

Identification of Inhibitors of HSF1 Functional Activity by High-Content Target-Based Screening

Au, Q., Zhang, Y., Barber, J. R., Ng, S. C., Zhang, B. — Mon, 12 Oct 2009 14:41:54 PDT

Cancer cells are known to experience a high level of stress and may require constant repair for survival and proliferation. Recent studies showed that inhibition of heat shock factor 1 (HSF1), the key regulator for the stress-activated transcription of heat shock protein (HSP), can reduce the tumorigenic potential of cancer cells. Such a "nononcogene addiction" phenomenon makes HSF1 an attractive cancer drug target. Here, the authors report an image-based high-content screening (HCS) assay for HSF1 functional inhibitors. A heat shock–based methodology was used to stimulate the stress response followed by quantitative measurement of HSF1/HSP70 granules for compound-induced inhibitory effects. The authors discovered a small molecule from a compound library that inhibits HSF1 granule formation substantially in heat-shocked HeLa cells with IC₅₀ at 80 nM. Electorphoretic mobility shift of HSF1 by this compound suggested significant inhibition of HSF1 phosphorylation, accompanied by reduced expression levels of HSP70 and HSP90 after heat induction. Importantly, HeLa cells stably transfected with HSF1 shRNA were more resistant to the compound treatment under lethal temperature than cells containing HSF1, further validating an HSF1-dependent mechanism of action. The HCS assay the authors developed was robust with a Z' factor of 0.65 in a 384-well plate format, providing a valuable method for identifying small-molecule functional inhibitors of HSF1 for potential cancer treatment. (Journal of Biomolecular Screening XXXX:xxx-xxx)

Analysis of Toxin-Induced Changes in Action Potential Shape for Drug Development

Akanda, N., Molnar, P., Stancescu, M., Hickman, J. J. — Fri, 02 Oct 2009 14:56:56 PDT

The generation of an action potential (AP) is a complex process in excitable cells that involves the temporal opening and closing of several voltage-dependent ion channels within the cell membrane. The shape of an AP can carry information concerning the state of the involved ion channels as well as their relationship to cellular processes. Alteration of these ion channels by the administration of toxins, drugs, and biochemicals can change the AP’s shape in a specific way, which can be characteristic for a given compound. Thus, AP shape analysis could be a valuable tool for toxin classification and the measurement of drug effects based on their mechanism of action. In an effort to begin classifying the effect of toxins on the shape of intracellularly recorded APs, patch-clamp experiments were performed on NG108-15 hybrid cells in the presence of veratridine, tetraethylammonium, and quinine. To analyze the effect, the authors generated a computer model of the AP mechanism to determine to what extent each ion channel was affected during compound administration based on the changes in the model parameters. This work is a first step toward establishing a new assay system for toxin detection and identification by AP shape analysis. (Journal of Biomolecular Screening XXXX:xx-xx)

Identification of Upregulators of BMP2 Expression via High-Throughput Screening of a Synthetic and Natural Compound Library

Tue, 22 Sep 2009 13:59:05 PDT

Bone morphogenetic protein II (BMP2), a member of the transforming growth factor–β (TGF-β) superfamily, is highly expressed in osteoblasts and is a crucial regulator of osteogenic differentiation. Many observations clearly indicate the high potency of BMP2 as an inducer of osteogenesis, and it may be a novel therapeutic target for diseases associated with bone loss, especially in menopausal and postmenopausal women. To discover new agents that enhance the expression of the mouse BMP2, the authors developed a high-throughput assay to screen a synthetic and natural compound library. The cell-based high-throughput screen was conducted in 96-well plates using the clonal murine calvarial MC3T3-E1 cells. These cells were stably transfected with mouse BMP2 promoter-luciferase and calibrated with the known antiosteoporosis compound genistein. Among 3192 compounds screened, 3 agents (daidzein, formononetin, and 2-Acetyldibenzothiophene) were picked up by the high-throughput screening assay, and those compounds were identified as upregulators of BMP2 expression by real-time quantitative reverse transcription–polymerase chain reaction and flow cytometry. Thus, it is demonstrated that this screening model is useful for identifying lead compounds to treat osteoporosis and maintain bone metabolism balance. (Journal of Biomolecular ScreeningXXXX:xx-xx)

Optimizing a Kinase Assay for IKKB on an HTS Station

Doti, N., Marasco, D., Pedone, C., Sabatella, M., Ruvo, M. — Tue, 22 Sep 2009 13:59:05 PDT

Using a commercially available time-resolved fluorescence resonance energy transfer (TR-FRET)–based assay for IKKβ, the authors have automated the assay procedure on a high-throughput screening station to carry out screening campaigns on multiwell plates. They have determined the Z' factor and optimized volumes, times, and time-resolved fluorescence parameters. They have also compared 2 kinases with different fusion tags, the influence of different enzyme/substrate ratios and of DMSO presence at different concentration. The authors found that glutathione S-transferase (GST)–fused IKKβ shows better signal-to-noise (S/N) ratios over the poly-histidine-tagged variant. The substrate can be used at 50 nM with optimal performances when the enzyme is used at 2 nM. DMSO at 0.2% and 1% only slightly affects the S/N ratio, whereas when used at 2%, the final concentration deriving from a 50-fold dilution from a 5-mM stock solution in pure solvent, S/N undergoes a decrease of about 15%. Under the optimized conditions, the assay Z' factor calculated over 192 data points has an optimized value of 0.881 and allows the testing of 94 molecules in quadruplicate in 140 min. (Journal of Biomolecular Screening 2009:000-000)

Book Review: Antisense Drug Technology, Principles, Strategies and Applications, 2nd Ed

Parker, C. — Tue, 12 Feb 2008 13:10:36 PST