Development of a high-throughput screening-amenable assay for human poly(ADP-ribose) polymerase inhibitors

INTRODUCTION: Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. Inhibitors of PARP have been shown to reduce infarct size in animal models of myocardial ischemia. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, head trauma, and neurodegenerative disease, and as an adjunct therapy with chemotherapeutic agents/radiation in cancer therapy. METHODS: Assays reported in the literature and commercially available PARP assay kits are labor-intensive, use radioactive reagents, use antibodies, and are not readily amenable to high throughput screening (HTS) [corrected]. Here we report the development and the validation of a nonradioactive PARP assay suitable for HTS. This is a biotinylated NAD-based colorimetric assay in a 96-well plate format. RESULTS: The assay is sensitive, reproducible, and easy to use. The IC(50) values generated for the known PARP inhibitors are in agreement with those generated using the commercial radioactive kit and those reported in the literature. DISCUSSION: The present study demonstrates a sensitive and reproducible methodology capable of screening human PARP inhibitors in high-throughput format.     

High throughput drug screening for human immunodeficiency virus type 1 reactivating compounds

The ability of human immunodeficiency virus type 1 (HIV-1) to persist in a latent stage in memory T cells in the presence of antiretroviral therapy poses a major obstacle to the development of an HIV-1 therapy with curative intent. As latently infected cells are phenotypically not distinguishable from uninfected cells, therapeutic reactivation of the latent infection, followed by the death of the host cell induced by viral cytopathicity, is considered the only means to eliminate this viral reservoir. To identify compounds with the potential to reactivate latent HIV-1, we have developed a series of latently HIV-1-infected reporter cell lines that allow for high throughput drug screening (HTS) in a 384-well plate-based format. The latent reporter cell lines use enhanced green fluorescence protein (eGFP) as a direct and quantitative marker of HIV-1 expression. To aid identification of specific compounds, the cells are engineered to constitutively express a second, red fluorescent protein that has no spectral overlap with eGFP, which allows for the simultaneous quantification of cell viability (inversely correlated to compound toxicity). Thus, these reporters enable prioritization of compounds most likely to have a favorable therapeutic window. The high dynamic signal range and the excellent reproducibility of the primary screening assay result in a Z' -factor of 0.89, which characterizes the HTS system as very robust. The assay has been implemented for automated drug screening, and we here discuss the advantages and limitations of the HTS system based on the data obtained for 1,600 compounds during a limited proof-of-concept drug screen.     

Development and characterization of a stable luciferase dengue virus for high-throughput screening

To facilitate dengue virus (DENV) drug discovery, we developed a stable luciferase reporter DENV-2. A renilla luciferase gene was engineered into the capsid-coding region of an infectious cDNA clone of DENV-2. Transfection of BHK-21 cells with the cDNA clone-derived RNA generated high titers (>10⁶ PFU/ml) of luciferase reporter DENV-2. The reporter virus was infectious to a variety of cells, producing robust luciferase signals. Compared with wild-type virus, the reporter virus replicated slower in both mammalian Vero and mosquito C6/36 cells. To examine the stability of the reporter virus, we continuously passaged the virus on Vero cells for five rounds. All passaged viruses stably maintained the luciferase gene, demonstrating the stability of the reporter virus. Furthermore, we found that the passaged virus accumulated a mutation (T108M) in viral NS4B gene that could enhance viral RNA replication in a cell-type specific manner. Using the reporter virus, we developed a HTS assay in a 384-well format. The HTS assay was validated with known DENV inhibitors and showed a robust Z′ factor of 0.79. The Luc-DENV-2 HTS assay allows screening for inhibitors of all steps of the viral life cycle. The reporter virus will also be a useful tool for studying DENV replication and pathogenesis.     

Using large-scale RNAi screens to identify novel drug targets for cancer

In recent years, the development and clinical implementation of targeted therapeutics have progressed significantly. The specific inhibition of components of signal transduction pathways controlling proliferation and survival has been a highly successful research strategy. However, cancer is a heterogeneous disease and, even within one type of cancer, different genetic alterations are associated with identical phenotypes. To advance the use of targeted therapeutics, it is not only essential to identify the crucial factors in the signal transduction networks that control cell proliferation and survival, but also to classify individual tumors according to genetic alterations that correlate with pathway activation. RNAi screening technologies have become established as an important strategy both to identify novel targets and to provide novel biomarkers that are crucial for the further development of personalized medicine. This feature review discusses different RNAi screening strategies and their contribution to the rapidly evolving field of targeted therapeutics.     

Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1- and Cmya1-ESTs): a preliminary cross-laboratory performance analysis

The Hand1- and Cmya1-ESTs are novel short-term tests for embryotoxic chemicals using genetically engineering mouse ES cells for luciferase reporter gene assays. These ESTs allow convenient determination of differentiation toxicity and cell viability in a short duration with high throughput 96-well microplates for prediction of embryotoxicity of chemicals. To assess the Hand1-EST technical protocol, we firstly compared reporter gene assay and cytotoxicity test data for a representative compound (hydroxyurea) from four different laboratories with tests carried out under the same experimental conditions. Extensive investigations of the Hand1- and Cmya1-ESTs were then performed to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, across the laboratories. The results gave good correspondence in all four laboratories, indicating that transferability, intra-laboratory variability and inter-laboratory variability of the present technical protocols of the ESTs were sufficient to conduct further validation studies.     

Development of a high throughput equilibrium dialysis method

The identification of large numbers of biologically active chemical entities during high throughput screening (HTS) necessitates the incorporation of new strategies to identify compounds with drug-like properties early during the lead prioritization and development processes. One of the major steps in lead prioritization is an assessment of compound binding to plasma proteins, because it affects both the pharmacokinetics and pharmacodynamics of the compound in vivo. Equilibrium dialysis is the preferred method to determine the free drug fraction, because it is less susceptible to experimental artifacts. However, even low-volume standard equilibrium dialysis is currently not amenable to the HTS format. Those considerations dictate the development of a high throughput equilibrium dialysis device, without compromising the analytical quality of the data. The present paper demonstrates successful development of a 96-well format equilibrium dialysis plate. Plasma protein binding of three drugs, propranolol, paroxetine, and losartan, with low, intermediate, and high binding properties, respectively, were chosen for assay validation. The data indicate that the apparent free fraction obtained by this method correlates with the published values determined by the traditional equilibrium dialysis techniques.     

A one-day, dispense-only IP-One HTRF assay for high-throughput screening of Galphaq protein-coupled receptors: towards cells as reagents

Abstract: Compared to biochemical high-throughput screening (HTS) assays, cell-based functional assays are generally thought to be more time consuming and complex because of additional efforts for running continuous cell cultures as well as the numerous assay steps when transferring media and compounds. A common strategy to compensate the anticipated reduction in overall throughput is to implement highly automated cell culture and screening systems. However, such systems require substantial investments in sophisticated hardware and highly specialized personnel. In trying to set up alternatives to increasing throughput in functional cell-based screening, we combined several approaches. By using (1) cryopreserved cell aliquots instead of continuous cell culture, (2) cells in suspension instead of adherent cells, and (3) "ready-to-screen" assay plates with nanoliter aliquots of test compounds, an assay procedure was developed that very much resembles a standard biochemical, enzymatic assay comprising only a few dispense steps. Chinese hamster ovary cells stably overexpressing a Galphaq-coupled receptor were used as a model system to measure receptor activation by detection of intracellular D-myo-inositol 1-phosphate with the help of homogeneous time-resolved fluorescence (HTRF, CISbio International, Bagnols-sur-Cèze, France). Initially established in 384-well adherent cell format, the assay was successfully transferred to 1,536-well format. The assay quality was sufficient to run HTS campaigns in both formats with good Z'-factors and excellent reproducibility of antagonists. Subsequently, the assay procedure was optimized for usage of suspension cells. The influences of cell culture media, plate type, cell number, and incubation time were assessed. Finally, the suspension cell assay was applied to pharmacological characterization of a small molecule antagonist by Schild plot analysis. Our data demonstrate not only the application of the IP-One HTRF assay (CISbio International) for HTS in a high-density format, but furthermore the successful use of cryopreserved and suspension cells in a one-day functional cell-based assay.     

Protein kinases as targets for cancer treatment

In various types of malignancies, conventional forms of therapy (surgery, radiation and chemotherapy) are often ineffective, as well as harmful. In the last few years, a convergence of scientific advances has enabled the identification of molecular targets and signaling pathways specific to cancer cells, resulting in therapies with enhanced selectivity and efficacy and reduced toxicity. Compound validation has relied on target validation first, although some of the most successful drugs often have effects outside of their postulated mechanism. Protein kinases represent such molecular targets; considerable research effort has been devoted to the development of targeted drugs that inhibit the action of pathogenic kinases, and clinical studies performed so far have validated the positive effects of kinase inhibitors for cancer treatment. In this review, the specificity, mechanism of action and antitumor activity of several new small-molecule inhibitors of tyrosine and serine/threonine kinases are discussed.     

Development of a cell-based HTS adaptable calcium fluorescent assay in FlexStation3 for the screening of TRPV3 inhibitors

To develop a cell-based high-throughput calcium fluorescent assay for screening of TRPV3 channel modulators, we employed the FlexStation3 microplate reader and optimized conditions of the high-throughput screening (HTS) adaptable calcium fluorescent assay, such as cell density, incubation time and concentration of Cal-520 calcium fluorescent dye. The optimized FlexStation3 assay included the cell density of 40 000 cells per well, 5.0% of Cal-520 calcium fluorescent dye and 1.5 h of incubation time. Using the FlexStation3 assay in a 96-well format, we screened and identified a natural neferine from Nymphaeaceae plant that inhibited TRPV3 channel. To further confirm the inhibitory effect of neferine on TRPV3, we utilized the whole-cell patch clamp recordings and determined the dose-dependent inhibition of TRPV3 current by neferine with an IC₅₀ at 24.5±4.1 μM (n = 6) without inhibition of TRPV1 or TRPV4 channels. Taken together, our data showed that this validated HTS adaptable calcium fluorescent assay in FlexStation3 format could be used for screening and identification of modulators for either TRPV3 channel or other calcium permeable TRP channels. The identified natural neferine could be used as a tool for pharmacological investigations of TRPV3 channel function.     

Automated high throughput human CYP isoform activity assay using SPE-LC/MS method: application in CYP inhibition evaluation

1. A high throughput screening (HTS) method for the evaluation of the seven major human hepatic CYP isoform activities was developed on a 96-well format, with automation. The method utilized pooled human liver microsomes and seven probe substrates, generic conditions for incubation, reaction termination and metabolite extraction with solid phase extraction (SPE) plates. Metabolites from the seven reactions were pooled and quantified using a generic liquid chromatography and tandem mass spectrometry (LCMS/MS) method. 2. The HTS method was validated based on Km values obtained, which were in agreement with literature data. 3. The isoform inhibition profiles of ketoconazole, quinidine, sulfaphenazole, tranylcypromine, alpha-naphthoflavone, and 4-methylpyrazole against CYPs 3A4, 2D6, 2C9, 2A6 land 2C19), 1A2 and 2E1, respectively, were obtained by this HTS method. Graphically obtained IC50 values are in agreement with literature reported values. 4. The HTS method represents a significant efficiency and selectivity improvement over traditional methods, and can be used for CYP inhibition assay and can be extended for liver activity profiling.     

Automated high throughput human CYP isoform activity assay using SPE-LC/MS method: application in CYP inhibition evaluation

1. A high throughput screening (HTS) method for the evaluation of the seven major human hepatic CYP isoform activities was developed on a 96-well format, with automation. The method utilized pooled human liver microsomes and seven probe substrates, generic conditions for incubation, reaction termination and metabolite extraction with solid phase extraction (SPE) plates. Metabolites from the seven reactions were pooled and quantified using a generic liquid chromatography and tandem mass spectrometry (LCMS/MS) method. 2. The HTS method was validated based on K_m values obtained, which were in agreement with literature data. 3. The isoform inhibition profiles of ketoconazole, quinidine, sulfaphenazole, tranylcypromine, alpha-naphthoflavone, and 4-methylpyrazole against CYPs 3A4, 2D6, 2C9, 2A6 (and 2C19), 1A2 and 2E1, respectively, were obtained by this HTS method. Graphically obtained IC₅₀ values are in agreement with literature reported values. 4. The HTS method represents a significant efficiency and selectivity improvement over traditional methods, and can be used for CYP inhibition assay and can be extended for liver activity profiling.     

RNA interference screening for the discovery of oncology targets

RNA interference (RNAi) mediated loss-of-function screens have the potential to delineate biological functions of genes and the proteins they encode. RNAi has proven to be a promising technology for identification and validation of new targets for the pharmacological treatment of many diseases including cancer. Here we review the use of high-throughput RNAi screens, examine the types of targets pursued for oncology indications, and discuss the integration of diverse datasets in both target discovery and drug discovery programs.     

慢病毒介导shRNA干扰前列腺癌PC3细胞Keap1基因表达的研究

目的：针对Keap1构建shRNA慢病毒干扰载体,并评价慢病毒介导的RNA干扰在人前列腺癌细胞PC3中的基因沉默效应。方法：利用生物信息学方法设计针对Keapl的RNAi寡聚核苷酸序列;采用慢病毒载体构建Keapl的shR-NA载体,利用大肠杆菌进行重组表达,利用293T细胞包装得到重组腺病毒;依据绿色荧光蛋白（GFP）示踪,逐孔稀释法确定转染效率及滴度;以实时荧光定量法比较各靶序列的基因干扰效果。结果：筛选了所构建的4个Keapl靶向序列,以慢病毒载体构建完成对应Keapl的shR-NA质粒。通过瞬时转染筛选得到效率最佳（干扰效率达到80％）的靶序列和工作条件。结论：本研究成功构建并筛选了针对Keapl的shRNA慢病毒载体,有效抑制PC3细胞中Keapl的表达。     

RNAi screening for therapeutic targets in human malignancies

The advent of RNA interference (RNAi) based library screening approaches has sparked a surge in loss-of-function genetic screens. Several recent screens have aimed to identify novel regulators of cancer-related phenotypes. These employ various tumor cell types to model malignant cell functions and use different RNAi effector library approaches to reveal a cache of novel tumor regulators. This review surveys recent RNAi screens conducted in transformed human cells.     

以微管蛋白为靶的高通量药物筛选方法的建立与应用

目的：以微管蛋白为靶建立高通量筛选（HTS）模型，以便有效地发现抗肿瘤化合物。方法：细胞培养与免疫荧光技术。结果：以常规的免疫组化（玻片）方法为基础优化实验条件，在96孔板上建立了以微管蛋白为靶点的高通量药物筛选模型；抗肿瘤药物紫杉醇和秋水仙碱作用于人肝癌HepG2细胞后，细胞的免疫荧光强度发生了明显的可检测的变化，间接反映药物对细胞徽管蛋白聚合／解聚作用的影响，与理论预测结果一致。结论：基于人肿瘤细胞的以徽管蛋白为靶点的高通量筛选方法可用于抗肿瘤化合物的筛选。     

High-throughput cell-based screening for hepatitis C virus NS3/4A protease inhibitors

Hepatitis C virus (HCV) encodes a viral protease, nonstructural (NS)3/4A, that is critical for virus maturation. Although NS3/4A has emerged as a promising target for anti-HCV drug discovery, no anti-HCV therapy has succeeded yet based on inhibition of NS3/4A. We have previously shown that EG(delta4AB)SEAP, a reporter consisting of enhanced green fluorescent protein (EG), the NS3-NS4A protease decapeptide recognition sequence (delta4AB), and secreted alkaline phosphatase (SEAP), is an efficient reporter for reflecting NS3/4A proteolytic activity inside cells. In this study, we describe the generation and characterization of a stable cell line, 293EEG(delta4AB)SEAP-NS3/4A, which constitutively expresses EG(delta4AB)SEAP reporter protein and NS3/4A protease. The reporter assay is validated with the compound BILN 2061, a specific and potent peptidomimetic inhibitor of the HCV NS3 protease. Additionally, we show here that this cell line allows screening for NS3/4A protease activity of living cells in 96-well plate format, with a Z factor >0.6. Thus, this cell-based assay may be used for high-throughput screening of chemical libraries.     

Development of a miniaturized assay for the high-throughput screening program for poly(ADP-ribose) polymerase-1

Poly(ADP-ribose) polymerase (PARP) plays a pivotal role in the repair of DNA strand breaks. However, excessive activation of PARP causes a rapid depletion of intracellular energy, leading to cell death. PARP inhibitors may have potential therapeutic benefit in the treatment of myocardial ischemia, stroke, and neurodegenerative disease. With these emerging medicinal interests, various screening programs have identified small molecules that inhibit PARP with reasonable potencies. However, the increasing numbers of diverse small molecules generated through combinatorial chemistry necessitate the use of robust assays with good sensitivity and specificity for use as a high-throughput screening (HTS) program. Here, we report the development and the validation of a nonisotopic PARP-1 assay suitable for HTS by converting a biotinylated NAD-based colorimetric assay to a miniaturized 384-well plate format. Comparing with the conventional methods, this miniaturized PARP-1 inhibition assay was equally sensitive with excellent reproducibility and cost-effectiveness. Because nonisotopic PARP-1 inhibition assays are widely used, the methodology described in this article can expand the feasibility of this assay as a high-throughput assay for screening of PARP-1 inhibitors from a random chemical library.     

Cryopreserved human hepatocytes in suspension are a convenient high throughput tool for the prediction of metabolic clearance.

Hepatocyte assays, routinely used to assess the metabolic stability of new chemical entities, were recently improved by using hepatocytes in suspension instead of primary cultures [N. Blanchard, L. Richert, B. Notter, F. Delobel, P. David, P. Coassolo, T. Lavé, Impact of serum on clearance predictions obtained from suspensions and primary cultures of rat hepatocytes, Eur. J. Pharm. Sci. 23 (2004) 189-199]. The aim of the present study was to investigate miniaturising the suspension assay by using cryopreserved human hepatocytes, i.e., 150,000 cells/well in 96-well plates, to predict hepatic clearance (CLH) in order to increase compound throughput and decrease cost and tissue requirements. For this, an evaluation was first carried out with rat hepatocytes. Then, human hepatocytes from various donors were used under these predetermined conditions, either immediately after isolation, either after a 20-h-cold storage period in UW or after cryopreservation. The values of CLint and CLH determined using human hepatocytes in suspension in 96-well plates, immediately after isolation, after cold storage or after cryopreservation, were comparable to those obtained with hepatocytes in primary culture. In particular, the use of cryopreserved human hepatocytes in suspension in a 96-well format appeared to be largely satisfactory as a tool for screening and ranking of compounds in the early phase of the drug discovery process.