A multiplexed siRNA screening strategy to identify genes in the PARP pathway.

Gene silencing by RNA interference has become a powerful tool to help identify genes that regulate biological processes. However, the complexity of the biology probed and the incomplete validation of the reagents used make it difficult to interpret the results of genome-wide siRNA screens. To address this challenge and maximize the return on the efforts required for validating genomic screen hits, the screening strategy must be designed to increase the robustness of the primary screening hits and include assays that inform on the mechanism of action of the knocked-down transcripts. Here, we describe the implementation of a small interfering RNA (siRNA) screen to identify genes that sensitize the effect of poly-(ADP ribose)-polymerase (PARP) inhibitor on cell survival. In the strategy we designed for the primary screen, two biological activities, apoptosis and cell viability, were measured simultaneously at different time points in the presence and absence of a PARP inhibitor (PARPi). The multiplexed assay allowed us to identify PARPi sensitizers induced by both caspase-dependent and independent mechanisms. The multiplexed screening strategy yielded robust primary hits with significant enrichment for DNA repair genes, which were further validated using relevant high-content imaging assays and confirmation of transcript knockdown by real-time PCR (rtPCR).

Induced pluripotent stem cells--opportunities for disease modelling and drug discovery.

The ability to generate induced pluripotent stem cells (iPSCs) from patients, and an increasingly refined capacity to differentiate these iPSCs into disease-relevant cell types, promises a new paradigm in drug development - one that positions human disease pathophysiology at the core of preclinical drug discovery. Disease models derived from iPSCs that manifest cellular disease phenotypes have been established for several monogenic diseases, but iPSCs can likewise be used for phenotype-based drug screens in complex diseases for which the underlying genetic mechanism is unknown. Here, we highlight recent advances as well as limitations in the use of iPSC technology for modelling a 'disease in a dish' and for testing compounds against human disease phenotypes in vitro. We discuss how iPSCs are being exploited to illuminate disease pathophysiology, identify novel drug targets and enhance the probability of clinical success of new drugs.

A 1,536-well ultra-high-throughput siRNA screen to identify regulators of the Wnt/beta-catenin pathway.

High-throughput siRNA screens are now widely used for identifying novel drug targets and mapping disease pathways. Despite their popularity, there remain challenges related to data variability, primarily due to measurement errors, biological variance, uneven transfection efficiency, the efficacy of siRNA sequences, or off-target effects, and consequent high false discovery rates. Data variability can be reduced if siRNA screens are performed in replicate. Running a large-scale siRNA screen in replicate is difficult, however, because of the technical challenges related to automating complicated steps of siRNA transfection, often with multiplexed assay readouts, and controlling environmental humidity during long incubation periods. Small-molecule screens have greatly benefited in the past decade from assay miniaturization to high-density plates such that 1,536-well nanoplate screenings are now a routine process, allowing fast, efficient, and affordable operations without compromising underlying biology or important assay characteristics. Here, we describe the development of a 1,536-well nanoplate siRNA transfection protocol that utilizes the instruments commonly found in small-molecule high throughput screening laboratories. This protocol was then successfully demonstrated in a triplicate large-scale siRNA screen for the identification of regulators of the Wnt/beta-catenin pathway.

Identification of small-molecule modulators of mouse SVZ progenitor cell proliferation and differentiation through high-throughput screening.

Adult mouse subventricular zone (SVZ) neural stem/progenitor cells are multipotent self-renewing cells that retain the capacity to generate the major cell types of the central nervous system in vitro and in vivo. The relative ease of expanding SVZ cells in culture as neurospheres makes them an ideal model for carrying out large-scale screening to identify compounds that regulate neural progenitor cell proliferation and differentiation. The authors have developed an adenosine triphosphate-based cell proliferation assay using adult SVZ cells to identify small molecules that activate or inhibit progenitor cell proliferation. This assay was miniaturized to a 1536-well format for high-throughput screening (HTS) of >1 million small-molecule compounds, and 325 and 581 compounds were confirmed as potential inducers of SVZ cell proliferation and differentiation, respectively. A number of these compounds were identified as having a selective proliferative and differentiation effect on SVZ cells versus mouse Neuro2a neuroblastoma cells. These compounds can potentially be useful pharmacological tools to modulate resident stem cells and neurogenesis in the adult brain. This study represents a novel application of primary somatic stem cells in the HTS of a large-scale compound library.

Genome-scale RNAi screen for host factors required for HIV replication.

Human immunodeficiency virus (HIV)-1 depends on the host cell machinery to support its replication. To discover cellular factors associated with HIV-1 replication, we conducted a genome-scale siRNA screen, revealing more than 311 host factors, including 267 that were not previously linked to HIV. Surprisingly, there was little overlap between these genes and the HIV dependency factors described recently. However, an analysis of the genes identified in both screens revealed overlaps in several of the associated pathways or protein complexes, including the SP1/mediator complex and the NF-kappaB signaling pathway. cDNAs for a subset of the identified genes were used to rescue HIV replication following knockdown of the cellular mRNA providing strong evidence that the following six genes are previously uncharacterized host factors for HIV: AKT1, PRKAA1, CD97, NEIL3, BMP2K, and SERPINB6. This study highlights both the power and shortcomings of large scale loss-of-function screens in discovering host-pathogen interactions.

Hit selection with false discovery rate control in genome-scale RNAi screens.

RNA interference (RNAi) is a modality in which small double-stranded RNA molecules (siRNAs) designed to lead to the degradation of specific mRNAs are introduced into cells or organisms. siRNA libraries have been developed in which siRNAs targeting virtually every gene in the human genome are designed, synthesized and are presented for introduction into cells by transfection in a microtiter plate array. These siRNAs can then be transfected into cells using high-throughput screening (HTS) methodologies. The goal of RNAi HTS is to identify a set of siRNAs that inhibit or activate defined cellular phenotypes. The commonly used analysis methods including median +/- kMAD have issues about error rates in multiple hypothesis testing and plate-wise versus experiment-wise analysis. We propose a methodology based on a Bayesian framework to address these issues. Our approach allows for sharing of information across plates in a plate-wise analysis, which obviates the need for choosing either a plate-wise or experimental-wise analysis. The proposed approach incorporates information from reliable controls to achieve a higher power and a balance between the contribution from the samples and control wells. Our approach provides false discovery rate (FDR) control to address multiple testing issues and it is robust to outliers.

A 1,536-well [(35)S]GTPgammaS scintillation proximity binding assay for ultra-high-throughput screening of an orphan galphai-coupled GPCR.

Members of the superfamily of seven transmembrane receptors, known as G protein-coupled receptors (GPCRs), are important targets for many therapeutic areas in drug discovery. A homogeneous guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPgammaS) scintillation proximity assay (SPA) binding assay targeting a Galphai-coupled GPCR recombinantly expressed in membranes of Chinese hamster ovary (CHO) cells was developed and miniaturized into 1,536-well plate format. The primary ultra-high-throughput screen of the entire compound collection was accomplished on the Kalypsys (San Diego, CA) robotic platform at a concentration of 8 muM using the 1,536-well [(35)S]GTPgammaS SPA binding functional assay. The signal-to-noise ratio of the primary screen was approximately 2.1-fold, and the plate coefficient of variation for the compound field was approximately 11%. The hit rate from the primary screen for receptor agonists at >35% activity was approximately 0.3%. Primary hits were cherry-picked, confirmed in triplicate, counterscreened against untransfected CHO cell membranes, and further analyzed in a cyclic AMP functional assay, resulting in 34 leads for optimization.

A fluorescence-based thiol quantification assay for ultra-high-throughput screening for inhibitors of coenzyme A production.

Here we report the development and miniaturization of a cell-free enzyme assay for ultra-high-throughput screening (uHTS) for inhibitors of two potential drug targets for obesity and cancer: fatty acid synthase (FAS) and acetyl-coenzyme A (CoA) carboxylase (ACC) 2. This assay detects CoA, a product of the FAS-catalyzed condensation of malonyl-CoA and acetyl-CoA. The free thiol of CoA can react with 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin (CPM), a profluorescent coumarin maleimide derivative that becomes fluorescent upon reaction with thiols. FAS produces long-chain fatty acid and CoA from the condensation of malonyl-CoA and acetyl-CoA. In our FAS assay, CoA released in the FAS reaction forms a fluorescence adduct with CPM that emits at 530 nm when excited at 405 nm. Using this detection method for CoA, we measured the activity of sequential enzymes in the fatty acid synthesis pathway to develop an ACC2/FAS-coupled assay where ACC2 produces malonyl-CoA from acetyl-CoA. We miniaturized the FAS and ACC2/FAS assays to 3,456- and 1,536-well plate format, respectively, and completed uHTSs for small molecule inhibitors of this enzyme system. This report shows the results of assay development, miniaturization, and inhibitor screening for these potential drug targets.

Miniaturization and HTS of a FRET-based membrane potential assay for K(ir) channel inhibitors.

The K(ir) family of potassium-selective ion channels is characterized by their inward (anomalous) rectifying current-voltage relationship. K(ir) channels are widely expressed in mammalian cells and through their role in regulation of the cell membrane potential have been implicated in diverse physiological functions. To enable the identification of novel K(ir) channel inhibitors, a fluorescence resonance energy transfer (FRET)-based membrane potential assay was developed using a Chinese hamster ovary cell line stably expressing a human K(ir) channel. The FRET-based assay incorporates the use of two dyes {N-(6-chloro-7-hydroxycoumarin-3-carbonyl)-dimyristoylphosphatidylethanolamine (CC2-DMPE) and bis(1,3-diethylthiobarbiturate)trimethine oxonol [DiSBAC(2)(3)]} to track changes in membrane potential, thus enabling all of the advantages of ratiometric readout: reduced inaccuracies arising from well-to-well variation in cell number, dye loading, signal intensities, and plate inconsistencies. The assay was miniaturized to a 1,536-well microtiter plate format and read on a fluorometric imaging plate reader (FLIPR(Tetra), Molecular Devices, Sunnyvale, CA). The assay was automated and utilized to perform a primary high-throughput screening campaign to identify novel inhibitors of the K(ir) channel.

Integrating experimental and analytic approaches to improve data quality in genome-wide RNAi screens.

RNA interference (RNAi) not only plays an important role in drug discovery but can also be developed directly into drugs. RNAi high-throughput screening (HTS) biotechnology allows us to conduct genome-wide RNAi research. A central challenge in genome-wide RNAi research is to integrate both experimental and computational approaches to obtain high quality RNAi HTS assays. Based on our daily practice in RNAi HTS experiments, we propose the implementation of 3 experimental and analytic processes to improve the quality of data from RNAi HTS biotechnology: (1) select effective biological controls; (2) adopt appropriate plate designs to display and/or adjust for systematic errors of measurement; and (3) use effective analytic metrics to assess data quality. The applications in 5 real RNAi HTS experiments demonstrate the effectiveness of integrating these processes to improve data quality. Due to the effectiveness in improving data quality in RNAi HTS experiments, the methods and guidelines contained in the 3 experimental and analytic processes are likely to have broad utility in genome-wide RNAi research.

An efficient and fully automated high-throughput transfection method for genome-scale siRNA screens.

RNA interference (RNAi), combined with the availability of genome sequences, provides an unprecedented opportunity for the massive and parallel investigations of gene function. Small interfering RNA (siRNA) represents a popular and quick approach of RNAi for in vitro loss-of-function genetic screens. Efficient transfection of siRNA is critical for unambiguous interpretation of screen results and thus overall success of any siRNA screen. A high-throughput, lipid-based transfection method for siRNA was developed that can process eighty 384-well microplates in triplicate (for a total of 30,720 unique transfections) in 8 h. Transfection throughput was limited only by the speed of robotics, whereas the cost of screening was reduced. As a proof of principle, a genome-scale screen with a library of 22,108 siRNAs was performed to identify the genes sensitizing cells to mitomycin C at concentrations of 0, 20, and 60 nM. Transfection efficiency, performances of control siRNAs, and other quality metrics were monitored and demonstrated that the new, optimized transfection protocol produced high-quality results throughout the screen.

Median absolute deviation to improve hit selection for genome-scale RNAi screens.

High-throughput screening (HTS) of large-scale RNA interference (RNAi) libraries has become an increasingly popular method of functional genomics in recent years. Cell-based assays used for RNAi screening often produce small dynamic ranges and significant variability because of the combination of cellular heterogeneity, transfection efficiency, and the intrinsic nature of the genes being targeted. These properties make reliable hit selection in the RNAi screen a difficult task. The use of robust methods based on median and median absolute deviation (MAD) has been suggested to improve hit selection in such cases, but mean and standard deviation (SD)-based methods are still predominantly used in many RNAi HTS. In an experimental approach to compare these 2 methods, a genome-scale small interfering RNA (siRNA) screen was performed, in which the identification of novel targets increasing the therapeutic index of the chemotherapeutic agent mitomycin C (MMC) was sought. MAD values were resistant to the presence of outliers, and the hits selected by the MAD-based method included all the hits that would be selected by SD-based method as well as a significant number of additional hits. When retested in triplicate, a similar percentage of these siRNAs were shown to genuinely sensitize cells to MMC compared with the hits shared between SD- and MAD-based methods. Confirmed hits were enriched with the genes involved in the DNA damage response and cell cycle regulation, validating the overall hit selection strategy. Finally, computer simulations showed the superiority and generality of the MAD-based method in various RNAi HTS data models. In conclusion, the authors demonstrate that the MAD-based hit selection method rescued physiologically relevant false negatives that would have been missed in the SD-based method, and they believe it to be the desirable 1st-choice hit selection method for RNAi screen results.

Miniaturization and automation of an ubiquitin ligase cascade enzyme-linked immunosorbent assay in 1,536-well format.

Enzyme-linked immunosorbent assays (ELISAs) are a long established and widely used assay format for drug discovery and diagnostics. They offer many advantages over homogeneous assay formats, including high sensitivity and separation (wash) steps that remove detection-interfering compounds. Many high-throughput screening assays are now performed in miniaturized formats (1,536- and 3,456-well plates) for higher throughput and lower reagent consumption. With miniaturization, separation steps in assays such as ELISA can become difficult to implement. Here we report on the implementation of the Kalypsys, Inc. (San Diego, CA) 1,536-well plate washer to enable the successful miniaturization and full automation of an ELISA that monitors ubiquitin ligase activity. The 1,536-well plate ELISA was robust and used for the high-throughput screening of a large screening collection (>1 million compounds).

A high-precision fluorogenic cholesteryl ester transfer protein assay compatible with animal serum and 3456-well assay technology.

Cholesteryl ester transfer protein (CETP) is a serum component responsible for both cholesteryl ester and triglyceride trafficking between high-density lipoprotein (HDL) and the apolipoprotein B (apoB)-containing very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL). Several fluorescence-based assays that monitor these transfers have been reported, but to date such assays have suffered from a low signal/background (S/B) ratio and have been described for use only in relatively purified in vitro systems. We have modified the more advanced of these assays to incorporate a noninterfering, nondiffusable fluorescence quencher into previously described cosonicate particles, often referred to as microemulsions. This simple improvement resulted in particles that had an average threefold enhanced S/B window over particles without quenchers but that continued to show the essential properties of a catalytic assay, including catalysis to a single endpoint, excellent linearity with protein and particle concentration, and an appropriate sensitivity to inhibition. This reduced assay noise allowed the subsequent development of protocols for the direct measure of cholesteryl ester (CE) transfer activity resident in human and animal serum as well as the development of 384- and 3456-well screening protocols with good precision and accuracy. Thus, by expanding the dynamic response window of the assay, we have created an assay generalizable to many settings.

The use of strictly standardized mean difference for hit selection in primary RNA interference high-throughput screening experiments.

RNA interference (RNAi) high-throughput screening (HTS) has been hailed as the 2nd genomics wave following the 1st genomics wave of gene expression microarrays and single-nucleotide polymorphism discovery platforms. Following an RNAi HTS, the authors are interested in identifying short interfering RNA (siRNA) hits with large inhibition/activation effects. For hit selection, the z-score method and its variants are commonly used in primary RNAi HTS experiments. Recently, strictly standardized mean difference (SSMD) has been proposed to measure the siRNA effect represented by the magnitude of difference between an siRNA and a negative reference group. The links between SSMD and d+-probability offer a clear interpretation of siRNA effects from a probability perspective. Hence, SSMD can be used as a ranking metric for hit selection. In this article, the authors investigated both the SSMD-based testing process and the use of SSMD as a ranking metric for hit selection in 2 primary siRNA HTS experiments. The analysis results showed that, as a ranking metric, SSMD was more stable and reliable than percentage inhibition and led to more robust hit selection results. Using the SSMD -based testing method, the false-negative rate can more readily be obtained. More important, the use of the SSMD-based method can result in a reduction in both the false-negative and false-positive rates. The applications presented in this article demonstrate that the SSMD method addresses scientific questions and fills scientific needs better than both percentage inhibition and the commonly used z-score method for hit selection.

Ultra-high-throughput screening for antagonists of a Gi-coupled receptor in a 2.2-microl 3,456-well plate format cyclicAMP assay.

3',5'-Cyclic adenosine monophosphate (cAMP) is a common intracellular second messenger that enables cells to respond to external stimuli. Measurement of intracellular cAMP concentrations is thus widely used for studying guanosine triphosphate binding protein-coupled receptors (GPCRs), which make up a large class of pharmaceutical drug targets. Although several assay technologies exist to measure cAMP, most are not suitable for ultra-high-throughput screening (uHTS), as is often required for screening large (greater than 1 million) chemical libraries for the identification of suitable leads for drug development. Here we report that the enzyme fragment complementation assay, a homogeneous gain of signal assay based on complementation of two fragments of a beta-galactosidase enzyme, is compatible with uHTS requirements of a 2.2-microl total assay volume in 3,456-well plate format. We describe the miniaturization of this assay into 3,456-well plate format exhibiting comparable sensitivity and plate statistics to those of a 384-well assay and the application of this assay in uHTS for the identification of antagonists of a Gi-coupled receptor.

LRRTM3 promotes processing of amyloid-precursor protein by BACE1 and is a positional candidate gene for late-onset Alzheimer's disease.

Rare familial forms of Alzheimer's disease (AD) are thought to be caused by elevated proteolytic production of the Abeta42 peptide from the beta-amyloid-precursor protein (APP). Although the pathogenesis of the more common late-onset AD (LOAD) is not understood, BACE1, the protease that cleaves APP to generate the N terminus of Abeta42, is more active in patients with LOAD, suggesting that increased amyloid production processing might also contribute to the sporadic disease. Using high-throughput siRNA screening technology, we assessed 15,200 genes for their role in Abeta42 secretion and identified leucine-rich repeat transmembrane 3 (LRRTM3) as a neuronal gene that promotes APP processing by BACE1. siRNAs targeting LRRTM3 inhibit the secretion of Abeta40, Abeta42, and sAPPbeta, the N-terminal APP fragment produced by BACE1 cleavage, from cultured cells and primary neurons by up to 60%, whereas overexpression increases Abeta secretion. LRRTM3 is expressed nearly exclusively in the nervous system, including regions affected during AD, such as the dentate gyrus. Furthermore, LRRTM3 maps to a region of chromosome 10 linked to both LOAD and elevated plasma Abeta42, and is structurally similar to a family of neuronal receptors that includes the NOGO receptor, an inhibitor of neuronal regeneration and APP processing. Thus, LRRTM3 is a functional and positional candidate gene for AD, and, given its receptor-like structure and restricted expression, a potential therapeutic target.

Small interfering RNA screens reveal enhanced cisplatin cytotoxicity in tumor cells having both BRCA network and TP53 disruptions.

RNA interference technology allows the systematic genetic analysis of the molecular alterations in cancer cells and how these alterations affect response to therapies. Here we used small interfering RNA (siRNA) screens to identify genes that enhance the cytotoxicity (enhancers) of established anticancer chemotherapeutics. Hits identified in drug enhancer screens of cisplatin, gemcitabine, and paclitaxel were largely unique to the drug being tested and could be linked to the drug's mechanism of action. Hits identified by screening of a genome-scale siRNA library for cisplatin enhancers in TP53-deficient HeLa cells were significantly enriched for genes with annotated functions in DNA damage repair as well as poorly characterized genes likely having novel functions in this process. We followed up on a subset of the hits from the cisplatin enhancer screen and validated a number of enhancers whose products interact with BRCA1 and/or BRCA2. TP53(+/-) matched-pair cell lines were used to determine if knockdown of BRCA1, BRCA2, or validated hits that associate with BRCA1 and BRCA2 selectively enhances cisplatin cytotoxicity in TP53-deficient cells. Silencing of BRCA1, BRCA2, or BRCA1/2-associated genes enhanced cisplatin cytotoxicity approximately 4- to 7-fold more in TP53-deficient cells than in matched TP53 wild-type cells. Thus, tumor cells having disruptions in BRCA1/2 network genes and TP53 together are more sensitive to cisplatin than cells with either disruption alone.

A cell-based ultra-high-throughput screening assay for identifying inhibitors of D-amino acid oxidase.

Enzymes are often considered less "druggable" targets than ligand-regulated proteins such as G-protein-coupled receptors, ion channels, or other hormone receptors. Reasons for this include cellular location (intracellular vs. cell surface), typically lower affinities for the binding of small molecules compared to ligand-specific receptors, and binding (catalytic) sites that are often charged or highly polar. A practical drawback to the discovery of compounds targeting enzymes is that screening of compound libraries is typically carried out in cell-free activity assays using purified protein in an inherently artificial environment. Cell-based assays, although often arduous to design for enzyme targets, are the preferred discovery tool for the screening of large compound libraries. The authors have recently described a novel cell-based approach to screening for inhibitors of a phosphatase enzyme and now report on the development and implementation of a homogeneous 3456-well plate assay for D-amino acid oxidase (DAO). Human DAO was stably expressed in Chinese hamster ovary (CHO) cells, and its activity was measured as the amount of hydrogen peroxide detected in the growth medium following feeding the cells with D-serine. In less than 12 weeks, the authors proved the concept in 96-and then 384-well formats, miniaturized the assay to the 3456-well (nanoplate) scale, and screened a library containing more than 1 million compounds. They have identified several cell-permeable inhibitors of DAO from this cell-based high-throughput screening, which provided the discovery program with a few novel and attractive lead structures.