High-throughput Screening Facilities Research Articles

HTS driven by fluorescence lifetime detection of FRET identifies activators and inhibitors of cardiac myosin.

Small molecules that bind to allosteric sites on target proteins to alter protein function are highly sought in drug discovery. High-throughput screening (HTS) assays are needed to facilitate the direct discovery of allosterically active compounds. We have developed technology for high-throughput time-resolved fluorescence lifetime detection of fluorescence resonance energy transfer (FRET), which enables the detection of allosteric modulators by monitoring changes in protein structure. We tested this approach at the industrial scale by adapting an allosteric FRET sensor of cardiac myosin to high-throughput screening (HTS), based on technology provided by Photonic Pharma and the University of Minnesota, and then used the sensor to screen 1.6 million compounds in the HTS facility at Bristol Myers Squibb. The results identified allosteric activators and inhibitors of cardiac myosin that do not compete with ATP binding, demonstrating high potential for FLT-based drug discovery.

Mycobacterium tuberculosis virulence inhibitors discovered by Mycobacterium marinum high-throughput screening

High-throughput screening facilities do not generally support biosafety level 3 organisms such as Mycobacterium tuberculosis. To discover not only antibacterials, but also virulence inhibitors with either bacterial or host cell targets, an assay monitoring lung fibroblast survival upon infection was developed and optimized for 384-plate format and robotic liquid handling. By using Mycobacterium marinum as surrogate organism, 28,000 compounds were screened at biosafety level 2 classification, resulting in 49 primary hits. Exclusion of substances with unfavourable properties and known antimicrobials resulted in 11 validated hits of which 7 had virulence inhibiting properties and one had bactericidal effect also in wild type Mycobacterium tuberculosis. This strategy to discover virulence inhibitors using a model organism in high-throughput screening can be a valuable tool for other researchers working on drug discovery against tuberculosis and other biosafety level 3 infectious agents.

Rational Design of Small Molecules Targeting Oncogenic Noncoding RNAs from Sequence.

The discovery of RNA catalysis in the 1980s and the dissemination of the human genome sequence at the start of this century inspired investigations of the regulatory roles of noncoding RNAs in biology. In fact, the Encyclopedia of DNA Elements (ENCODE) project has shown that only 1-2% of the human genome encodes protein, yet 75% is transcribed into RNA. Functional studies both preceding and following the ENCODE project have shown that these noncoding RNAs have important roles in regulating gene expression, developmental timing, and other critical functions. RNA's diverse roles are often a consequence of the various folds that it adopts. The single-stranded nature of the biopolymer enables it to adopt intramolecular folds with noncanonical pairings to lower its free energy. These folds can be scaffolds to bind proteins or to form frameworks to interact with other RNAs. Not surprisingly, dysregulation of certain noncoding RNAs has been shown to be causative of disease. Given this as the background, it is easy to see why it would be useful to develop methods that target RNA and manipulate its biology in rational and predictable ways. The antisense approach has afforded strategies to target RNAs via Watson-Crick base pairing and has typically focused on targeting partially unstructured regions of RNA. Small molecule strategies to target RNA would be desirable not only because compounds could be lead optimized via medicinal chemistry but also because structured regions within an RNA of interest could be targeted to directly interfere with RNA folds that contribute to disease. Additionally, small molecules have historically been the most successful drug candidates. Until recently, the ability to design small molecules that target non-ribosomal RNAs has been elusive, creating the perception that they are "undruggable". In this Account, approaches to demystify targeting RNA with small molecules are described. Rather than bulk screening for compounds that bind to singular targets, which is the purview of the pharmaceutical industry and academic institutions with high throughput screening facilities, we focus on methods that allow for the rational design of small molecules toward biological RNAs. One enabling and foundational technology that has been developed is two-dimensional combinatorial screening (2DCS), a library-versus-library selection approach that allows the identification of the RNA motif binding preferences of small molecules from millions of combinations. A landscape map of the 2DCS-defined and annotated RNA motif-small molecule interactions is then placed into Inforna, a computational tool that allows one to mine these interactions against an RNA of interest or an entire transcriptome. Indeed, this approach has been enabled by tools to annotate RNA structure from sequence, an invaluable asset to the RNA community and this work, and has allowed for the rational identification of "druggable" RNAs in a target agnostic fashion.

Identification of small-molecule modulators of the circadian clock.

Chemical biology or chemical genetics has emerged as an interdisciplinary research area applying chemistry to understand biological systems. The development of combinatorial chemistry and high-throughput screening technologies has enabled large-scale investigation of the biological activities of diverse small molecules to discover useful chemical probes. This approach is applicable to the analysis of the circadian clock mechanisms through cell-based assays to monitor circadian rhythms using luciferase reporter genes. We and others have established cell-based high-throughput circadian assays and have identified a variety of novel small-molecule modulators of the circadian clock by phenotype-based screening of hundreds of thousands of compounds. The results demonstrated the effectiveness of chemical biology approaches in clock research field. This technique will become more and more common with propagation of high-throughput screening facilities. This chapter describes assay development, screening setups, and their optimization for successful screening campaigns.

Abstract 31: A Novel Fluorescence-Based Assay is Used to Investigate the Triglyceride Hydrolytic Activity of Lipases and to Identify Synthetic Peptides With Strong Lipase-Stimulating Activities

Hypertriglyceridemia is estimated to affect about one third of Americans and is an independent risk factor for cardiovascular disease, pancreatitis and xanthomatosis. Lipoprotein lipase (LPL) is a key enzyme involved in the hydrolysis and clearance of triglycerides from plasma and its enzymatic activity is regulated by specific apolipoproteins; apoC-III, which acts as a potent inhibitor and apoC-II and apoA-V, which act as stimulators. Their importance in hypertriglyceridemia is underscored by GWAS analyses, which have identified the APOA1/C3/A4/A5 gene cluster (chromosome 11q23) as an important determinant of dylipidemia. We have developed a fluorescence-based lipase assay that utilizes a fluorogenic triglyceride analog (EnzChek®, Molecular Probes) and facilitates the rapid analysis of lipase enzyme kinetics in a microplate format. This assay has made it possible for us to undertake the screening of the Prestwick (1,200 FDA-approved drugs) and Natural Product (~3,000) libraries at the University of Illinois high-throughput screening facility, in order to identify lipase activating compounds. To validate the assay we have characterized the influence of heparin, different metals and apolipoproteins on the hydrolytic activities of LPL in vitro, and hepatic lipase in HepG2 cell culture. As expected, enzyme activities were supported by heparin and calcium, and both apoC-II and apoC-III when complexed with VLDL, stimulated and inhibited lipase activity, respectively. Interestingly, the heparin/Fe2+ complex was a strong inhibitor of lipase activity, possibly explaining the high incidence of hypertriglyceridemia associated with iron supplementation to treat iron-deficiency. Finally, using a synthetic peptide approach we have localized apoA-V’s lipase stimulating and heparin binding domains to a short amphipathic, alpha-helical peptide. When included in the assays this peptide could increase lipase hydrolytic rates by 3-6 fold both in vitro, and in cell culture. Given that on a molar basis apoA-V is about 1200-fold less abundant than apoC-II, we anticipate that apoA-V will provide valuable sequence information for the development of potent triglyceride lowering therapeutics.

UCSF Small Molecule Discovery Center: Innovation, Collaboration and Chemical Biology in the Bay Area

The Small Molecule Discovery Center (SMDC) at the University of California, San Francisco, works collaboratively with the scientific community to solve challenging problems in chemical biology and drug discovery. The SMDC includes a high throughput screening facility, medicinal chemistry, and research labs focused on fundamental problems in biochemistry and targeted drug delivery. Here, we outline our HTS program and provide examples of chemical tools developed through SMDC collaborations. We have an active research program in developing quantitative cell-based screens for primary cells and whole organisms; here, we describe whole-organism screens to find drugs against parasites that cause neglected tropical diseases. We are also very interested in target-based approaches for so-called "undruggable", protein classes and fragment-based lead discovery. This expertise has led to several pharmaceutical collaborations; additionally, the SMDC works with start-up companies to enable their early-stage research. The SMDC, located in the biotech-focused Mission Bay neighborhood in San Francisco, is a hub for innovative small-molecule discovery research at UCSF.

A Perspective on 10-Years HTS Experience at the Walter and Eliza Hall Institute of Medical Research – Eighteen Million Assays and Counting

The Walter and Eliza Hall Institute of Medical Research (WEHI) is Australia's longest serving medical research institute. WEHI's High Throughput Screening (HTS) Facility was established in 2003 with $5 million of infrastructure funds invested by WEHI, and the Victorian State Government's Strategic Technology Initiative through Bio21 Australia Ltd. The Facility was Australia's first truly academic HTS facility and was one of only a handful operating in publicly funded institutions worldwide at that time. The objectives were to provide access to enabling HTS technologies, such as assay design, liquid handling automation, compound libraries and expertise to promote translation of basic research in a national setting that has a relatively young biotech sector and does not have a big Pharma research presence. Ten years on and the WEHI HTS Facility has participated in over 92 collaborative projects, generated over 18 million data points, and most importantly, projects that began in the Facility have been commercialized successfully (due to strong ties with Business Development and emphasis on intellectual property management) and now have molecules progressing in clinical trials.

Signature Motifs Identify an Acinetobacter Cif Virulence Factor with Epoxide Hydrolase Activity

Endocytic recycling of the cystic fibrosis transmembrane conductance regulator (CFTR) is blocked by the CFTR inhibitory factor (Cif). Originally discovered in Pseudomonas aeruginosa, Cif is a secreted epoxide hydrolase that is transcriptionally regulated by CifR, an epoxide-sensitive repressor. In this report, we investigate a homologous protein found in strains of the emerging nosocomial pathogens Acinetobacter nosocomialis and Acinetobacter baumannii ("aCif"). Like Cif, aCif is an epoxide hydrolase that carries an N-terminal secretion signal and can be purified from culture supernatants. When applied directly to polarized airway epithelial cells, mature aCif triggers a reduction in CFTR abundance at the apical membrane. Biochemical and crystallographic studies reveal a dimeric assembly with a stereochemically conserved active site, confirming our motif-based identification of candidate Cif-like pathogenic EH sequences. Furthermore, cif expression is transcriptionally repressed by a CifR homolog ("aCifR") and is induced in the presence of epoxides. Overall, this Acinetobacter protein recapitulates the essential attributes of the Pseudomonas Cif system and thus may facilitate airway colonization in nosocomial lung infections.

Rethinking Molecular Similarity: Comparing Compounds on the Basis of Biological Activity

Since the advent of high-throughput screening (HTS), there has been an urgent need for methods that facilitate the interrogation of large-scale chemical biology data to build a mode of action (MoA) hypothesis. This can be done either prior to the HTS by subset design of compounds with known MoA or post HTS by data annotation and mining. To enable this process, we developed a tool that compares compounds solely on the basis of their bioactivity: the chemical biological descriptor "high-throughput screening fingerprint" (HTS-FP). In the current embodiment, data are aggregated from 195 biochemical and cell-based assays developed at Novartis and can be used to identify bioactivity relationships among the in-house collection comprising ~1.5 million compounds. We demonstrate the value of the HTS-FP for virtual screening and in particular scaffold hopping. HTS-FP outperforms state of the art methods in several aspects, retrieving bioactive compounds with remarkable chemical dissimilarity to a probe structure. We also apply HTS-FP for the design of screening subsets in HTS. Using retrospective data, we show that a biodiverse selection of plates performs significantly better than a chemically diverse selection of plates, both in terms of number of hits and diversity of chemotypes retrieved. This is also true in the case of hit expansion predictions using HTS-FP similarity. Sets of compounds clustered with HTS-FP are biologically meaningful, in the sense that these clusters enrich for genes and gene ontology (GO) terms, showing that compounds that are bioactively similar also tend to target proteins that operate together in the cell. HTS-FP are valuable not only because of their predictive power but mainly because they relate compounds solely on the basis of bioactivity, harnessing the accumulated knowledge of a high-throughput screening facility toward the understanding of how compounds interact with the proteome.

121 Identification, validation and mechanisms of action of novel therapeutic compounds modulating angiogenesis

Abnormal angiogenesis is a signature feature in over 70 health conditions and is critical for tumour growth and metastasis. Therefore, discovering novel therapeutic compounds, that stimulate or abrogate this biological process would be, potentially, clinically invaluable. The zebrafish has emerged as an important model for studying vascular development and angiogenesis. Advantages over other models include rapid embryonic development and optical clarity of the embryos. The small body size of the zebrafish embryo and accessibility of embryonic developmental structures within water make zebrafish embryos suitable for high through-put drug screens. We performed an angiogenesis chemical genetic screen with two compound libraries (LOPAC and Spectrum Collection) using Flk1:EGFP (fluorescently labelled endothelial cells) transgenic zebrafish embryos. The compound bioactivity was measured by numbers of the zebrafish trunk intersegmental vessel (ISV) number. The screens were performed in 96 well plates and a total of ten antiangiogenic compounds and three proangiogenic compounds were identified. A CFI-funded high throughput screening facility will be use for identification of more lead compounds. The goal of the current study was to validate the potential therapeutic efficacy of selected compounds and investigate their molecular mechanisms. Retinoic acid p-hydroxyanilide (4HPR; Fenretinide) was identified as a pro-angiogenic compound. At 4 μM, 4HPR promoted ISV development with increased ISV numbers (ISV = 32.82 ± 0.98; control ISV = 27.58 ± 0.76; P < 0.05). In HUVEC cell proliferation assay, 4HPR stimulated human endothelial cell proliferation at low molar concentrations (0.06-0.12 μM). The effects of this compound on physiological angiogenesis are currently being tested on larvae and adult zebrafish fin regeneration models. Indirubin-3-oxime (IO) was identified as a very potent antiangiogenic compound, which inhibited zebrafish ISV development in a dose-dependent manner (Fig. 1). At 16 μM, IO almost completely inhibited ISV development. In tissue regeneration models, IO inhibited larval and adult zebrafish fin regeneration. The efficacy of IO in cancer therapy will be further studied in a zebrafish cancer metastasis model. Possible molecular mechanisms responsible for IO and 4HPR action such as VEGF and notch signaling will be studied. Using the zebrafish chemical genetic screen, we demonstrated that indirubin-3-oxime is a potent angiogenic inhibitor which inhibits zebrafish angiogenesis and fin regeneration. IO may prove to be effective as an anti-cancer drug. Retinoic acid p-hydroxyanilide was identified as a pro-angiogenic effector, which may be clinically effective in promoting tissue regeneration and in treatment of myocardial infarction.

Screensaver: an open source lab information management system (LIMS) for high throughput screening facilities.

BackgroundShared-usage high throughput screening (HTS) facilities are becoming more common in academe as large-scale small molecule and genome-scale RNAi screening strategies are adopted for basic research purposes. These shared facilities require a unique informatics infrastructure that must not only provide access to and analysis of screening data, but must also manage the administrative and technical challenges associated with conducting numerous, interleaved screening efforts run by multiple independent research groups.ResultsWe have developed Screensaver, a free, open source, web-based lab information management system (LIMS), to address the informatics needs of our small molecule and RNAi screening facility. Screensaver supports the storage and comparison of screening data sets, as well as the management of information about screens, screeners, libraries, and laboratory work requests. To our knowledge, Screensaver is one of the first applications to support the storage and analysis of data from both genome-scale RNAi screening projects and small molecule screening projects.ConclusionsThe informatics and administrative needs of an HTS facility may be best managed by a single, integrated, web-accessible application such as Screensaver. Screensaver has proven useful in meeting the requirements of the ICCB-Longwood/NSRB Screening Facility at Harvard Medical School, and has provided similar benefits to other HTS facilities.

Simulating Henipavirus Multicycle Replication in a Screening Assay Leads to Identification of a Promising Candidate for Therapy

Nipah (NiV) and Hendra (HeV) viruses are emerging zoonotic paramyxoviruses that cause encephalitis in humans, with fatality rates of up to 75%. We designed a new high-throughput screening (HTS) assay for inhibitors of infection based on envelope glycoprotein pseudotypes. The assay simulates multicycle replication and thus identifies inhibitors that target several stages of the viral life cycle, but it still can be carried out under biosafety level 2 (BSL-2) conditions. These features permit a screen for antivirals for emerging viruses and select agents that otherwise would require BSL-4 HTS facilities. The screening of a small compound library identified several effective molecules, including the well-known compound chloroquine, as highly active inhibitors of pseudotyped virus infection. Chloroquine inhibited infection with live HeV and NiV at a concentration of 1 microM in vitro (50% inhibitory concentration, 2 microM), which is less than the plasma concentrations present in humans receiving chloroquine treatment for malaria. The mechanism for chloroquine's antiviral action likely is the inhibition of cathepsin L, a cellular enzyme that is essential for the processing of the viral fusion glycoprotein and the maturation of newly budding virions. Without this processing step, virions are not infectious. The identification of a compound that inhibits a known cellular target that is important for viral maturation but that had not previously been shown to have antiviral activity for henipaviruses highlights the validity of this new screening assay. Given the established safety profile and broad experience with chloroquine in humans, the results described here provide an option for treating individuals infected by these deadly viruses.

Natural diversity of aminotransferases and dehydrogenase activity in a large collection of Lactococcus lactis strains

The natural diversity of Lactococcus lactis was studied with respect to the flavour-generating enzymes leucine (Leu-AT) and aromatic (AraT) aminotransferase, and hydroxyisocaproic acid dehydrogenase (HicDH). The screening of 175 L. lactis strains required the development of enzyme assays to enable the use of high throughput screening facilities. Natural diversity was expressed as the difference between 10% of the strains with the highest and lowest enzyme activity. The natural diversity found for Leu-AT, AraT and HicDH activity was 13-, 49- and 30-fold, respectively. Leu-AT activity showed a weak correlation with the AraT and HicDH activity, whereas there was no correlation between AraT and HicDH activity. In the group of strains with the highest Leu-AT activity, 5- and 6-fold differences of AraT and HicDH activities were found, respectively. These results demonstrated that it is possible to select strains with specific combinations of important ripening enzymes that could enhance cheese flavour formation.

Future trends in screening technology for drug discovery

The last decade showed a further upsurge in screening technology advance and innovation. Notably, the establishment of ultra high-throughput screening facilities led to an explosion of screening capacities. However, in the last 2 years, a turning point in screening philosophy can be observed worldwide. Increasingly more companies are reducing the size of screening campaigns, while increasing the emphasis on data quality and relevance. This article tries to investigate how screening technologies will develop in the ever-changing landscape of drug discovery.

Automation highlights from literature

Automation highlights from literature

Genetic optimization of combinatorial libraries

Most agrochemical and pharmaceutical companies have set up high-throughput screening programs which require large numbers of compounds to screen. Combinatorial libraries provide an attractive way to deliver these compounds. A single combinatorial library with four variable positions can yield more than 10(12) potential compounds, if one assumes that about 1000 reagents are available for each position. This is far more than any high-throughput screening facility can afford to screen. We have proposed a method for iterative compound selection from large databases, which identifies the most active compounds by examining only a small fraction of the database. In this article, we describe the extension of this method to the problem of selecting compounds from large combinatorial libraries. Copyright 1998 John Wiley & Sons, Inc.

Approaches to automation for high-throughput screening

Automation of high-throughput screening (HTS) assays can occur by one of two approaches. One involves design and implementation of systems employing robot arms for the tasks that comprise an assay. While this approach may offer excellent capacity, it usually takes time and large capital outlays to implement, debug, and maintain. Such systems also require specialists for operation and maintenance. The other approach is more stepwise in nature. It involves acquisition of automated workstations from different vendors and installing them in a concatenated fashion to comprise an assay protocol. This method is a more straightforward approach to establishing a turnkey HTS facility. As the fully automated approach is termed robotic, the stepwise method may be termed hubotic, insofar as it reflects the synergies obtainable by allowing people to provide the work flow between optimized laboratory workstations. Specific examples of these approaches will be described for different aspects of HTS, particularly for running complex cell-based assays. In addition, emerging robotic systems that are capable of ultrahigh-throughput screening capacities and utilize novel design principles will be discussed. We term these ubotic HTS approaches. © 1996 John Wiley & Sons, Inc.

Approaches to automation for high‐throughput screening

Automation of high-throughput screening (HTS) assays can occur by one of two approaches. One involves design and implementation of systems employing robot arms for the tasks that comprise an assay. While this approach may offer excellent capacity, it usually takes time and large capital outlays to implement, debug, and maintain. Such systems also require specialists for operation and maintenance. The other approach is more stepwise in nature. It involves acquisition of automated workstations from different vendors and installing them in a concatenated fashion to comprise an assay protocol. This method is a more straightforward approach to establishing a turnkey HTS facility. As the fully automated approach is termed robotic, the stepwise method may be termed hubotic, insofar as it reflects the synergies obtainable by allowing people to provide the work flow between optimized laboratory workstations. Specific examples of these approaches will be described for different aspects of HTS, particularly for running complex cell-based assays. In addition, emerging robotic systems that are capable of ultrahigh-throughput screening capacities and utilize novel design principles will be discussed. We term these ubotic HTS approaches. © 1996 John Wiley & Sons, Inc.