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High content analysis (HCA) has become a leading methodology in phenotypic drug discovery efforts. Typical HCA workflows include imaging cells using an automated microscope and analyzing the data using algorithms designed to quantify one or more specific phenotypes of interest. Due to the richness of high content data, unappreciated phenotypic changes may be discovered in existing image sets using interactive machine-learning based software systems. Primary postnatal day four retinal cells from the photoreceptor (PR) labeled QRX-EGFP reporter mice were isolated, seeded, treated with a set of 234 profiled kinase inhibitors and then cultured for 1 week. The cells were imaged with an Acumen plate-based laser cytometer to determine the number and intensity of GFP-expressing, i.e. PR, cells. Wells displaying intensities and counts above threshold values of interest were re-imaged at a higher resolution with an INCell2000 automated microscope. The images were analyzed with an open source HCA analysis tool, PhenoRipper (Rajaram et al., Nat Methods 9:635-637, 2012), to identify the high GFP-inducing treatments that additionally resulted in diverse phenotypes compared to the vehicle control samples. The pyrimidinopyrimidone kinase inhibitor CHEMBL-1766490, a pan kinase inhibitor whose major known targets are p38α and the Src family member lck, was identified as an inducer of photoreceptor neuritogenesis by using the open-source HCA program PhenoRipper. This finding was corroborated using a cell-based method of image analysis that measures quantitative differences in the mean neurite length in GFP expressing cells. Interacting with data using machine learning algorithms may complement traditional HCA approaches by leading to the discovery of small molecule-induced cellular phenotypes in addition to those upon which the investigator is initially focusing.

The structural integrity of myelin formed by Schwann cells in the peripheral nervous system (PNS) is required for proper nerve conduction and is dependent on adequate expression of myelin genes including peripheral myelin protein 22 (PMP22). Consequently, excess PMP22 resulting from its genetic duplication and overexpression has been directly associated with the peripheral neuropathy called Charcot-Marie-Tooth disease type 1A (CMT1A), the most prevalent type of CMT. Here, in an attempt to identify transcriptional inhibitors with therapeutic value towards CMT1A, we developed a cross-validating pair of orthogonal reporter - firefly luciferase (FLuc) and β-lactamase (βLac) - assays capable of recapitulating PMP22 expression, utilizing the intronic regulatory element of the human PMP22 gene. Each compound from a collection of approximately 3,000 approved drugs was tested at multiple titration points to achieve a pharmacological endpoint in a 1536-well plate quantitative high-throughput screen (qHTS) format. In conjunction with an independent counter-screen for cytotoxicity, the design of our orthogonal screen platform effectively contributed to selection and prioritization of active compounds, among which three drugs (fenretinide, olvanil, and bortezomib) exhibited marked reduction of endogenous Pmp22 mRNA and protein. Overall, the findings of this study provide a strategic approach to assay development for gene-dosage diseases such as CMT1A.

Quinazolin-4-one 1 was identified as an inhibitor of the HIF-1α transcriptional factor from a high-throughput screen. HIF-1α up-regulation is common in many cancer cells. In this Letter, we describe an efficient one-pot sequential reaction for the synthesis of quinazolin-4-one 1 analogues. The structure-activity relationship (SAR) study led to the 5-fold more potent analogue, 16.

Malaria remains a devastating disease largely because of widespread drug resistance. New drugs and a better understanding of the mechanisms of drug action and resistance are essential for fulfilling the promise of eradicating malaria. Using high-throughput chemical screening and genome-wide association analysis, we identified 32 highly active compounds and genetic loci associated with differential chemical phenotypes (DCPs), defined as greater than or equal to fivefold differences in half-maximum inhibitor concentration (IC(50)) between parasite lines. Chromosomal loci associated with 49 DCPs were confirmed by linkage analysis and tests of genetically modified parasites, including three genes that were linked to 96% of the DCPs. Drugs whose responses mapped to wild-type or mutant pfcrt alleles were tested in combination in vitro and in vivo, which yielded promising new leads for antimalarial treatments.

The thyroid hormone receptors (TR) are members of the nuclear hormone receptor (NHR) superfamily that regulate development, growth, and metabolism. Upon ligand binding, TR releases bound corepressors and recruits coactivators to modulate target gene expression. Steroid receptor coactivator 2 (SRC2) is an important coregulator that interacts with TRβ to activate gene transcription. To identify novel inhibitors of the TRβ and SRC2 interaction, the authors performed a quantitative high-throughput screen (qHTS) of a TRβ-SRC2 fluorescence polarization assay against more than 290 000 small molecules. The qHTS assayed compounds at 6 concentrations up to 92 µM to generate titration-response curves and determine the potency and efficacy of all compounds. The qHTS data set enabled the characterization of actives for structure-activity relationships as well as for potential artifacts such as fluorescence interference. Selected qHTS actives were tested in the screening assay using fluoroprobes labeled with Texas Red or fluorescein. The retest identified 19 series and 4 singletons as active in both assays with 40% or greater efficacy, free of compound interference, and not toxic to mammalian cells. Selected compounds were tested as independent samples, and a methylsulfonylnitrobenzoate series inhibited the TRβ-SRC2 interaction with 5 µM IC(50). This series represents a new class of thyroid hormone receptor-coactivator modulators.

In an effort to expand the stereochemical and structural complexity of chemical libraries used in drug discovery, the Center for Chemical Methodology and Library Development at Boston University has established an infrastructure to translate methodologies accessing diverse chemotypes into arrayed libraries for biological evaluation. In a collaborative effort, the NIH Chemical Genomics Center determined IC 50 ’s for Plasmodium falciparum viability for each of 2,070 members of the CMLD-BU compound collection using quantitative high-throughput screening across five parasite lines of distinct geographic origin. Three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified, and the nascent structure activity relationships (SAR) from this experiment used to initiate optimization of these chemotypes for further development.

Thyroid hormone receptors (TRs) are members of the nuclear hormone receptor (NR) superfamily and regulate development, growth, and metabolism. Upon binding thyroid hormone, TR undergoes a conformational change that allows the release of corepressors and the recruitment of coactivators, which in turn regulate target gene transcription. Although a number of TR antagonists have been developed, most are analogs of the endogenous hormone that inhibit ligand binding. In a screen for inhibitors that block the association of TRβ with steroid receptor coactivator 2 (SRC2), we identified a novel methylsulfonylnitrobenzoate (MSNB)-containing series that blocks this interaction at micromolar concentrations. Here we have studied a series of MSNB analogs and characterized their structure activity relationships. MSNB members do not displace thyroid hormone T3 but instead act by direct displacement of SRC2. MSNB series members are selective for the TR over the androgen, vitamin D, and PPARγ NR members, and they antagonize thyroid hormone-activated transcription action in cells. The methylsulfonylnitro group is essential for TRβ antagonism. Side-chain alkylamine substituents showed better inhibitory activity than arylamine substituents. Mass spectrum analysis suggested that MSNB inhibitors bind irreversibly to Cys-298 within the AF-2 cleft of TRβ to disrupt SRC2 association.

Activation of Gq protein-coupled receptors can be monitored by measuring the increase in intracellular calcium with fluorescent dyes. Recent advances in fluorescent kinetic plate readers and liquid-handling technology have made it possible to follow these transient changes in intracellular calcium in a 1,536-well plate format for high-throughput screening (HTS). Here, we have applied the latest generation of fluorescence kinetic plate readers to multiplex the agonist and antagonist screens of a G protein-coupled receptor (GPCR). This multiplexed assay format provides an efficient and cost-effective method for HTS of Gq-coupled GPCR targets.

Surfactin-type phosphopantetheinyl transferases (Sfp-PPTases) are responsible for modifying type I polyketide and non-ribosomal peptide synthases of prokaryotes and have been implicated in the activation of a variety of pathogen-associated virulence factors. As such, inhibitors of this enzyme class represent enticing leads for antibiotic development and can serve as tools in studies of bacterial metabolism. Currently, no small molecule inhibitors of Sfp-PPTase are known, highlighting the need for efficient methods for PPTase inhibitor identification and development. Herein, we present the design and implementation of a robust and miniaturized high-throughput kinetic assay for inhibitors of Sfp-PPTase using the substrate combination of rhodamine-labeled coenzyme A and Black Hole Quencher-2 labeled consensus acceptor peptide YbbR. Upon PPTase-catalyzed transfer of the rhodamine-labeled phosphopantetheinyl arm onto the acceptor peptide, the fluorescent donor and quencher are covalently joined and the fluorescence signal is reduced. This assay was miniaturized to a low 4 microL volume in 1536-well format and was used to screen the library of pharmacologically active compounds (LOPAC(1280)). Top inhibitors identified by the screen were further characterized in secondary assays, including protein phosphopantetheinylation detected by gel electrophoresis. The present assay enables the screening of large compound libraries against Sfp-PPTase in a robust and automated fashion and is applicable to designing assays for related transferase enzymes.

Assays for ATPases have been enabled for high-throughput screening (HTS) by employing firefly luciferase to detect the remaining ATP in the assay. However, for any enzyme assay, measurement of product formation is a more sensitive assay design. Recently, technologies that allow detection of the ADP product from ATPase reactions have been described using fluorescent methods of detection. We describe here the characterization of a bioluminescent assay that employs firefly luciferase in a coupled-enzyme assay format to enable detection of ADP levels from ATPase assays (ADP-Glo, Promega Corp.). We determined the performance of the ADP-Glo assay in 1,536-well microtiter plates using the protein kinase Clk4 and a 1,352 member kinase focused combinatorial library. The ADP-Glo assay was compared to the Clk4 assay performed using a bioluminescence ATP-depletion format (Kinase-Glo, Promega Corp). We performed this analysis using quantitative HTS (qHTS) where we determined potency values for all library members and identified approximately 300 compounds with potencies ranging from as low as 50 nM to10 microM, yielding a robust dataset for the comparison. Both assay formats showed high performance (Z'-factors approximately 0.9) and showed a similar potency distribution for the actives. We conclude that the bioluminescence ADP detection assay system is a viable generic alternative to the widely used ATP-depletion assay for ATPases and discuss the advantages and disadvantages of both approaches.

A series of substituted 6-arylquinazolin-4-amines were prepared and analyzed as inhibitors of Clk4. Synthesis, structure-activity relationships and the selectivity of a potent analogue against a panel of 402 kinases are presented. Inhibition of Clk4 by these agents at varied concentrations of assay substrates (ATP and receptor peptide) highly suggests that this chemotype is an ATP competitive inhibitor. Molecular docking provides further evidence that inhibition is the result of binding at the kinase hinge region. Selected compounds represent novel tools capable of potent and selective inhibition of Clk1, Clk4, and Dyrk1A.

The perceived and actual burden of false positives in high-throughput screening has received considerable attention; however, few studies exist on the contributions of distinct mechanisms of non-specific effects like chemical reactivity, assay signal interference, and colloidal aggregation. Here, we analyze the outcome of a screen of 197,861 diverse compounds in a concentration-response format against the cysteine protease cruzain, a target expected to be particularly sensitive to reactive compounds and using an assay format with light detection in the short-wavelength region where significant compound autofluorescence is typically encountered. Approximately 1.9% of all compounds screened were detergent-sensitive inhibitors. The contribution from autofluorescence and compounds bearing reactive functionalities was dramatically lower: of all hits, only 1.8% were autofluorescent and 1.48% contained reactive or undesired functional groups. The distribution of false positives was relatively constant across library sources. The simple step of including detergent in the assay buffer suppressed the nonspecific effect of approximately 93% of the original hits.

The cytochrome P450 (CYP) gene family catalyzes drug metabolism and bioactivation and is therefore relevant to drug development. We determined potency values for 17,143 compounds against five recombinant CYP isozymes (1A2, 2C9, 2C19, 2D6 and 3A4) using an in vitro bioluminescent assay. The compounds included libraries of US Food and Drug Administration (FDA)-approved drugs and screening libraries. We observed cross-library isozyme inhibition (30-78%) with important differences between libraries. Whereas only 7% of the typical screening library was inactive against all five isozymes, 33% of FDA-approved drugs were inactive, reflecting the optimized pharmacological properties of the latter. Our results suggest that low CYP 2C isozyme activity is a common property of drugs, whereas other isozymes, such as CYP 2D6, show little discrimination between drugs and unoptimized compounds found in screening libraries. We also identified chemical substructures that differentiated between the five isozymes. The pharmacological compendium described here should further the understanding of CYP isozymes. Refereed/Peer-reviewed

Here, we report the results of a quantitative high-throughput screen (qHTS) measuring the endocytosis and translocation of a beta-lactamase-fused-lethal factor and the identification of small molecules capable of obstructing the process of anthrax toxin internalization. Several small molecules protect RAW264.7 macrophages and CHO cells from anthrax lethal toxin and protected cells from an LF-Pseudomonas exotoxin fusion protein and diphtheria toxin. Further efforts demonstrated that these compounds impaired the PA heptamer pre-pore to pore conversion in cells expressing the CMG2 receptor, but not the related TEM8 receptor, indicating that these compounds likely interfere with toxin internalization.

Cellular metabolism depends on the availability of oxygen and the major regulator of oxygen homeostasis is hypoxia-inducible factor 1 (HIF-1), a highly conserved transcription factor that plays an essential role in cellular and systemic homeostatic responses to hypoxia. HIF-1 is a heterodimeric transcription factor composed of hypoxia-inducible HIF-1alpha and constitutively expressed HIF-1beta. Under hypoxic conditions, the two subunits dimerize, allowing translocation of the HIF-1 complex to the nucleus where it binds to hypoxia-response elements (HREs) and activates expression of target genes implicated in angiogenesis, cell growth, and survival. The HIF-1 pathway is essential to normal growth and development, and is involved in the pathophysiology of cancer, inflammation, and ischemia. Thus, there is considerable interest in identifying compounds that modulate the HIF-1 signaling pathway. To assess the ability of environmental chemicals to stimulate the HIF-1 signaling pathway, we screened a National Toxicology Program collection of 1408 compounds using a cell-based beta-lactamase HRE reporter gene assay in a quantitative high-throughput screening (qHTS) format. Twelve active compounds were identified. These compounds were tested in a confirmatory assay for induction of vascular endothelial growth factor, a known hypoxia target gene, and confirmed compounds were further tested for their ability to mimic the effect of a reduced-oxygen environment on hypoxia-regulated promoter activity. Based on this testing strategy, three compounds (o-phenanthroline, iodochlorohydroxyquinoline, cobalt sulfate heptahydrate) were confirmed as hypoxia mimetics, whereas two compounds (7-diethylamino-4-methylcoumarin and 7,12-dimethylbenz(a)anthracence) were found to interact with HIF-1 in a manner different from hypoxia. These results demonstrate the effectiveness of qHTS in combination with secondary assays for identification of HIF-1alpha inducers and for distinguishing among inducers based on their pattern of activated hypoxic target genes. Identification of environmental compounds having HIF-1alpha activation activity in cell-based assays may be useful for prioritizing chemicals for further testing as hypoxia-response inducers in vivo.

Hsp90 has emerged as an important anticancer drug target because of its essential role in promoting the folding and maturation of many oncogenic proteins. The authors describe the development of the first high-throughput screen, based on AlphaScreen technology, to identify a novel type of Hsp90 inhibitors that interrupt its interaction with the cochaperone HOP. The assay used the 20-mer C-terminal peptide of Hsp90 and the TPR2A domain of HOP. Assay specificity was demonstrated by measuring different interactions using synthetic peptides, with measured IC50s in good agreement with reported values. The assay was stable over 12 h and tolerated DMSO up to 5%. The authors first validated the assay by screening against 20,000 compounds in a 384-well format. After further optimization into a 1536-well format, it was screened against an NIH Chemical Genomics Center library of 76,134 compounds, with a signal-to-background ratio of 78 and Z' factor of 0.77. The present assay can be used for discovery of novel small-molecule Hsp90 inhibitors that can be used as chemical probes to investigate the role of cochaperones in Hsp90 function. Such molecules have the potential to be developed into novel anticancer drugs, for use alone or in combination with other Hsp90 inhibitors.

Many studies have implicated the cAMP Response Element Binding (CREB) protein signaling pathway in long-term memory. To identify small molecule enhancers of CREB activation of gene expression, we screened approximately 73,000 compounds, each at 7-15 concentrations in a quantitative high-throughput screening (qHTS) format, for activity in cells by assaying CREB mediated beta-lactamase reporter gene expression. We identified 1,800 compounds that potentiated CREB mediated gene expression, with potencies as low as 16 nM, comprising 96 structural series. Mechanisms of action were systematically determined, and compounds that affect phosphodiesterase 4, protein kinase A, and cAMP production were identified, as well as compounds that affect CREB signaling via apparently unidentified mechanisms. qHTS followed by interrogation of pathway targets is an efficient paradigm for lead generation for chemical genomics and drug development.

Nuclear translocation is an important step in glucocorticoid receptor (GR) signaling and assays that measure this process allow the identification of nuclear receptor ligands independent of subsequent functional effects. To facilitate the identification of GR-translocation agonists, an enzyme fragment complementation (EFC) cell-based assay was scaled to a 1536-well plate format to evaluate 9,920 compounds using a quantitative high throughput screening (qHTS) strategy where compounds are assayed at multiple concentrations. In contrast to conventional assays of nuclear translocation the qHTS assay described here was enabled on a standard luminescence microplate reader precluding the requirement for imaging methods. The assay uses beta-galactosidase alpha complementation to indirectly detect GR-translocation in CHO-K1 cells [Fung, P., et al. Assay Drug Devel. Technol. 2006, 4(3): 263–272]. 1536-well assay miniaturization included the elimination of a media aspiration step, and the optimized assay displayed a Z′ of 0.55. qHTS yielded EC50 values for all 9,920 compounds and allowed us to retrospectively examine the dataset as a single concentration-based screen to estimate the number of false positives and negatives at typical activity thresholds. For example, at a 9 μM screening concentration the assay showed an accuracy that is comparable to typical cell-based assays as judged by the occurrence of false positives that we determined to be 1.3% or 0.3%, for a 3σ or 6σ threshold, respectively. This corresponds to a confirmation rate of ~30% or ~50%, respectively. The assay was consistent with glucocorticoid pharmacology as scaffolds with close similarity to dexamethasone were identified as active, while, for example, steroids that act as ligands to other nuclear receptors such as the estrogen receptor were found to be inactive.

Cell-based functional assays used for compound screening and lead optimization play an important role in drug discovery for G-protein coupled receptors (GPCRs). Cell-based assays can define the role of a compound as an agonist, antagonist or inverse agonist and can provide detailed information about the potency and efficacy of a compound. In addition, cell-based screens can be used to identify allosteric modulators that interact with sites other than the binding site of the endogenous ligand. Intracellular calcium assays which use a fluorescent calcium binding dye (such as Fluo-3, Fluo-4 or Fura-2) have been used in compound screening campaigns to measure the activity of Gq-coupled GPCRs. However, such screening methodologies require a special instrumentation to record the rapid change in intracellular free calcium concentration over time. The radioactive inositol 1,4,5- triphosphate (IP(3)) assay measures (3)H-inositol incorporation and is another traditional assay for the assessment of Gq-coupled GPCR activity, but it is not suitable for screening of large size compound collections because it requires a cell wash step and generates radioactive waste. To avoid these limitations, we have optimized and miniaturized a TR-FRET based IP-One assay that measures inositol monophosphate in a 1536-well plate format. This assay is homogenous, non-radioactive and does not require a kinetic readout. It has been tested with the cell lines expressing M(1) acetylcholine, FFAR1, vasopressin V1b, or Neuropeptide S receptors. The activities of antagonists determined in the IP-One assay correlated well with these measured in the intracellular calcium assay while the correlation of agonist activities might vary from cell line to cell line. This IP-One assay offers an alternative method for high throughput screening of Gq-coupled GPCRs without using costly kinetic plate readers.

The importance of bioluminescence in enabling a broad range of high-throughput screening (HTS) assay formats is evidenced by widespread use in industry and academia. Therefore, understanding the mechanisms by which reporter enzyme activity can be modulated by small molecules is critical to the interpretation of HTS data. In this Perspective, we provide evidence for stabilization of luciferase by inhibitors in cell-based luciferase reporter-gene assays resulting in the counterintuitive phenomenon of signal activation. These data were derived from our analysis of luciferase inhibitor compound structures and their prevalence in the Molecular Libraries Small Molecule Repository using 100 HTS experiments available in PubChem. Accordingly, we found an enrichment of luciferase inhibitors in luciferase reporter-gene activation assays but not in assays using other reporters. In addition, for several luciferase inhibitor chemotypes, we measured reporter stabilization and signal activation in cells that paralleled the inhibition determined using purified luciferase to provide further experimental support for these contrasting effects.

The GoLoco motif is a short Galpha-binding polypeptide sequence. It is often found in proteins that regulate cell-surface receptor signaling, such as RGS12, as well as in proteins that regulate mitotic spindle orientation and force generation during cell division, such as GPSM2/LGN. Here, we describe a high throughput fluorescence polarization (FP) assay using fluorophore-labeled GoLoco motif peptides for identifying inhibitors of the GoLoco motif interaction with the G-protein alpha subunit Galpha (i1). The assay exhibits considerable stability over time and is tolerant to DMSO up to 5%. The Z'-factors for robustness of the GPSM2 and RGS12 GoLoco motif assays in a 96-well plate format were determined to be 0.81 and 0.84, respectively; the latter assay was run in a 384-well plate format and produced a Z'-factor of 0.80. To determine the screening factor window (Z-factor) of the RGS12 GoLoco motif screen using a small molecule library, the NCI Diversity Set was screened. The Z-factor was determined to be 0.66, suggesting that this FP assay would perform well when developed for 1,536-well format and scaled up to larger libraries. We then miniaturized to a 4 microL final volume a pair of FP assays utilizing fluorescein- (green) and rhodamine- (red) labeled RGS12 GoLoco motif peptides. In a fully-automated run, the Sigma-Aldrich LOPAC(1280) collection was screened three times with every library compound being tested over a range of concentrations following the quantitative high throughput screening (qHTS) paradigm; excellent assay performance was noted with average Z-factors of 0.84 and 0.66 for the green- and red-label assays, respectively.

Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic "release" assays which measure the extra-cellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known compounds from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.

An efficient and versatile Compound Management operation is essential for the success of all downstream processes in high-throughput screening (HTS) and small molecule lead development. Staff, equipment, and processes need to be not only reliable, but remain flexible and prepared to incorporate paradigm changes. In the present report, we describe a system and associated processes that enable handling of compounds for both screening and follow-up purposes at the NIH Chemical Genomics Center (NCGC), a recently established HTS and probe development center within the Molecular Libraries Initiative of the NIH Roadmap. Our screening process, termed quantitative HTS (qHTS), involves assaying the complete compound library, currently containing > 200,000 members, at a series of dilutions to construct a full concentration—response profile. As such, Compound Management at the NCGC has been uniquely tasked to prepare, store, register, and track a vertically developed plate dilution series (i.e., inter-plate titrations) in the 384-well format. These are compressed into a series of 1536-well plates and are registered to track all subsequent plate storage. Here, we present details on the selection of equipment to enable automated, reliable, and parallel compound manipulation in 384- and 1536-well formats, protocols for preparation of inter-plate dilution series for qHTS, as well as qHTS-specific processes and issues.

Chromo/fluorophoric properties often accompany the heterocyclic scaffolds and impurities that comprise libraries used for high-throughput screening (HTS). These properties affect assay outputs obtained with optical detection, thus complicating analysis and leading to false positives and negatives. Here, we report the fluorescence profile of more than 70,000 samples across spectral regions commonly utilized in HTS. The quantitative HTS paradigm was utilized to test each sample at seven or more concentrations over a 4-log range in 1,536-well format. Raw fluorescence was compared with fluorophore standards to compute a normalized response as a function of concentration and spectral region. More than 5% of library members were brighter than the equivalent of 10 nM 4-methyl umbelliferone, a common UV-active probe. Red-shifting the spectral window by as little as 100 nm was accompanied by a dramatic decrease in autofluorescence. Native compound fluorescence, fluorescent impurities, novel fluorescent compounds, and the utilization of fluorescence profiling data are discussed.

The thyroid-stimulating hormone (TSH; thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of 7-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates the growth and function of thyroid follicular cells. Recombinant TSH is used in diagnostic screens for thyroid cancer, especially in patients after thyroid cancer surgery. Currently, no selective small-molecule agonists of the TSHR are available. To screen for novel TSHR agonists, the authors miniaturized a commercially available cell-based cyclic adenosine 3',5' monophosphate (cAMP) assay into a 1536-well plate format. This assay uses an HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel as a biosensor. From a quantitative high-throughput screen of 73,180 compounds in parallel with a parental cell line (without the TSHR), 276 primary active compounds were identified. The activities of the selected active compounds were further confirmed in an orthogonal homogeneous time-resolved fluorescence cAMP-based assay. Forty-nine compounds in several structural classes have been confirmed as the small-molecule TSHR agonists that will serve as a starting point for chemical optimization and studies of thyroid physiology in health and disease.

A series of substituted 3,6-diphenyl-7H-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazines were prepared and analyzed as inhibitors of phosphodiesterase 4 (PDE4). Synthesis, structure-activity relationships, and the selectivity of a highly potent analogue against related phosphodiesterase isoforms are presented.

A series of 1,3,5-triazine-2,4,6-triamines were prepared and analyzed as inhibitors of glucocerebrosidase. Synthesis, structure activity relationships and the selectivity of chosen analogues against related sugar hydrolases enzymes are described.

Publications reporting results of small-molecule screens are becoming more common as academic researchers increasingly make use of high-throughput screening (HTS) facilities. However, no standards have been formally established for reporting small-molecule screening data, and often key information important for the evaluation and interpretation of results is omitted in published HTS protocols. Here, we propose concise guidelines for reporting small-molecule HTS data.

High-throughput screening (HTS) is the primary technique for new lead identification in drug discovery and chemical biology. Unfortunately, it is susceptible to false-positive hits. One common mechanism for such false-positives is the congregation of organic molecules into colloidal aggregates, which nonspecifically inhibit enzymes. To both evaluate the feasibility of large-scale identification of aggregate-based inhibition and quantify its prevalence among screening hits, we tested 70,563 molecules from the National Institutes of Health Chemical Genomics Center (NCGC) library for detergent-sensitive inhibition. Each molecule was screened in at least seven concentrations, such that dose-response curves were obtained for all molecules in the library. There were 1274 inhibitors identified in total, of which 1204 were unambiguously detergent-sensitive. We identified these as aggregate-based inhibitors. Thirty-one library molecules were independently purchased and retested in secondary low-throughput experiments; 29 of these were confirmed as either aggregators or nonaggregators, as appropriate. Finally, with the dose-response information collected for every compound, we could examine the correlation between aggregate-based inhibition and steep dose-response curves. Three key results emerge from this study: first, detergent-dependent identification of aggregate-based inhibition is feasible on the large scale. Second, 95% of the actives obtained in this screen are aggregate-based inhibitors. Third, aggregate-based inhibition is correlated with steep dose-response curves, although not absolutely. The results of this screen are being released publicly via the PubChem database.

A cell-sensor assay for stabilization of IkappaBalpha was developed in the activated B cell-like diffuse large B-cell lymphoma cell line OCI-Ly3. This cell line expresses known nuclear factor kappaB (NFkappaB) target genes due to high constitutive activity of IkappaB kinase (IKK), which phosphorylates the protein IkappaBalpha leading to proteasomal degradation of IkappaBalpha and activation of NFkappaB. The cell-sensor assay uses green and red light-emitting beetle luciferases, with the green luciferase fused to IkappaBalpha (IkappaBalpha-CBG68) and the red luciferase (CBR) present in its native state. The IkappaBalpha-CBG68 reporter functions as a sensor of IKK and proteasome activity, while CBR serves to normalize for cell number and nonspecific effects. Both reporter constructs were stably integrated and placed under the control of an inducible promoter system, which increased fold responsiveness to inhibitors when assay incubations were performed simultaneous to reporter induction by doxycycline. The assay was miniaturized to a 1,536-well plate format and showed a Z' of 0.6; it was then used to panel 2,677 bioactive compounds by a concentration-response-based screening strategy. The concentration-effect curves for the IkappaBalpha-CBG68 and CBR signals were then used to identify specific stabilizers of IkappaBalpha, such as IKK inhibitors or proteasome inhibitors, which increased the doxycycline-induced rise in IkappaBalpha-CBG68 without affecting the rise in CBR. Known and unexpected inhibitors of NFkappaB signaling were identified from the bioactive collection. We describe here the development and performance of this assay, and discuss the merits of its specific features.

Assays in which the detection of a biological phenomenon is coupled to the production of bioluminescence by luciferase have gained widespread use. As firefly luciferases (FLuc) and kinases share a common substrate (ATP), coupling of a kinase to FLuc allows for the amount of ATP remaining following a kinase reaction to be assessed by quantitating the amount of luminescence produced. Alternatively, the amount of ADP produced by the kinase reaction can be coupled to FLuc through a two-step process. This chapter describes the bioluminescent assays that were developed for three classes of kinases (lipid, protein, and metabolic kinases) and miniaturized to 1536-well format, enabling their use for quantitative high-throughput (qHTS) of small-molecule libraries.

Drug resistance in Plasmodium parasites is a constant threat. Novel therapeutics, especially new drug combinations, must be identified at a faster rate. In response to the urgent need for new antimalarial drug combinations we screened a large collection of approved and investigational drugs, tested 13,910 drug pairs and identified many promising antimalarial drug combinations. The activity of known antimalarial drug regimens was confirmed and a myriad of new classes of positively interacting drug pairings were discovered. Network and clustering analyses reinforced established mechanistic relationships for known drug combinations and identified several novel mechanistic hypotheses. From eleven screens comprising >4,600 combinations per parasite strain (including duplicates) we further investigated interactions between approved antimalarials, calcium homeostasis modulators and inhibitors of phosphatidylinositide 3-kinases (PI3K) and the mammalian target of rapamycin (mTOR). These studies highlight important targets and pathways and provide promising leads for clinically actionable antimalarial therapy.

The number of academic drug discovery centres has grown considerably in recent years, providing new opportunities to couple the curiosity-driven research culture in academia with rigorous preclinical drug discovery practices used in industry. To fully realize the potential of these opportunities, it is important that academic researchers understand the risks inherent in preclinical drug discovery, and that translational research programmes are effectively organized and supported at an institutional level. In this article, we discuss strategies to mitigate risks in several key aspects of preclinical drug discovery at academic drug discovery centres, including organization, target selection, assay design, medicinal chemistry and preclinical pharmacology.

Parkin, an E3 ubiquitin ligase, is a central mediator of mitochondrial quality control and is linked to familial forms of Parkinson's disease (PD). Removal of dysfunctional mitochondria from the cell by Parkin is thought to be neuroprotective, and pharmacologically increasing Parkin levels may be a novel therapeutic approach. We used genome-editing to integrate a coincidence reporter into the PARK2 gene locus of a neuroblastoma-derived cell line and developed a quantitative high-throughput screening (qHTS) assay capable of accurately detecting subtle compound-mediated increases in endogenous PARK2 expression. Interrogation of a chemogenomic library revealed diverse chemical classes that up-regulate the PARK2 transcript, including epigenetic agents, drugs controlling cholesterol biosynthesis, and JNK inhibitors. Use of the coincidence reporter eliminated wasted time pursuing reporter-biased false positives accounting for ∼2/3 of the actives and, coupled with titration-based screening, greatly improves the efficiency of compound selection. This approach represents a strategy to revitalize reporter-gene assays for drug discovery.

Guanosine triphosphate binding protein (G protein)-coupled receptors (GPCRs) are a large class of pharmaceutical drug targets. With the increasing popularity of functional assays for high throughput screening, there arises an increasing need for robust second messenger assays that reflect GPCR activation and are readily amenable for miniaturization. GPCRs that upon agonist stimulation modulate adenylyl cyclase activity, and, consequently, cellular cyclic adenosine monophosphate (cAMP) levels, via the G protein Gs or Gi, form a subset of therapeutic targets. While there are several cAMP assays currently available, most are not scalable for miniaturization into the 1536-well format employed for automated high throughput screening of large chemical libraries. Here, we describe a cAMP assay based on the enzyme fragmentation complementation (EFC) of beta-galactosidase. In this assay, recombinant cells expressing Gs- or Gi-coupled receptors exhibit robust and reproducible pharmacology for agonists and antagonists, as measured by cAMP levels. Furthermore, the EFC cAMP assay offers sufficient sensitivity to be used with cells expressing endogenous GPCRs. We demonstrate the miniaturization of this assay into a 1536-well format with comparable sensitivity and plate statistics to those of the 384-well assay for both Gs- and Gi-coupled receptors, and its suitability for miniaturized high throughput screening.

Estrogen action is mediated via two estrogen receptor (ER) subtypes, ERalpha and ERbeta. Selective ER modulators with balanced high affinity for ERalpha and ERbeta have been developed as therapeutics for the treatment of a variety of diseases, including hormone-responsive breast cancer and osteoporosis. Recent data based primarily on the evaluation of ER-knockout mice have revealed that ERalpha and ERbeta may regulate separate and distinct biological processes. The identification of ERbeta specific ligands could further enhance our understanding of ERbeta biology. In addition, compounds targeting ERbeta may prove useful as therapeutic agents with activity profiles distinguishable from that of estradiol. To discover novel selective ligands for ERbeta, we developed and characterized a cell-based Gal4-ERbeta beta-lactamase reporter gene assay (GERTA) in CHO cells for the ligand-induced activation of the human ERbeta. The sensitivity and selectivity of this assay were found to be comparable to those of an ER ligand-binding assay. The assay was optimized for screening in an ultra high throughput 3456-well nanoplate format and was successfully used to screen a large compound collection for ERbeta agonists. Compounds identified in a primary screen were tested in an in vitro ligand-binding assay to characterize further the selectivity and potency for ERbeta.

An epitope-unmasking, homogeneous time-resolved fluorescence (HTRF) assay has been developed for measuring carboxypeptidase B (CPB) activity in a miniaturized high-throughput screening format. The enzyme substrate (biotin-RYRGLMVGGVVR- oh ) is cleaved by CPB at the C terminus, causing release of the C-terminal Arg residue. The product (biotin-RYRGLMVGGVV- oh ) is recognized specifically by a monoclonal antibody (G2-10) which is labeled with Eu3+-cryptate ([Eu3+]G2-10 mAb), and the complex is detected by fluorescence resonance energy transfer using streptavidin labeled with allophycocyanin ([XL665]SA). The CPB HTRF assay is readily adapted from 96- to 1536-well format as a robust (Z′>0.5) assay for high-throughput screening.

The diversity of physiological functions mediated by the GPCR superfamily provides a rich source of molecular targets for drug discovery programs. Consequently, a variety of assays have been designed to identify lead molecules based on ligand binding or receptor function. In one of these, the binding of [(35)S]GTPgammaS, a nonhydrolyzable analogue of GTP, to receptor-activated G-protein alpha subunits represents a unique functional assay for GPCRs and is well suited for use with automated HTS. Here we compare [(35)S]GTPgammaS scintillation proximity binding assays for two different G(i)-coupled GPCRs, and describe their implementation with automated high-throughput systems.

We have recently described a biochemical detection method for peptide products of enzymatic reactions based on the formation of PDZ domain*peptide ligand complexes. The product sensor is based on using masked or cryptic PDZ domain peptide ligands as enzyme substrates. Upon enzymatic processing, a PDZ-binding motif is exposed, and the product sequence bound specifically by a Eu(3+)chelate-labeled GST-PDZ ([Eu(3+)]GST-PDZ). The practical applicability of this PDZ-based detection method is determined by the affinity of the PDZ domain*peptide ligand interaction, and the efficiency of the enzyme to process the masked peptide ligand. To expand the use of this PDZ-based detection strategy to a broader range of enzymatic assays, we have taken advantage of the plasticity in ligand recognition by the variety of PDZ domains found in nature. In the original work, the PDZ3 of PSD-95 was used, which preferentially recognizes the consensus sequence Ser-X-Val-COOH. Here, we show that NHERF PDZ1, which binds to the consensus sequence Thr/Ser-X-Leu-COOH, can be used to extend the flexibility in the recognition of the carboxy-terminal amino acid of the ligand, and monitor the enzymatic activity of HIV protease. The choices of detection format, for example, TRET or ALPHA, were also investigated and influenced assay design.

A time-resolved fluorescence resonance energy transfer (TR-FRET) detection method based on the formation of a PDZ domain · peptide ligand complex has been developed for enzymatic assays as an alternative to immuno-based detection strategies. The enzyme substrate is a “masked” biotinylated PDZ domain peptide ligand containing the consensus sequence Ser–X–Val-COOH. The critical residues in the binding consensus sequence of the ligand have been modified, for example, by phosphorylation of Ser or C-terminal extensions, providing binding-incompetent PDZ domain peptides. On processing by the corresponding enzyme, the binding epitope is exposed, and the product sequence is recognized specifically by Eu 3+ chelate-labeled GST–PDZ ([Eu 3+ ]GST–PDZ) (GST–PDZ-glutathione S -transferase fused to PDZ domain). A ternary complex is subsequently formed by addition of allophycocyanin-labeled streptavidin ([XL665]SA), which binds to the biotinylated N terminus of the peptide, and detected by TR-FRET. Reported here are examples of the applicability of this detection strategy to three enzymatic systems, an endoprotease, an exoprotease, and a Ser/Thr phosphatase.

The structure and biological properties of RNAs are a function of changing cellular conditions, but comprehensive, simultaneous investigation of the effect of multiple interacting environmental variables is not easily achieved. We have developed an efficient, high-throughput method to characterize RNA structure and thermodynamic stability as a function of multiplexed solution conditions using Förster resonance energy transfer (FRET). In a single FRET experiment using conventional quantitative PCR instrumentation, 19,400 conditions of MgCl2, ligand and temperature are analysed to generate detailed empirical conformational and stability landscapes of the cyclic diguanylate (c-di-GMP) riboswitch. The method allows rapid comparison of RNA structure modulation by cognate and non-cognate ligands. Landscape analysis reveals that kanamycin B stabilizes a non-native, idiosyncratic conformation of the riboswitch that inhibits c-di-GMP binding. This demonstrates that allosteric control of folding, rather than direct competition with cognate effectors, is a viable approach for pharmacologically targeting riboswitches and other structured RNA molecules., The structure and biological properties of RNAs are a function of changing cellular conditions. Here, Baird et al. report a high-throughput Förster resonance energy transfer (FRET) method to rapidly compare RNA structure modulation by cognate and non-cognate ligands across multiplexed solution conditions.

Retinal degenerations are a heterogeneous group of diseases in which there is slow but progressive loss of photoreceptors (PR). There are currently no approved therapies for treating retinal degenerations. In an effort to identify novel small molecules that are (1) neuroprotective and (2) promote PR differentiation, we have developed microscale (1,536 well) cell culture assays using primary retinal neurons.Primary murine retinal cells are isolated, seeded, treated with a 1,280 compound chemical library in a 7 point titration and then cultured under conditions developed to assay protection against an introduced stress or enhance PR differentiation. In the protection assays a chemical insult is introduced and viability assessed after 72 h using CellTiterGlo, a single-step chemiluminescent reagent. In the differentiation assay, cells are isolated from the rhodopsin-GFP knock-in mouse and PR differentiation is assessed by fixing cells after 21 days in culture and imaging with the Acumen plate-based laser cytometer (TTP Labtech) to determine number and intensity of GFP-expressing cells. Positive wells are re-imaged at higher resolution with an INCell2000 automated microscope (GE). Concentration-response curves are generated to pharmacologically profile each compound and hits identified by xx.We have developed PR differentiation and neuroprotection assays with a signal to background (S/B) ratios of 11 and 3, and a coefficient of variation (CV) of 20 and 9 %, suitable for chemical screening. Staurosporine has been shown in our differentiation assay to simultaneously increase the number of rhodopsin positive objects while decreasing the mean rhodopsin intensity and punctate rhodopsin fluorescent objects.Using primary murine retinal cells, we developed high throughput assays to identify small molecules that influence PR development and survival. By screening multiple compound concentrations, dose-response curves can be generated, and the false negative rate minimized. It is hoped that this work will identify both potential preclinical candidates as well as molecular probes that will be useful for analysis of the molecular mechanisms that promote PR differentiation and survival.

The G protein-coupled receptor kinase GRK6 undergoes posttranslational modification by palmitoylation. Palmitoylated GRK6 is associated with the membrane, while nonpalmitoylated GRK6 remains cytosolic. We have separated palmitoylated from nonpalmitoylated GRK6 to assess their relative kinase activity. Palmitoylated GRK6 is 10-fold more active at phosphorylating beta2-adrenergic receptor than nonpalmitoylated wild-type GRK6 or a nonpalmitoylatable mutant GRK6. A nonpalmitoylatable mutant GRK6 which has been further mutated to undergo posttranslational geranylgeranylation is also more active, recovering most of the activity of the palmitoylated enzyme. This activity increase by lipid modification is expected, as the lipid helps GRK6 localize to cellular membranes where its receptor substrates are found. However, when assayed using a soluble protein (casein) as a substrate, both palmitoylated and prenylated GRK6 display significantly higher activity than nonpalmitoylated wild-type or nonpalmitoylatable mutant GRK6 kinases. This increased activity is not altered by addition of exogenous palmitate or phosphatidycholine vesicles, arguing that it is not due to direct activation of GRK6 by binding palmitate, nor to nonspecific association of the GRK6 with casein. Further, chemical depalmitoylation reduces the casein phosphorylation activity of the palmitoylated, but not prenylated, GRK6 kinase. Thus, palmitoylation of GRK6 appears to play a dual role in increasing the activity of GRK6: it increases the hydrophobicity and membrane association of the GRK6 protein, which helps bring the GRK6 to its membrane-bound substrates, and it increases the kinase catalytic activity of GRK6.

Phosducin, which tightly binds betagamma-subunits of heterotrimeric G-proteins, has been conjectured to play a role in regulating second messenger signaling cascades, but to date its specific function has not been elucidated. Here we demonstrate a potential role for phosducin in regulating olfactory signal transduction. In isolated olfactory cilia certain odorants elicit a rapid and transient cAMP response, terminated by a concerted process which requires the action of two protein kinases, protein kinase A (PKA) and a receptor-specific kinase (GRK3) (Schleicher, S., Boekhoff, I. Arriza, J., Lefkowitz, R. J., and Breer, H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 1420-1424). The mechanism of action of GRK3 involves a Gbetagamma-mediated translocation of the kinase to the plasma membrane bound receptors (Pitcher, J. A., Inglese, J., Higgins, J. B. , Arriza, J. L., Casey, P. J., Kim, C., Benovic, J. L., Kwatra, M. M. , Caron, M. G., and Lefkowitz, R. J. (1992) Science 257, 1264-1267). A protein with a molecular mass of 33 kDa that comigrates on SDS gels with recombinant phosducin and which is immunoreactive with phosducin antibodies is present in olfactory cilia. Recombinant phosducin added to permeabilized olfactory cilia preparations strongly inhibits termination of odorant-induced cAMP response and odorant-induced membrane translocation of GRK3. In addition, the cAMP analogue dibutyryl cAMP stimulates membrane targeting of the receptor kinase. This effect is presumably due to PKA-mediated phosphorylation of phosducin, which diminishes its affinity for binding to the Gbetagamma-subunit, thereby making Gbetagamma available to function as a membrane anchor for GRK3. A specific PKA inhibitor blocks the odorant-induced translocation of the receptor kinase. Consistent with this formulation, a non-phosphorylatable mutant of phosducin (phosducin Ser-73 --Ala) is an even more effective inhibitor of desensitization and membrane targeting of GRK3 than the wild-type protein. A phosducin mutant that mimics phosphorylated phosducin (phosducin Ser-73 --Asp) lacks this property and in fact recruits GRK3 to the membrane and potentiates desensitization. These results suggest that phosducin may act as a phosphorylation-dependent switch in second messenger signaling cascades, regulating the kinetics of desensitization processes by controlling the activity of Gbetagamma-dependent GRKs.

With the development of new technologies in genome sequencing, gene expression profiling, genotyping, and high-throughput screening of chemical compound libraries, small molecules are playing increasingly important roles in studying gene expression regulation, gene–gene interaction, and gene function. Here we briefly review and discuss some recent advancements in drug target identification and phenotype characterization using combinations of high-throughput screening of small-molecule libraries and various genome-wide methods such as whole-genome sequencing, genome-wide association studies (GWAS), and genome-wide expression analysis. These approaches can be used to search for new drugs against parasite infections, to identify drug targets or drug resistance genes, and to infer gene function.

Glaucoma, a major cause of blindness worldwide, is a neurodegenerative optic neuropathy in which vision loss is caused by loss of retinal ganglion cells (RGCs). To better define the pathways mediating RGC death and identify targets for the development of neuroprotective drugs, we developed a high-throughput RNA interference screen with primary RGCs and used it to screen the full mouse kinome. The screen identified dual leucine zipper kinase (DLK) as a key neuroprotective target in RGCs. In cultured RGCs, DLK signaling is both necessary and sufficient for cell death. DLK undergoes robust posttranscriptional up-regulation in response to axonal injury in vitro and in vivo. Using a conditional knockout approach, we confirmed that DLK is required for RGC JNK activation and cell death in a rodent model of optic neuropathy. In addition, tozasertib, a small molecule protein kinase inhibitor with activity against DLK, protects RGCs from cell death in rodent glaucoma and traumatic optic neuropathy models. Together, our results establish a previously undescribed drug/drug target combination in glaucoma, identify an early marker of RGC injury, and provide a starting point for the development of more specific neuroprotective DLK inhibitors for the treatment of glaucoma, nonglaucomatous forms of optic neuropathy, and perhaps other CNS neurodegenerations.

A novel human G protein-coupled receptor kinase was recently identified by positional cloning in the search for the Huntington's disease locus (Ambrose, C., James, M., Barnes, G., Lin, C., Bates, G., Altherr, M., Duyao, M., Groot, N., Church, D., Wasmuth, J. J., Lehrach, H., Housman, D., Buckler, A., Gusella, J. F., and MacDonald, M. E. (1993) Hum. Mol. Genet. 1, 697-703). Comparison of the deduced amino acid sequence of GRK4 with those of the closely related GRK5 and GRK6 suggested the apparent loss of 32 codons in the amino-terminal domain and 46 codons in the carboxyl-terminal domain of GRK4. These two regions undergo alternative splicing in the GRK4 mRNA, resulting from the presence or absence of exons filling one or both of these apparent gaps. Each inserted sequence maintains the open reading frame, and the deduced amino acid sequences are similar to corresponding regions of GRK5 and GRK6. Thus, the GRK4 mRNA and the GRK4 protein can exist as four distinct variant forms. The human GRK4 gene is composed of 16 exons extending over 75 kilobase pairs of DNA. The two alternatively spliced exons correspond to exons II and XV. The genomic organization of the GRK4 gene is completely distinct from that of the human GRK2 gene, highlighting the evolutionary distance since the divergence of these two genes. Human GRK4 mRNA is expressed highly only in testis, and both alternative exons are abundant in testis mRNA. The four GRK4 proteins have been expressed, and all incorporate [3H]palmitate. GRK4 is capable of augmenting the desensitization of the rat luteinizing hormone/chorionic gonadotropin receptor upon coexpression in HEK293 cells and of phosphorylating the agonist-occupied, purified beta2-adrenergic receptor, indicating that GRK4 is a functional protein kinase.

Parasitic diseases continue to have a devastating impact on human populations worldwide. Lack of effective treatments, the high cost of existing ones, and frequent emergence of resistance to these agents provide a strong argument for the development of novel therapies. Here we report the results of a hybrid approach designed to obtain a dual acting molecule that would demonstrate activity against a variety of parasitic targets. The antimalarial drug amodiaquine has been covalently joined with a nitric oxide-releasing furoxan to achieve multiple mechanisms of action. Using in vitro and ex vivo assays, the hybrid molecule shows activity against three parasites – Plasmodium falciparum, Schistosoma mansoni, and Ancylostoma ceylanicum.

A library of 367 protein kinase inhibitors, the GSK Published Kinase Inhibitor Set (PKIS), which has been annotated for protein kinase family activity and is available for public screening efforts, was assayed against the commonly used luciferase reporter enzymes from the firefly, Photinus pyralis (FLuc) and marine sea pansy, Renilla reniformis (RLuc). A total of 22 compounds (∼6% of the library) were found to inhibit FLuc with 10 compounds showing potencies ≤1 µM. Only two compounds were found to inhibit RLuc, and these showed relatively weak potency values (∼10 µM). An inhibitor series of the VEGFR2/TIE2 protein kinase family containing either an aryl oxazole or benzimidazole-urea core illustrate the different structure activity relationship profiles FLuc inhibitors can display for kinase inhibitor chemotypes. Several FLuc inhibitors were broadly active toward the tyrosine kinase and CDK families. These data should aid in interpreting the results derived from screens employing the GSK PKIS in cell-based assays using the FLuc reporter. The study also underscores the general need for strategies such as the use of orthogonal reporters to identify kinase or non-kinase mediated cellular responses.