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ACS Chemical Biology	A Specific Mechanism for Nonspecific Activation in Reporter-Gene Assays. Auld DS, Thorne N, Nguyen DT, Inglese J. 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.
Journal of Biomolecular Screening	A Cell-Based PDE4 Assay in 1536-Well Plate Format for High-Throughput Screening. Titus SA, Xiao L, Southall N, Lu J, Inglese J, Brasch M, Austin CP, Zheng W. The cyclic nucleotide phosphodiesterases (PDEs) are intracellular enzymes that catalyze the hydrolysis of 3,'5'-cyclic nucleotides, such as cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), to their corresponding 5'nucleotide monophosphates. These enzymes play an important role in controlling cellular concentrations of cyclic nucleotides and thus regulate a variety of cellular signaling events. PDEs are emerging as drug targets for several diseases, including asthma, cardiovascular disease, attention-deficit hyperactivity disorder, Parkinson's disease, and Alzheimer's disease. Although biochemical assays with purified recombinant PDE enzymes and cAMP or cGMP substrate are commonly used for compound screening, cell-based assays would provide a better assessment of compound activity in a more physiological context. The authors report the development and validation of a new cell-based PDE4 assay using a constitutively active G-protein-coupled receptor as a driving force for cAMP production and a cyclic nucleotide-gated cation channel as a biosensor in 1536-well plates.
Nature Medicine	Identification of oxadiazoles as new drug leads for the control of schistosomiasis Sayed AA, Simeonov A, Thomas CJ, Inglese J, Austin CP, Williams DL. Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease, and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principal components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to glutathione via a TGR-catalyzed reaction and then to hydrogen peroxide via a Prx-catalyzed step. A fully automated quantitative high-throughput (qHTS) experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 microL reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC(50)s ranging from micromolar to the assay response limit ( approximately 25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease, and provides a paradigm that can be used to jump-start development of novel therapeutics for other neglected tropical diseases.
Toxicology in Vitro	A bioluminescent cytotoxicity assay for assessment of membrane integrity using a proteolytic biomarker. Cho MH, Niles A, Huang R, Inglese J, Austin CP, Riss T, Xia M. 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.
Proceedings of the National Academy of Sciences	Three classes of glucocerebrosidase inhibitors identified by quantitative high-throughput screening are chaperone leads for Gaucher disease Zheng W, Padia J, Urban D, Jadhav A, Simeonov A, Goldin E, Auld DS, LaMarca ME, Inglese J, Austin CP, Sidransky E. Gaucher disease is an autosomal recessive lysosomal storage disorder caused by mutations in the glucocerebrosidase gene. Missense mutations result in reduced enzyme activity that may be due to misfolding, raising the possibility of small-molecule chaperone correction of the defect. Screening large compound libraries by quantitative high-throughput screening (qHTS) provides comprehensive information on the potency, efficacy, and structure-activity relationships (SAR) of active compounds directly from the primary screen, facilitating identification of leads for medicinal chemistry optimization. We used qHTS to rapidly identify three structural series of potent, selective, nonsugar glucocerebrosidase inhibitors. The three structural classes had excellent potencies and efficacies and, importantly, high selectivity against closely related hydrolases. Preliminary SAR data were used to select compounds with high activity in both enzyme and cell-based assays. Compounds from two of these structural series increased N370S mutant glucocerebrosidase activity by 40-90% in patient cell lines and enhanced lysosomal colocalization, indicating chaperone activity. These small molecules have potential as leads for chaperone therapy for Gaucher disease, and this paradigm promises to accelerate the development of leads for other rare genetic disorders.
PLoS Neglected Tropical Diseases	*Quantitative High-Throughput Screen Identifies Inhibitors of the Schistosoma mansoni* Redox Cascade. Simeonov S, Jadhav A, Sayed AA, Wang Y, Nelson ME, Inglese J, Williams DL, Austin CP.** Schistosomiasis is a tropical disease associated with high morbidity and mortality, currently affecting over 200 million people worldwide. Praziquantel is the only drug used to treat the disease and with its increased use the probability of developing drug resistance has grown significantly. The Schistosoma parasites can survive for up to decades in the human host due in part to a unique set of antioxidant enzymes that continuously degrade the reactive oxygen species produced by the host's innate immune response. Two principle components of this defense system have been recently identified in S. mansoni as thioredoxin/glutathione reductase (TGR) and peroxiredoxin (Prx) and as such these enzymes present attractive new targets for anti-schistosomiasis drug development. Inhibition of TGR/Prx activity was screened in a dual-enzyme format with reducing equivalents being transferred from NADPH to a glutathione intermediate via a TGR-catalyzed reaction and then to hydrogen peroxide via Prx-catalyzed step. A fully-automated qHTS experiment (Inglese et al, PNAS, 103, 1147 (2006)) was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 5 ?L reaction volume in 1536-well plate format. In order to generate a robust data set and to minimize the effect of compound autofluorescence, apparent reaction rates derived from a kinetic read were utilized instead of end-point measurements. Actives identified from the screen, along with previously-untested analogues, were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in secondary assays. Several novel active series were identified which inhibited TGR at a range of potencies, with IC50s ranging from micromolar to the assay response limit (~25 nM). This is, to our knowledge, the first report of a large-scale HTS to identify lead compounds for a helminthic disease.
Proceedings of the National Academy of Sciences	Selective targeting of ITK blocks multiple steps of HIV replication Readinger JA, Schirali GM, Jiang JK, Thomas CJ, August A, Henderson AJ, Schwartzberg PL. Treatment for HIV has relied on the use of antiretroviral agents that can be subject to the development of resistant viruses. The study of inhibitors directed against cellular proteins required for HIV replication is therefore of growing interest. Inducible T cell kinase (ITK) is a Tec family tyrosine kinase that regulates T cell receptor (TCR)-induced activation of PLC-1, Ca2 mobilization and transcription factor activation, and actin rearrangement downstream of both TCR and chemokine receptors. Because productive infection of T cells with HIV requires T cell activation, chemokine receptors and actin reorganization, we asked whether ITK affects HIV infection using ITK-specific siRNA, a kinase-inactive ITK mutant or an ITK inhibitor. We demonstrate that loss of ITK function resulted in marked reductions in intracellular p24 levels upon HIV infection. Loss of ITK function after establishment of HIV infection also decreased virus spread within the culture. Inhibition of ITK did not affect expression of the HIV coreceptors CD4 or CXCR4 but partially blocked HIV viral entry, an effect that correlated with decreased actin polarization to gp120. Additionally, ITK was required for efficient HIV transcription, and overexpression of ITK increased both viral transcription and virus-like particle formation. Our data suggest that inhibition of ITK blocks HIV infection by affecting multiple steps of HIV replication.
Journal of Medicinal Chemistry	Characterization of Chemical Libraries for Luciferase Inhibitory Activity. Auld DS, Southall N, Jadhav A, Johnson RL, Diller D, Simeonov S, Austin CP, Inglese J. To aid in the interpretation of HTS results derived from luciferase-based assays we used quantitative HTS (qHTS), an approach that defines the concentration-response behavior of each library sample, to profile the ATP-dependent luciferase from Photinus pyralis against >70,000 samples. We found approximately 3% of the library was active, containing only compounds with inhibitory concentration-responses of which 681 (0.9%) exhibited IC50s < 10 uM. Representative compounds were shown to inhibit purified P. pyralis as well as several commercial luciferase-based detection reagents but were found to be largely inactive against Renilla reniformis luciferase. Light attenuation by the samples was also examined and found to be more prominent in the blue-shifted bioluminescence produced by R. reniformas luciferase than with bioluminescence produced by P. pyralis luciferase. We describe the SAR of the luciferase inhibitors and discuss the use of this data in the interpretation of HTS results, and configuration of luciferase-based assays.
Journal of Medicinal Chemistry	Fluorescence Spectroscopic Profiling of Compound Libraries. Simeonov S, Jadhav A, Thomas CJ, Wang Y, Huang R, Southall N, Shinn P, Smith J, Austin CP, Inglese J. Chromo/fluorophoric properties often accompany the conjugated, aromatic and heterocyclic features of many of the scaffolds and impurities that make up library samples used for high throughput screening (HTS). These properties impart highly variable effects on assay outputs employing optical detection, thus complicating the interpretation of data and leading to false positives and negatives. Here, we report the comprehensive fluorescence profile of >70,000 samples across multiple spectral regions commonly utilized in HTS assays. The quantitative HTS (qHTS) paradigm was utilized to test each sample at seven or more concentration points over a 4-log concentration range in 1536-well format, with raw fluorescence response collected using a CCD-based imager. The resulting output was compared with fluorophore standards to compute a normalized fluorescence response (termed fluorophore-equivalent concentration, FEC) for each sample, concentration, and relevant spectral region. The greatest fraction of fluorescent compounds appeared in the UV-end of the light spectrum, where over 5% of library members matched or exceeded 10 nM FEC of 4-methylumbelliferone and AlexaFluor 350, while approximately 1.8% of the library matched or exceeded 100 nM FEC of these standards. Red-shifting the spectral window by as little as 100 nm was accompanied by a dramatic decrease in autofluorescence. Native compound fluorescence, scaffold overlap with known fluorophores, fluorescent impurities, novel fluorescent compounds, and the ability to discriminate generalities of fluorescent interferences and devise strategies to identify them are discussed.
Journal of the American Chemical Society	A virtual screen for diverse ligands: discovery of selective G protein-coupled receptor antagonists. Engel S, Skoumbourdis AP, Childress J, Neumann S, Deschamps JR, Thomas CJ, Colson AO, Costanzi S, Gershengorn MC. Virtual screening has become a major focus of bioactive small molecule lead identification, and reports of agonists and antagonists discovered via virtual methods are becoming more frequent. G protein-coupled receptors (GPCRs) are the one class of protein targets for which success with this approach has been limited. This is likely due to the paucity of detailed experimental information describing GPCR structure and the intrinsic function-associated structural flexibility of GPCRs which present major challenges in the application of receptor-based virtual screening. Here we describe an in silico methodology that diminishes the effects of structural uncertainty, allowing for more inclusive representation of a potential docking interaction with exogenous ligands. Using this approach, we screened one million compounds from a virtual database, and a diverse subgroup of 100 compounds was selected, leading to experimental identification of five structurally diverse antagonists of the thyrotropin-releasing hormone receptors (TRH-R1 and TRH-R2). The chirality of the most potent chemotype was demonstrated to be important in its binding affinity to TRH receptors; the most potent stereoisomer was noted to have a 13-fold selectivity for TRH-R1 over TRH-R2. A comprehensive mutational analysis of key amino acid residues that form the putative binding pocket of TRH receptors further verified the binding modality of these small molecule antagonists. The described virtual screening approach may prove applicable in the search for novel small molecule agonists and antagonists of other GPCRs.
Bioorganic & Medicinal Chemistry Letters	Identification of a potent new chemotype for the selective inhibition of PDE4. Skoumbourdis AP, Huang R, Southall N, Leister W, Guo V, Cho MH, Inglese J, Nirenberg M, Austin CP, Xia M, Thomas CJ. 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.
Chemical Research Toxicology	*Characterization of Diversity in Toxicity Mechanism Using In Vitro* Cytotoxicity Assays in Quantitative High Throughput Screening. Huang R, Southall N, Cho MH, Xia M, Inglese J, Austin CP.** Assessing the potential health risks of environmental chemical compounds is an expensive undertaking which has motivated the development of new alternatives to traditional in vivo toxicological testing. One approach is to stage the evaluation, beginning with less expensive and higher throughput in vitro testing before progressing to more definitive trials. In vitro testing can be used to generate a hypothesis about a compound's mechanism of action, which can then be used to design an appropriate in vivo experiment. Here we begin to address the question of how to design such a battery of in vitro cell-based assays by combining data from two different types of assays, cell viability and caspase activation, with the aim of elucidating mechanism of action. Because caspase activation is a transient event during apoptosis, it is not possible to design a single end-point assay protocol that would identify all instances of compound-induced caspase activation. Nevertheless, useful information about compound mechanism of action can be obtained from these assays in combination with cell viability data. Unsupervised clustering in combination with Dunn's cluster validity index is a robust method for identifying mechanisms of action without requiring any a priori knowledge about mechanisms of toxicity. The performance of this clustering method is evaluated by comparing the clustering results against literature annotations of compound mechanisms.
Combinatorial Chemistry & High Throughput Screening	A High Throughput Fluorescence Polarization Assay for Inhibitors of the GoLoco Motif/G-alpha Interaction. Kimple AJ, Yasgar A, Hughes M, Jadhav A, Willard FS, Muller RE, Austin CP, Inglese J, Ibeanu GC, Siderovski DP, Simeonov A. 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.
Analytical Biochemistry	Dual-fluorophore quantitative high-throughput screen for inhibitors of BRCT-phosphoprotein interaction. Simeonov A, Yasgar A, Jadhav A, Lokesh GL, Klumpp C, Michael S, Inglese J, Austin CP, Natarajan A. Finding specific small-molecule inhibitors of protein-protein interactions remains a significant challenge. Recently, attention has grown toward "hot spot" interactions where binding is dominated by a limited number of amino acid contacts, theoretically offering an increased opportunity for disruption by small molecules. Inhibitors of the interaction between BRCT (the C-terminal portion of BRCA1, a key tumor suppressor protein with various functions) and phosphorylated proteins (Abraxas/BACH1/CtIP), implicated in DNA damage response and repair pathways, should prove to be useful in studying BRCA1's role in cancer and in potentially sensitizing tumors to chemotherapeutic agents. We developed and miniaturized to a 1536-well format and 3ul final volume a pair of fluorescence polarization (FP) assays using fluorescein- and rhodamine-labeled pBACH1 fragment. To minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members all were assayed against both the fluorescein- and rhodamine-labeled probe-protein complexes in separate but interleaved reactions. In addition, every library compound was tested over a range of concentrations following the quantitative high-throughput screening (qHTS) paradigm. Analyses of the screening results led to the selection and subsequent confirmation of 16 compounds active in both assays. Faced with a traditionally difficult protein-protein interaction assay, by performing two-fluorophore qHTS, we were able to confidently select a number of actives for further studies.
Analytical Biochemistry	A Quantitative High-Throughput Screen Identifies Potential Epigenetic Modulators of Gene Expression. Johnson RL, Huang W, Jadhav A, Austin CP, Inglese J, Martinez ED. Epigenetic regulation of gene expression is essential in embryonic development and contributes to cancer pathology. We used a cell-based imaging assay that measures derepression of a silenced GFP reporter to identify novel classes of compounds involved in epigenetic regulation. This Locus Derepression (LDR) assay was screened against a 69,137-member chemical library using quantitative high-throughput screening (qHTS), a titration-response method that assays compounds at multiple concentrations. From structure-activity relationships of the 411 actives recovered from the qHTS, six distinct chemical series were chosen for further study. Forty-eight qHTS actives and analogs were counter screened using the parental line of the LDR cells, which lack the GFP reporter. Three series, 8-hydroxy quinoline, quinoline-8-thiol and 1,3,5- thiadiazinane-2-thione, were not fluorescent and re-confirmed activity in the LDR cells. The three active series did not inhibit histone deacetylase activity in nuclear extracts or reactivate the expression of the densely methylated p16 gene in cancer cells. However, one series induced expression of the methylated CDH13 gene and inhibited the viability of several lung cancer lines at submicromolar concentrations. These results suggest that the identified small molecules act on epigenetic or transcriptional components and validate our approach of using a cell-based imaging assay in conjunction with qHTS.
Journal of Medicinal Chemistry	A Comprehensive Mechanistic Analysis of Hits from High-Throughput and Docking Screens Against Beta-Lactamase. Babaoglu K, Simeonov A, Irwin, J, Nelson M, Feng BY, Thomas C, Cancian L, Costi MP, Maltby D, Jadhav A, Inglese J, Austin CP, Shoichet BK. High-throughput screening (HTS) is widely used in drug discovery. Especially for screens of unbiased libraries, false positives can dominate "hit lists"; their origins are much debated. Here we determine the mechanism of every active hit from a screen of 70,563 unbiased molecules against beta-lactamase using quantitative HTS (qHTS). Of the 1,274 initial inhibitors, 95% were detergent-sensitive and were classified as aggregators. Among the 70 remaining were 25 potent, covalent-acting beta-lactams. Mass spectra, counter-screens, and crystallography identified 12 as promiscuous covalent inhibitors. The remaining 33 were either aggregators or irreproducible. No specific reversible inhibitors were found. We turned to molecular docking to prioritize molecules from the same library for testing at higher concentrations. Of 16 tested, 2 were modest inhibitors. Subsequent X-ray structures corresponded to the docking prediction. Analog synthesis improved affinity to 8 microM. These results suggest that it may be the physical behavior of organic molecules, not their reactivity, that accounts for most screening artifacts. Structure-based methods may prioritize weak-but-novel chemotypes in unbiased library screens.
Journal of Biomolecular Screening	Quantitative High Throughput Screening Using a Live Cell cAMP Assay Identifies Small Molecule Agonists of the TSH Receptor. Titus S, Neumann S, Zheng W, Southall N, Michael S, Klumpp C, Shinn P, Thomas CJ, Inglese J, Gershengorn MC, Austin CP. The thyroid stimulating hormone (TSH, thyrotropin) receptor belongs to the glycoprotein hormone receptor subfamily of seven-transmembrane spanning receptors. TSH receptor (TSHR) is expressed mainly in thyroid follicular cells and is activated by TSH, which regulates 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 TSH receptor are available. To screen for novel TSH receptor agonists, we miniaturized a cell-based cAMP assay into 1536-well plate format. This assay uses a HEK293 cell line stably transfected with the TSHR coupled to a cyclic nucleotide gated ion channel (CNG) 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 HTRF cAMP-based assay. 49 compounds in several structural classes have been confirmed as the small molecule TSHR agonists that will serve as starting point for chemical optimization and studies of thyroid physiology in health and disease.
Journal of the Association for Laboratory Automation	Compound Management for Quantitative High-Throughput Screening. Yasgar A, Shinn P, Jadhav A, Auld DS, Michael S, Zheng W, Austin CP, Inglese J, Simeonov A. 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 which 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 1,536- 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 1,536-well formats, protocols for preparation of inter-plate dilution series for qHTS, as well as qHTS-specific processes and issues.
ASSAY and Drug Development Technologies	Evaluation of Micro-Parallel Liquid Chromatography (uPLC) as a Method for HTS-coupled Actives Verification. Simeonov A, Yasgar A, Klumpp C, Zheng W, Shafqat N, Oppermann U, Austin CP, Inglese J. The identification of biologically active compounds from HTS can involve considerable post-screening analysis to verify the nature of the sample activity. In this study we evaluated the performance of Micro Parallel Liquid Chromatography (uPLC) as a separation-based enzyme assay platform for follow-up of compound activities found in qHTS of two different targets, a hydrolase and an oxidoreductase. In an effort to couple secondary analysis to primary screening we explored the application of uPLC immediately after a primary screen. In a proof-of-concept experiment for screen-coupled actives verification, we identified, selected and consolidated the contents of "active" wells from a 1536-well format HTS experiment into a 384-well plate, and subsequently analyzed these samples by a 24-channel uPLC system. The method utilized 0.6% of the original 6 uL 1536-well assay for the analysis. The analysis revealed several nonbiological based "positive" samples. The main examples included "false" enzyme activators resulting from an increase in well-fluorescence due to fluorescent compound or impurity. The uPLC analysis also provided a verification of the activity of two activators of glucocerebrosidase. We discuss the benefits of uPLC and its limitations from the standpoint of ease of use and integration into a seamless post-screen workflow.
Combinatorial Chemistry & High Throughput Screening	A Miniaturized Glucocorticoid Receptor Translocation Assay using Enzymatic Fragment Complementation Evaluated with qHTS. Zhu PJ, Zheng W, Auld DS, Jadhav A, MacArthur R, Olson KR, Peng K, Dotimas H, Austin CP, Inglese J. 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 uM 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 3? or 6? 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.
Environmental Health Perspectives	Compound Cytotoxicity Profiling Using Quantitative High-Throughput Screening. Xia M, Huang R, Witt KL, Southall N, Fostel J, Cho MH, Jadhav A, Smith CS, Inglese J, Portier CJ, Tice RR, Austin CP. Background: The propensity of compounds to produce adverse health effects in humans is generally evaluated using animal-based test methods. Such methods can be relatively expensive, low-throughput, and associated with pain suffered by the treated animals. In addition, differences in species biology may confound extrapolation to human health effects. Objective: The U.S. National Toxicology Program and the NIH Chemical Genomics Center are collaborating to identify a battery of cell-based screens to prioritize compounds for further toxicological evaluation. Methods: 1,408 compounds previously tested in one or more traditional toxicological assays were profiled for cytotoxicity using quantitative high-throughput screening (qHTS) in 13 human and rodent cell types derived from six common targets of xenobiotic toxicity (liver, blood, kidney, nerve, lung, skin). Selected cytotoxicants were further tested to define response kinetics. Results: qHTS of these compounds produced robust and reproducible results which allowed cross-compound, cross-cell type, and cross-species comparisons. Some compounds were cytotoxic to all cell types at similar concentrations, while others exhibited species- or cell typespecific cytotoxicity. Closely related cell types and analogous cell types in human and rodent frequently showed different patterns of cytotoxicity. Some compounds inducing similar levels of cytotoxicity showed distinct time-dependence in kinetic studies, consistent with known mechanisms of toxicity. Conclusions: The generation of high-quality cytotoxicity data on this large library of known compounds using qHTS demonstrates the potential of this methodology to profile a much broader array of assays and compounds, which, in aggregate, may be valuable for prioritizing compounds for further toxicological evaluation, identifying compounds with particular mechanisms of action, and potentially predicting in vivo biological response
Journal of the American Chemical Society	Distinctive Inhibition of O-GlcNAcase Isoforms by an alpha-GlcNAc Thiolsulfonate. Kim, E. J.; Amorelli, B.; Abdo, M.; Thomas, C. J.; Love, D. C.; Knapp, S.; Hanover, J. A. O-GlcNAcase (OGA) promotes O-GlcNAc removal, and thereby plays a key role in O-GlcNAc metabolism, a feature of a variety of vital cellular processes. Two splice transcripts of human OGA encode "long OGA", which contains a distinct N-terminal O-GlcNAcase domain and a C-terminal histoneacetylferase (HAT) domain, and "short OGA", which lacks the HAT domain. The functional roles of long OGA are only beginning to be unraveled, and the characteristics of short OGA remain almost unknown. We find that short OGA, which possesses O-GlcNAcase catalysis machinery like that of long OGA, exhibits comparative resistance to previously described potent inhibitors of long OGA and lysosomal hexosaminidases, including PUGNAc and NAG-thiazoline, suggesting a role for the HAT domain in O-GlcNAcase catalysis. We also find that -GlcNAc thiolsulfonate (2) is the most potent inhibitor of short OGA yet described (Ki = 10 M), and exhibits some degree of selectivity versus long OGA and lysosomal hexosaminidases. In contrast to its mode of inhibition of short OGA, 2 acts as a irreversible inhibitor of long OGA by covalently modifying the enzyme as an S-GlcNAc derivative. Covalent attachment of GlcNAc to the HAT domain of long OGA dramatically changes its properties with respect to enzymatic activity and caspase-3 cleavage.
Tetrahedron Letters	Synthesis of substituted 2-phenylhistamines via a microwave promoted Suzuki coupling. Skoumbourdis, A. P., Moore, S., Landsman, M., Thomas, C. J. Substitutions on the 2-position of the imidazole ring of histamine have proven useful in a number of biochemical settings. Current art for the synthesis of these constructs relies upon a cumbersome and low-yielding condensation reaction. Here-in we report a new procedure for the synthesis of a series of substituted 2-phenylhistamines utilizing a microwave-promoted Suzuki coupling.
Bioorganic and Medicinal Chemistry Letters	Identification of N-(quinolin-8-yl)benzenesulfonamides as agents capable of down-regulating NFkappaB activity within two separate high-throughput screens of NFkappaB activation. Xie Y, Deng S, Thomas CJ, Liu Y, Zhang YQ, Rinderspacher A, Huang W, Gong G, Wyler M, Cayanis E, Aulner N, T�bben U, Chung C, Pampou S, Southall N, Vidovic D, Sch�rer S, Branden L, Davis RE, Staudt LM, Inglese J, Austin CP, Landry DW, Smith DH, Auld DS. We describe here a series of N-(quinolin-8-yl)benzenesulfonamides capable of suppressing the NFkappaB pathway identified from two high-throughput screens run at two centers of the NIH Molecular Libraries Initiative. These small molecules were confirmed in both primary and secondary assays of NFkappaB activation and expanded upon through analogue synthesis. The series exhibited potencies in the cell-based assays at as low as 0.6muM, and several indications suggest that the targeted activity lies within a common region of the NFkappaB pathway.
Bioorganic and Medicinal Chemistry Letters	N4-phenyl modifications of N2-(2-hydroxyl)ethyl-6-(pyrrolidin-1-yl)-1,3,5-triazine-2,4-diamines enhance glucocerebrosidase inhibition by small molecules with potential as chemical chaperones for Gaucher disease. Huang W, Zheng W, Urban DJ, Inglese J, Sidransky E, Austin CP, Thomas CJ. 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.
Journal of Medicinal Chemistry	Bidirectional, Iterative Approach to the Structural Delineation of the Functional "Chemoprint" in GPR40 for Agonist Recognition. Tikhonova IG, Sum CS, Neumann S, Thomas CJ, Raaka BM, Costanzi S, Gershengorn MC. GPR40, free fatty acid receptor 1 (FFAR1), is a member of the GPCR superfamily and a possible target for the treatment of type 2 diabetes. In this work, we conducted a bidirectional iterative investigation, including computational modeling and site-directed mutagenesis, aimed at delineating amino acid residues forming the functional "chemoprint" of GPR40 for agonist recognition. The computational and experimental studies revolved around the recognition of the potent synthetic agonist GW9508. Our experimentally supported model suggested that H137(4.56), R183(5.39), N244(6.55), and R258(7.35) are directly involved in interactions with the ligand. We have proposed a polarized NH-pi interaction between H137(4.56) and GW9508 as one of the contributing forces leading to the high potency of GW9508. The modeling approach presented in this work provides a general strategy for the exploration of receptor-ligand interactions in G-protein coupled receptors beginning prior to acquisition of experimental data.
Neurobiology of Disease	Differentiating Alzheimer Disease-Associated Aggregates with Small Molecules Honson NS, Johnson RL, Huang W, Inglese J, Austin CP, Kuret J. Alzheimer disease is diagnosed postmortem by the density and spatial distribution of b-amyloid plaques and tau-bearing neurofibrillary tangles. The major protein component of each lesion adopts cross-b-sheet conformation capable of binding small molecules with submicromolar affinity. In many cases, however, Alzheimer pathology overlaps with Lewy body disease, characterized by the accumulation of a third cross-b-sheet forming protein, a-synuclein. To determine the feasibility of distinguishing tau aggregates from b-amyloid and a-synuclein aggregates with small molecule probes, a library containing 71,975 small molecules was screened for antagonists of tau-aggregate mediated changes in Thioflavin S fluorescence, followed by secondary screens to distinguish the relative affinity for each substrate protein. Results showed that >10-fold binding selectivity among substrates could be achieved, with molecules selective for tau aggregates containing at least three aromatic or rigid moieties connected by two rotatable bonds.
Nature Chemical Biology	High-throughput screening assays for the identification of chemical probes Inglese J, Johnson RL, Simeonov A, Xia M, Zheng W, Austin CP, Auld DS. High-throughput screening (HTS) assays enable the testing of large numbers of chemical substances for activity in diverse areas of biology. The biological responses measured in HTS assays span isolated biochemical systems containing purified receptors or enzymes to signal transduction pathways and complex networks functioning in cellular environments. This Review addresses factors that need to be considered when implementing assays for HTS and is aimed particularly at investigators new to this field. We discuss assay design strategies, the major detection technologies and examples of HTS assays for common target classes, cellular pathways and simple cellular phenotypes. We conclude with special considerations for configuring sensitive, robust, informative and economically feasible HTS assays.
Nature Chemical Biology	Reporting data from high-throughput screening of small-molecule libraries Inglese J, Shamu CE, Guy RK. 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.
European Pharmaceutical Review	HTS technologies to facilitate chemical genomics Auld DS, Inglese J, Jadhav A, Austin CP, Sittampalam GS, Montrose-Rafizadeh C, Mcgee JE Iversen PW. Industrial scale technologies developed and applied within the pharmaceutical industry for the purpose of drug discovery have recently been adopted by many research laboratories for the purpose of facilitating chemical genomics. Taking full advantage of these technologies will require education in highthroughput screening assay systems as well as new methods that exploit the capabilities of existing technologies.
Journal of Medicinal Chemistry	A high-throughput screen for aggregation-based inhibition in a large compound library. Feng BY, Simeonov A, Jadhav A, Babaoglu K, Inglese J, Shoichet BK, Austin CP. 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.
ASSAY and Drug Development Technologies	A cell-based assay for IkappaBalpha stabilization using a two-color dual luciferase-based sensor. Davis RE, Zhang YQ, Southall N, Staudt LM, Austin CP, Inglese J, Auld DS. 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.
Methods in Enzymology	Fluorescent protein-based cellular assays analyzed by laser-scanning microplate cytometry in 1536-well plate format. Auld DS, Johnson RL, Zhang YQ, Veith H, Jadhav A, Yasgar A, Simeonov A, Zheng W, Martinez ED, Westwick JK, Austin CP, Inglese J. Microtiter plate readers have evolved from photomultiplier and charged-coupled device-based readers, where a population-averaged signal is detected from each well, to microscope-based imaging systems, where cellular characteristics from individual cells are measured. For these systems, speed and ease of data analysis are inversely proportional to the amount of data collected from each well. Microplate laser cytometry is a technology compatible with a 1536-well plate format and capable of population distribution analysis. Microplate cytometers such as the Acumen Explorer can monitor up to four fluorescent signals from single objects in microtiter plates with densities as high as 1536 wells. These instruments can measure changes in fluorescent protein expression, cell shape, or simple cellular redistribution events such as cytoplasmic to nuclear translocation. To develop high-throughput screening applications using laser-scanning microplate cytometry, we used green fluorescent protein- and yellow fluorescent protein-expressing cell lines designed to measure diverse biological functions such as nuclear translocation, epigenetic signaling, and G protein-coupled receptor activation. This chapter illustrates the application of microplate laser cytometry to these assays in a manner that is suitable for screening large compound collections in high throughput.
Drug Discovery Today	Measure, mine, model, and manipulate: the future for HTS and chemoinformatics? Parker CN, Shamu CE, Kraybill B, Austin CP, Bajorath J.
Proceedings of the National Academy of Sciences	Quantitative high-throughput screening: A titration-based approach that efficiently identifies biological activities in large chemical libraries Inglese J, Auld DS, Jadhav A, Johnson RL, Simeonov A, Yasgar A, Zheng W, Austin CP. High-throughput screening (HTS) of chemical compounds to identify modulators of molecular targets is a mainstay of pharmaceutical development. Increasingly, HTS is being used to identify chemical probes of gene, pathway, and cell functions, with the ultimate goal of comprehensively delineating relationships between chemical structures and biological activities. Achieving this goal will require methodologies that efficiently generate pharmacological data from the primary screen and reliably profile the range of biological activities associated with large chemical libraries. Traditional HTS, which tests compounds at a single concentration, is not suited to this task, because HTS is burdened by frequent false positives and false negatives and requires extensive follow-up testing. We have developed a paradigm, quantitative HTS (qHTS), tested with the enzyme pyruvate kinase, to generate concentration-response curves for >60,000 compounds in a single experiment. We show that this method is precise, refractory to variations in sample preparation, and identifies compounds with a wide range of activities. Concentration-response curves were classified to rapidly identify pyruvate kinase activators and inhibitors with a variety of potencies and efficacies and elucidate structure-activity relationships directly from the primary screen. Comparison of qHTS with traditional single-concentration HTS revealed a high prevalence of false negatives in the single-point screen. This study demonstrates the feasibility of qHTS for accurately profiling every compound in large chemical libraries (>10(5) compounds). qHTS produces rich data sets that can be immediately mined for reliable biological activities, thereby providing a platform for chemical genomics and accelerating the identification of leads for drug discovery.
Science	NIH Molecular Libraries Initiative Austin CP, Brady LS, Insel TR, Collins FS. The MLI is a bold initiative to catalyze science in the genome era. By providing a new path for discovery, this program aims to accelerate science and its translation into benefits for the health of the public.
Nature Genetics	The knockout mouse project. Austin CP, et al. Mouse knockout technology provides a powerful means of elucidating gene function in vivo, and a publicly available genome-wide collection of mouse knockouts would be significantly enabling for biomedical discovery. To date, published knockouts exist for only about 10% of mouse genes. Furthermore, many of these are limited in utility because they have not been made or phenotyped in standardized ways, and many are not freely available to researchers. It is time to harness new technologies and efficiencies of production to mount a high-throughput international effort to produce and phenotype knockouts for all mouse genes, and place these resources into the public domain.
Annual Review of Medicine	The impact of the completed human genome sequence on the development of novel therapeutics for human disease. Austin CP. With the official completion of the Human Genome Project in April 2003, we have both the opportunity and the imperative to translate this unprecedented scientific accomplishment into tangible improvements in human health. Medical benefits from the genome will come in stages and can be conceptualized as occurring in three areas: improved understanding of disease causation at the molecular level, improved diagnosis and disease classification based on genetic profiles, and new therapeutics based on targets identified in the genome. These improvements will require increased physician understanding of genetic principles applied to common diseases.
Drug Discovery Today	Expanding the HTS paradigm. Inglese J.