Firefly luciferase (FLuc) is frequently used as a reporter in high-throughput screening assays owing to the exceptional sensitivity, dynamic range, and rapid measurement that bioluminescence affords. However, interaction of small molecules with FLuc has, to some extent, confounded its use in chemical biology and drug discovery. To identify and characterize chemotypes interacting with FLuc, we determined potency values for 360,864 compounds, found in the NIH Molecular Libraries Small Molecule Repository, available in PubChem. FLuc inhibitory activity was observed for 12% of this library with discernible SAR. Characterization of 151 inhibitors demonstrated a variety of inhibition modes including FLuc-catalyzed formation of multisubstrate-adduct enzyme inhibitor complexes. As in some cell-based FLuc reporter assays compounds acting as FLuc inhibitors yield paradoxical luminescence increases, data on compounds acquired from FLuc-dependent assays requires careful analysis as described in this report.
profiling; PubChem; luciferase; quantitative high-throughput screening; qHTS; firefly luciferase; reporter-gene assays; adenylate forming enzymes
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.
Cancer cells engage in a metabolic program to enhance biosynthesis and support cell proliferation. The regulatory properties of pyruvate kinase M2 (PKM2) influence altered glucose metabolism in cancer. PKM2 interaction with phosphotyrosine-containing proteins inhibits enzyme activity and increases availability of glycolytic metabolites to support cell proliferation. This suggests that high pyruvate kinase activity may suppress tumor growth. We show that expression of PKM1, the pyruvate kinase isoform with high constitutive activity, or exposure to published small molecule PKM2 activators inhibit growth of xenograft tumors. Structural studies reveal that small molecule activators bind PKM2 at the subunit interaction interface, a site distinct from that of the endogenous activator fructose-1,6-bisphosphate (FBP). However, unlike FBP, binding of activators to PKM2 promotes a constitutively active enzyme state that is resistant to inhibition by tyrosine-phosphorylated proteins. These data support the notion that small molecule activation of PKM2 can interfere with anabolic metabolism.
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 paper, 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.
hypoxia-inducible factor-1α; quinazolin-4-ones; parallel synthesis
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.
The chemical diversity of nature has tremendous potential for discovery of new molecular probes and medicinal agents. However, sensitivity of HTS assays to interfering components of crude extracts derived from plants, macro- and microorganisms has curtailed their use in lead discovery efforts. Here we describe a process for leveraging the concentration-response curves (CRCs) obtained from quantitative HTS to improve the initial selection of “actives” from a library of partially fractionated natural product extracts derived from marine actinomycetes and fungi. By using pharmacological activity, the first-pass CRC paradigm aims to improve the probability that labor-intensive subsequent steps of re-culturing, extraction and bioassay-guided isolation of active component(s) target the most promising strains and growth conditions. We illustrate how this process identified a family of fungal metabolites as potent inhibitors of firefly luciferase, subsequently resolved in molecular detail by x-ray crystallography.
Ubiquitin-specific proteases (USPs) have in recent years emerged as a promising therapeutic target class. We identified selective small-molecule inhibitors against a deubiquitinase complex, the human USP1/UAF1, through quantitative high throughput screening (qHTS) of a collection of bioactive molecules. The top inhibitors, pimozide and GW7647, inhibited USP1/UAF1 noncompetitively with a Ki of 0.5 and 0.7 μM respectively, and displayed selectivity against a number of deubiquitinases, deSUMOylase and cysteine proteases. The USP1/UAF1 inhibitors act synergistically with cisplatin in inhibiting cisplatin-resistant non-small cell lung cancer (NSCLC) cell proliferation. USP1/UAF1 represents a promising target for drug intervention because of its involvement in translesion synthesis and Fanconi anemia pathway important for normal DNA damage response. Our results support USP1/UAF1 as a potential therapeutic target and provide the first example of targeting the USP/WD40 repeat protein complex for inhibitor discovery.
Deubiquitinase; ubiquitin-specific protease; DNA damage response; translesion synthesis; Fanconi anemia; cisplatin
We herein describe the rapid synthesis of a diverse set of dihydroquinazolin-4-ones and quinazolin-4-ones, their biological evaluation as thyroid stimulating hormone receptor (TSHR) agonists, and SAR analysis. Among the compounds screened, 8b was 60-fold more potent than the hit compound 1a, which was identified from a high throughput screen of over 73,000 compounds.
We describe how room temperature storage of a 1,120 member compound library prepared in either DMSO or in a hydrated DMSO/water (67/33) mixture affects the reproducibility of potency values as monitored using cytochrome P450 1A2 and 2D6 isozyme assays. The bioluminescent assays showed Z′-factors of 0.71 and 0.62, with 18% and 32% of the library found as active against the CYP 1A2 and 2D6 isozymes respectively. We tested the library using quantitative high-throughput screening to generate potency values for every library member which was measured at seven time intervals spanning 37 weeks. We calculated the minimum significant ratio (MSR) from these potency values at each time interval and we found that for the library stored in DMSO, the CYP 1A2 and 2D6 assay MSRs progressed from approximately 2.0 to 5.0. The hydrated conditions showed similar performance in both MSR progression and analytical QC results. Based on this study we recommend that DMSO samples be stored in 1,536-well plates for < 4 months at room temperature. Further, the study shows the magnitude of potency changes that can occur in a robust bioassay due to compound sample storage.
HTS; compound storage; DMSO; quantitative HTS
We measured the “druggability” of the ATP-dependent luciferase derived from the firefly Photuris pennsylvanica that was optimized using directed evolution (Ultra-Glo™, Promega). Quantitative high throughput screening (qHTS) was used to determine IC50’s of 198,899 samples against a formulation of Ultra-Glo luciferase (Kinase-Glo™). We found that only 0.1% of the Kinase-Glo inhibitors showed an IC50 < 10 μM compared to 0.9% found from a previous qHTS against the firefly luciferase from Photinus pyralis (lucPpy). Further, the maximum affinity identified in the lucPpy qHTS was 50 nM while for Kinase-Glo this value increased to 600 nM. Compounds with interactions stretching outside the luciferin binding pocket were largely lost with Ultra-Glo luciferase. Therefore, Ultra-Glo luciferase will show less compound interference when used as an ATP sensor compared to lucPpy. This study demonstrates the power of large-scale quantitative analysis of structure-activity relationships (>100K compounds) in addressing important questions such as a target's druggability.
chemical profiling; enzyme assay; PubChem; luciferase; quantitative high-throughput screening
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 and genes associated with differential chemical phenotypes (DCPs), defined as ≥5-fold differences in half-maximum inhibitor concentration (IC50) 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, yielding promising new leads for antimalarial treatments.
Plasmodium falciparum; high-throughput screening; genetic mapping; chemical genomics; phenotype
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 (C-terminal portion of BRCA1, a key tumor suppressor protein with various functions), and phosphorylated protein (Abraxas, BACH1, CtIP) implicated in DNA damage response and repair pathways, should prove useful in studies of BRCA1’s role in cancer and to potentially sensitize tumors to chemotherapeutic agents. We developed and miniaturized to 1536-well format and 3 μL final volume a pair of fluorescence polarization (FP) assays utilizing fluorescein- and rhodamine-labeled pBACH1 fragment. In order to minimize the effect of fluorescence artifacts and to increase the overall robustness of the screen, the 75,552 compound library members were each 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 qHTS paradigm (Inglese et al, PNAS, 103, 1147 (2006)). 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.
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, we 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 six concentrations up to 92 uM to generate titration-response curves and determine the potency and efficacy of all compounds. The qHTS dataset 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 uM IC50. This series represents a new class of thyroid hormone receptor-coactivator modulators.
thyroid receptor; small molecule; HTS; coactivator; protein-protein interaction
Understanding luciferase enzymology and the structure of compounds that modulate luciferase activity can be used to improve the design of luminescence-based assays. This review provides an overview of these popular reporters with an emphasis on the commonly used firefly luciferase from Photinus pyralis (FLuc). Large-scale chemical profile studies have identified a variety of scaffolds that inhibit FLuc. In some cell-based assays these inhibitors can act in a counter-intuitive way –leading to a gain in luminescent signal. Although formerly attributed to transcriptional activation, intracellular stabilization of FLuc is the primary mechanism underlying this observation. FLuc inhibition/stabilization can be complex, as illustrated by the compound PTC124, which is converted by FLuc in the presence of ATP to a high affinity multi-substrate-adduct inhibitor, PTC124-AMP. The potential influence these findings can have on drug discovery efforts is provided here.
Cancer cells have distinct metabolic needs that are different from normal cells and can be exploited for development of anti-cancer therapeutics. Activation of the tumor specific M2 form of pyruvate kinase (PKM2) is a potential strategy for returning cancer cells to a metabolic state characteristic of normal cells. Here, we describe activators of PKM2 based upon a substituted thieno[3,2-b]pyrrole[3,2-d]pyridazinone scaffold. The synthesis of these agents, structure activity relationships, analysis of activity at related targets (PKM1, PKR and PKL) and examination of aqueous solubility are investigated. These agents represent the second reported chemotype for activation of PKM2.
Warburg effect; pyruvate kinase; cellular metabolism; anti-cancer strategies; small molecule activators
Summary of recent advances
Expansive compound collections made up of structurally heterogeneous chemicals, the activities of which are largely undefined, present challenging problems for high-throughput screening (HTS). Foremost is differentiating whether the activity for a given compound in an assay is directed against the targeted biology, or is the result of surreptitious compound activity involving the assay detection system. Such compound interference can be especially difficult to identify if it is reproducible and concentration-dependent – characteristics generally attributed to compounds with genuine activity. While reactive chemical groups on compounds were once thought to be the primary source of compound interference in assays used in HTS, recent work suggests that other factors, such as compound aggregation, may play a more significant role in many assay formats. Considerable progress has been made to profile representative compound libraries in an effort to identify chemical classes susceptible to producing compound interference, such as compounds commonly found to inhibit the reporter enzyme firefly luciferase. Such work has also led to the development of practices that have the potential to significantly reduce compound interference, for example, through the addition of non-ionic detergent to assay buffer to reduce aggregation-based inhibition.
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.
Glutathione S-transferases (GSTs) constitute a family of detoxification enzymes that catalyze the conjugation of glutathione with a variety of hydrophobic compounds, including drugs and their metabolites, to yield water-soluble derivatives that are excreted in urine or bile. Profiling the effect of small molecules on GST activity is an important component in the characterization of drug candidates and compound libraries. Additionally, specific GST isozymes have been implicated in drug resistance, especially in cancer, and thus represent potential targets for intervention. To date, there are no sensitive miniaturized high-throughput assays available for GST activity detection. A series of GST substrates containing a masked luciferin moiety have been described recently, offering the potential for configuring a sensitive screening assay via coupled luciferase reaction and standard luminescence detection. We report on the optimization and miniaturization of this homogeneous method to 1,536-well format using GSTs from 3 different species: mouse isozyme A4-4, human isozymes A1-1, M1-1, and P1-1, and the major GST from the parasitic worm Schistosoma japonicum.
Hsp90 has emerged as an important anti-cancer drug target because of its essential role in promoting the folding and maturation of many oncogenic proteins. Here we 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 uses 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 is stable over 12 hours and tolerates DMSO up to 5%. We first validated the assay by screening against 20,000 compounds in 384-well format. After further optimization into a 1536-well format, it was screened against a NCGC library of 76,134 compounds, with a signal-to-background (S/B) 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 anti-cancer drugs, for use alone or in combination with other Hsp90 inhibitors.
heat shock protein 90 (Hsp90); Hsp organizing protein (HOP); tetratricopeptide repeat (TPR); AlphaScreen™; high-throughput screening (HTS)
The metabolism of cancer cells is altered to support rapid proliferation. Pharmacological activators of a tumor cell specific pyruvate kinase isozyme (PKM2) may be an approach for altering the classic Warburg effect characteristic of aberrant metabolism in cancer cells yielding a novel anti-proliferation strategy. In this manuscript we detail the discovery of a series of substituted N,N′-diarylsulfonamides as activators of PKM2. The synthesis of numerous analogues and the evaluation of structure activity relationships are presented as well as assessments of mechanism and selectivity. Several agents are found that have good potencies and appropriate solubility for use as chemical probes of PKM2 including 55 (AC50 = 43 nM, maximum response = 84%; solubility = 7.3 μg/mL), 56 (AC50 = 99 nM, maximum response = 84%; solubility = 5.7 μg/mL) and 58 (AC50 = 38 nM, maximum response = 82%; solubility = 51.2 μg/mL). The small molecules described here represent first-in-class activators of PKM2
Warburg effect; pyruvate kinase; cellular metabolism; high-throughput screening; small molecule activators
Trypanosoma cruzi and Trypanosoma brucei are parasites that cause Chagas’ disease and African sleeping sickness, respectively. Both parasites rely on essential cysteine proteases for survival, cruzain for T. cruzi and TbCatB/rhodesain for T. brucei. A recent quantitative high-throughput screen of cruzain identified triazine nitriles, which are known inhibitors of other cysteine proteases, as reversible inhibitors of the enzyme. Structural modifications detailed herein, including core scaffold modification from triazine to purine, improved the in vitro potency against both cruzain and rhodesain by 350-fold, while also gaining activity against T. brucei parasites. Selected compounds were screened against a panel of human cysteine and serine proteases to determine selectivity, and a co-crystal was obtained of our most potent analog bound to Cruzain.
In support of the U.S. Tox21 program, we have developed a simple and chemically intuitive model we call weighted feature significance (WFS) to predict the toxicological activity of compounds, based on the statistical enrichment of structural features in toxic compounds. We trained and tested the model on the following: (1) data from quantitative high–throughput screening cytotoxicity and caspase activation assays conducted at the National Institutes of Health Chemical Genomics Center, (2) data from Salmonella typhimurium reverse mutagenicity assays conducted by the U.S. National Toxicology Program, and (3) hepatotoxicity data published in the Registry of Toxic Effects of Chemical Substances. Enrichments of structural features in toxic compounds are evaluated for their statistical significance and compiled into a simple additive model of toxicity and then used to score new compounds for potential toxicity. The predictive power of the model for cytotoxicity was validated using an independent set of compounds from the U.S. Environmental Protection Agency tested also at the National Institutes of Health Chemical Genomics Center. We compared the performance of our WFS approach with classical classification methods such as Naive Bayesian clustering and support vector machines. In most test cases, WFS showed similar or slightly better predictive power, especially in the prediction of hepatotoxic compounds, where WFS appeared to have the best performance among the three methods. The new algorithm has the important advantages of simplicity, power, interpretability, and ease of implementation.
modeling; toxicity prediction; structural features; cell viability; caspase-3,7 activation; in vivo toxicity
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.
kinase inhibition; pre-mRNA splicing; Clk; Dyrk1A
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 ∼300 compounds with potencies ranging from as low as 50 nM to >10 µM, yielding a robust dataset for the comparison. Both assay formats showed high performance (Z′-factors ∼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.
Surfactin-type phosphopantetheinyl transferases (Sfp-PPTases) are responsible for modifying type I polyketide and nonribosomal 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 μL volume in 1,536-well format and was used to screen the Library of Pharmacologically Active Compounds (LOPAC1280). 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.