A novel high-throughput screening assay for discovery of molecules that increase cellular tetrahydrobiopterin

Tetrahydrobiopterin (BH(4)) is an essential cofactor for the nitric oxide (NO) synthases and the aromatic amino acid hydroxylases. Insufficient BH(4) has been implicated in various cardiovascular and neurological disorders. GTP cyclohydrolase 1 (GTPCH-1) is the rate-limiting enzyme for de novo biosynthesis of BH(4). The authors have recently shown that the interaction of GTPCH-1 with GTP cyclohydrolase feedback regulatory protein (GFRP) inhibits endothelial GTPCH-1 enzyme activity, BH(4) levels, and NO production. They propose that agents that disrupt the GTPCH-1/GFRP interaction can increase cellular GTPCH-1 activity, BH(4) levels, and NO production. They developed and optimized a novel time-resolved fluorescence resonance energy transfer (TR-FRET) assay to monitor the interaction of GTPCH-1 and GFRP. This assay is highly sensitive and stable and has a signal-to-background ratio (S/B) greater than 12 and a Z' factor greater than 0.8. This assay was used in an ultra-high-throughput screening (uHTS) format to screen the Library of Pharmacologically Active Compounds. Using independent protein-protein interaction and cellular activity assays, the authors identified compounds that disrupt GTPCH-1/GFRP binding and increase endothelial cell biopterin levels. Thus, this TR-FRET assay could be applied in future uHTS of additional libraries to search for molecules that increase GTPCH-1 activity and BH(4) levels.     

A novel multiplex cell viability assay for high-throughput RNAi screening

Cell-based high-throughput RNAi screening has become a powerful research tool in addressing a variety of biological questions. In RNAi screening, one of the most commonly applied assay system is measuring the fitness of cells that is usually quantified using fluorescence, luminescence and absorption-based readouts. These methods, typically implemented and scaled to large-scale screening format, however often only yield limited information on the cell fitness phenotype due to evaluation of a single and indirect physiological indicator. To address this problem, we have established a cell fitness multiplexing assay which combines a biochemical approach and two fluorescence-based assaying methods. We applied this assay in a large-scale RNAi screening experiment with siRNA pools targeting the human kinome in different modified HEK293 cell lines. Subsequent analysis of ranked fitness phenotypes assessed by the different assaying methods revealed average phenotype intersections of 50.7±2.3%-58.7±14.4% when two indicators were combined and 40-48% when a third indicator was taken into account. From these observations we conclude that combination of multiple fitness measures may decrease false-positive rates and increases confidence for hit selection. Our robust experimental and analytical method improves the classical approach in terms of time, data comprehensiveness and cost.     

An image-based, high-throughput screening assay for molecules that induce excess DNA replication in human cancer cells

Previous studies have shown DNA re-replication can be induced in cells derived from human cancers under conditions in which it is not possible for cells derived from normal tissues. Because DNA re-replication induces cell death, this strategy could be applied to the discovery of potential anticancer therapeutics. Therefore, an imaging assay amenable to high-throughput screening was developed that measures DNA replication in excess of four genomic equivalents in the nuclei of intact cells and indexes cell proliferation. This assay was validated by screening a library of 1,280 bioactive molecules on both normal and tumor-derived cells where it proved more sensitive than current methods for detecting excess DNA replication. This screen identified known inducers of excess DNA replication, such as inhibitors of microtubule dynamics, and novel compounds that induced excess DNA replication in both normal and cancer cells. In addition, two compounds were identified that induced excess DNA replication selectively in cancer cells and one that induced endocycles selectively in cancer cells. Thus, this assay provides a new approach to the discovery of compounds useful for investigating the regulation of genome duplication and for the treatment of cancer.     

A novel screening assay for hydroxynitrile lyases suitable for high-throughput screening

Hydroxynitrile lyases (Hnls) are important biocatalysts for the synthesis of optically pure cyanohydrins, which are used as precursors and building blocks for a wide range of high price fine chemicals. Although two Hnl enzymes, from the tropical rubber tree Hevea brasiliensis and from the almond tree Prunus amygdalus, are already used for large scale industrial applications, the enzymes still need to be improved and adapted to the special demands of industrial processes. In many cases directed evolution has been the method of choice to improve enzymes, which are applied as industrial biocatalysts. The screening procedure is the most crucial point in every directed evolution experiment. Herein, we describe the successful development of a novel screening assay for Hnls and its application in high-throughput screening of Escherichia coli mutant libraries. The new assay allows rapid screening of mutant libraries and facilitates the discovery of improved enzyme variants. Hnls catalyze the cleavage of cyanohydrins to hydrocyanic acid and the corresponding aldehyde or ketone. The enzyme assay is based on the detection of hydrocyanic acid produced, making it an all-purpose screening assay, without restriction to any kind of substrate. The gaseous HCN liberated within the Hnl reaction is detected by a visible colorimetric reaction. The facile, highly sensitive and reproducible screening method was validated by identifying new enzyme variants with novel substrate specificities.     

A miniaturized high-throughput screening assay for fucosyltransferase VII

Fucosyltransferase VII (FucTVII) is a very promising drug target for treatment of inflammatory skin diseases. Its activity is required for synthesis of the sialyl-Lewis X glycoepitopes on the E- and P-selectin ligands, necessary for lymphocyte migration into the skin. High-throughput screening (HTS) of large chemical libraries has become the main source of novel chemical entities for the pharmaceutical industry. The screening of very large compound collections requires the use of specialized assay techniques that minimize time and costs. We describe the development of a miniaturized scintillation proximity assay for human FucTVII based on a oligosaccharide acceptor substrate that is identical to the glycosylation of the physiological substrate. In addition to assay development, the assay performance in a HTS campaign is shown. We screened 798,131 compounds from the Schering AG HTS library and identified 233 IC50 hits; 229 hits were FucTVII specific in so far as they did not inhibit either alpha-fucosidase or galactosyltransferase. In addition to screening a drug-like small-molecule collection, we worked on rational approaches to develop inhibitors or glycosidic decoys based on oligosaccharide-substrate analogues. The structure-activity relationship observed thereby is very narrow and shows strict requirements that are consistent with the described substrate specificity of FucTVII.     

A rapid transglutaminase assay for high-throughput screening applications

Transglutaminases (TGs) are widely distributed enzymes that catalyze posttranslational modification of proteins by Ca(2+)-dependent cross-linking reactions. The family members of TGs participate in many significant processes of biological functions such as tissue regeneration, cell differentiation, apoptosis, and certain pathologies. A novel technique for TG activity assay was developed in this study. It was based on the rapid capturing, fluorescence quenching, and fast separation of the unreacted fluorescent molecules from the macromolecular product with magnetic dextran-coated charcoal. As few as 3 ng of guinea pig liver transglutaminase (gpTG) could be detected by the method; activities of 96 TG samples could be measured within an hour. The K(m) of gpTG determined by this method for monodansylcadaverine (dansyl-CAD) and N, N-dimethylcasein was 14 and 5 muM, respectively. A typical competitive inhibition pattern of cystamine on dansyl-CAD for gpTG activity was also demonstrated. The application of this technique is not limited to the use of dansyl-CAD as the fluorescent substrate of TG; other small fluor-labeled TG substrates may substitute dansyl-CAD. Finally, this method is rapid, highly sensitive, and inexpensive. It is suitable not only for high-throughput screening of enzymes or enzyme inhibitors but also for enzyme kinetic analysis.     

A fluorescence lifetime-based assay for serine and threonine kinases that is suitable for high-throughput screening

We describe the development of a novel method for the assay of serine/threonine protein kinases based on fluorescence lifetime. The assay consists of three generic peptides (which have been used by others in the assay of >140 protein kinases in various assay formats) labeled with a long lifetime fluorescent dye (14 or 17 ns) that act as substrates for protein kinases and an iron(III) chelate that modulates the fluorescence lifetime of the peptide only when it is phosphorylated. The decrease in average fluorescence lifetime as measured in a recently developed fluorescence lifetime plate reader (Edinburgh Instruments) is a measure of the degree of phosphorylation of the peptide. We present data showing that the assay performs as well as, and in some cases better than, the “gold standard” radiometric kinase assays with respect to Z′ values, demonstrating its utility in high-throughput screening applications. We also show that the assay gives nearly identical results in trial screening to those obtained by radiometric assays and that it is less prone to interference than simple fluorescence intensity measurements.     

Inhibition of thioredoxin reductase 1 by porphyrins and other small molecules identified by a high-throughput screening assay

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K_i = 2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k_inact) of (0.73 ± 0.07) × 10^? 3 μM^? 1 min^? 1. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.     

A fluorometric assay for high-throughput screening targeting nicotinamide phosphoribosyltransferase

Nicotinamide adenine dinucleotide (NAD) plays a crucial role in many cellular processes. As the rate-limiting enzyme of the predominant NAD biosynthesis pathway in mammals, nicotinamide phosphoribosyltransferase (Nampt) regulates the cellular NAD level. Tumor cells are more sensitive to the NAD levels, making them more susceptible to Nampt inhibition than their nontumorigenic counterparts. Experimental evidence has indicated that Nampt might have proangiogenic activity and supports the growth of some tumors, so Nampt inhibitors may be promising as antitumor agents. However, only four Nampt inhibitors have been reported, and no high-throughput screening (HTS) strategy for Nampt has been proposed to date, largely limiting the drug discovery targeting Nampt. Therefore, the development of a robust HTS strategy for Nampt is both imperative and significant. Here we developed a fluorometric method for a Nampt activity assay by measuring the fluorescence of nicotinamide mononucleotide (NMN) derivative resulting from the enzymatic product NMN through simple chemical reactions. Then we set up an HTS system after thorough optimizations of this method and validated that it is feasible and effective through a pilot screening on a small library. This HTS system should expedite the discovery of Nampt inhibitors as antitumor drug candidates.     

A novel assay for monoacylglycerol hydrolysis suitable for high-throughput screening

A simple assay for monoacylglycerol hydrolysis suitable for high-throughput screening is described. The assay uses [(3)H]2-oleoylglycerol as substrate, with the tritium label in the glycerol part of the molecule and the use of phenyl sepharose gel to separate the hydrolyzed product ([(3)H]glycerol) from substrate. Using cytosolic fractions derived from rat cerebella as a source of hydrolytic activity, the assay gives the appropriate pH profile and sensitivity to inhibition with compounds known to inhibit hydrolysis of this substrate. The assay could also be adapted to a 96-well plate format, using C6 cells as the source of hydrolytic activity. Thus the assay is simple and appropriate for high-throughput screening of inhibitors of monoacylglycerol hydrolysis.     

A rapid enzymatic assay for high-throughput screening of adenosine-producing strains

Adenosine is a major local regulator of tissue function and industrially useful as precursor for the production of medicinal nucleoside substances. High-throughput screening of adenosine overproducers is important for industrial microorganism breeding. An enzymatic assay of adenosine was developed by combined adenosine deaminase (ADA) with indophenol method. The ADA catalyzes the cleavage of adenosine to inosine and NH₃, the latter can be accurately determined by indophenol method. The assay system was optimized to deliver a good performance and could tolerate the addition of inorganic salts and many nutrition components to the assay mixtures. Adenosine could be accurately determined by this assay using 96-well microplates. Spike and recovery tests showed that this assay can accurately and reproducibly determine increases in adenosine in fermentation broth without any pretreatment to remove proteins and potentially interfering low-molecular-weight molecules. This assay was also applied to high-throughput screening for high adenosine-producing strains. The high selectivity and accuracy of the ADA assay provides rapid and high-throughput analysis of adenosine in large numbers of samples.     

High-throughput assay for small molecules that modulate zebrafish embryonic heart rate

To increase the facility and throughput of scoring phenotypic traits in embryonic zebrafish, we developed an automated micro-well assay for heart rate using automated fluorescence microscopy of transgenic embryos expressing green fluorescent protein in myocardium. The assay measures heart rates efficiently and accurately over a large linear dynamic range, and it rapidly characterizes dose dependence and kinetics of small molecule-induced changes in heart rate. This is the first high-throughput micro-well assay for organ function in an intact vertebrate.     

A high-throughput assay for mitochondrial membrane potential in permeabilized yeast cells

A fluorometric assay for mitochondrial membrane potential in permeabilized yeast cells has been developed. This method involves permeabilizing the plasma membrane and measuring the distribution of a mitochondrial membrane potential sensitive probe 3,3'-dipropylthiadicarbocyanine iodide (DiSC(3)(5); DiSC(3)). In permeabilized cells, DiSC(3) fluorescence decreased when introduced into energized mitochondria and increased three- to sixfold when the mitochondrial membrane potential was dissipated by the chemical uncoupler carbonylcyanide m-chlorophenyl hydrazone. Plasma membrane potential was abolished by permeabilization, as shown by a lack of polarization of the plasma membrane induced by K(+) and glucose. Uncoupling protein 1 (UCP1), a mitochondrial H(+) transporter, was used as a model for method validation. The fluorescence intensity responded vigorously to specific modulators in UCP1-expressing cells. This method has been adapted as a high-throughput assay to screen for modulators of mitochondrial membrane potential.     

A solid-phase glycosyltransferase assay for high-throughput screening in drug discovery research

Glycosyltransferases mediate changes in glycosylation patterns which, in turn, may affect the function of glycoproteins and/or glycolipids and, further downstream, processes of development, differentiation, transformation and cell-cell recognition. Such enzymes, therefore, represent valid targets for drug discovery. We have developed a solid-phase glycosyltransferase assay for use in a robotic high-throughput format. Carbohydrate acceptors coupled covalently to polyacrylamide are coated onto 96-well plastic plates. The glycosyltransferase reaction is performed with recombinant enzymes and radiolabeled sugar-nucleotide donor at 37°C, followed by washing, addition of scintillation counting fluid, and measurement of radioactivity using a 96-well -counter. Glycopolymer construction and coating of the plastic plates, enzyme and substrate concentrations, and linearity with time were optimized using recombinant Core 2 1-6-N-acetylglucosaminyltransferase (Core 2 GlcNAc-T). This enzyme catalyzes a rate-limiting reaction for expression of polylactosamine and the selectin ligand sialyl-Lewis^x in -glycans. A glycopolymer acceptor for 1-6-N-acetylglucosaminyltransferase V was also designed and shown to be effective in the solid-phase assay. In a high-throughput screen of a microbial extract library, the coefficient of variance for positive controls was 9.4%, and high concordance for hit validation was observed between the Core 2 GlcNAc-T solid-phase assay and a standard solution-phase assay. The solid-phase assay format, which can be adapted for a variety of glycosyltransferase enzymes, allowed a 5–6 fold increase in throughput compared to the corresponding solution-phase assay.     

Differential assay for high-throughput screening of antibacterial compounds

The previously described Bacillus subtilis reporter strain BAU-102 is capable of detecting cell wall synthesis inhibitors that act at all stages of the cell wall synthesis pathway. In addition, this strain is capable of detecting compounds with hydrophobic/surfactant activity and alternative mechanisms of cell wall disruption. BAU-102 sequesters preformed beta-gal in the periplasm, suggesting leakage of beta-gal as the means by which this assay detects compound activities. A model is proposed according to which beta-gal release by BAU-102 reflects activation of pathways leading to autolysis. The authors also report a simplified high-throughput assay using BAU-102 combined with the fluorogenic substrate N-methylumbelliferyl-beta-D-galactoside as a single reagent. Cell wall inhibitors release beta-gal consistently only after 60 min of incubation, whereas compounds with surfactant activity show an almost immediate release. A high-throughput screen of a 480-compound library of known bioactives yielded 8 compounds that cause beta-gal release. These results validate the BAU-102 assay as an effective tool in antimicrobial drug discovery.     

A high-throughput pH indicator assay for screening glycosyltransferase saturation mutagenesis libraries

Protein engineering using directed evolution or saturation mutagenesis at hot spots is often used to improve enzyme properties such as their substrate selectivity or stability. This requires access to robust high-throughput assays to facilitate the analysis of enzyme libraries. However, relatively few studies on directed evolution or saturation mutagenesis of glycosyltransferases have been reported in part due to a lack of suitable screening methods. In the present study we report a general screening assay for glycosyltransferases that has been developed using the blood group α-(1→3)-galactosyltransferase (GTB) as a model. GTB utilizes UDP-Gal as a donor substrate and α-L-Fucp-(1→2)-β-D-Galp-O-R (H antigen) as an acceptor substrate and synthesizes the blood group B antigen α-D-Galp-(1→3)-[α-L-Fucp-(1→2)]-β-D-Galp-O-R. A closely related α-(1→3)-N-acetylgalactosaminyltransferase (GTA) uses UDP-GalNAc as a donor with the same H acceptor, yielding the A antigen α-D-Galp-NAc-(1→3)-[α-L-Fuc(1→2)]-β-D-Gal-O-R. GTA and GTB are highly homologous enzymes differing in only 4 of 354 amino acids, Arg/Gly-176, Gly/Ser-235, Leu/Met-266, and Gly/Ala-268. The screening assay is based on the color change of the pH indicator bromothymol blue when a proton is released during the transfer of Gal/GalNAc from UDP-Gal/UDP-GalNAc to the acceptor substrate. Saturation mutagenesis of GTB enzyme at M214, a hot spot adjacent to the ²¹¹DVD²¹³ metal binding motif, was performed and the resulting library was screened for increases in UDP-GalNAc transfer activity. Two novel mutants, M214G and M214S, identified by pH indicator screening, were purified and kinetically characterized. M214S and M214G both exhibited two-fold higher k_cat and specific activity than wild-type GTB for UDP-GalNAc. The results confirm the importance of residue M214 for donor enzyme specificity.     

A high-throughput screening assay for the carboxyltransferase subunit of acetyl-CoA carboxylase

One consequence of the dramatic rise of antibiotic-resistant pathogenic bacteria is the need for new targets for antibiotics. Because membrane lipid biogenesis is essential for bacterial growth, enzymes of the fatty acid biosynthetic pathway offer attractive possibilities for the development of new antibiotics. Acetyl-coenzyme A carboxylase (ACC) catalyzes the first committed and regulated step in fatty acid biosynthesis in bacteria and thus is a prime target for development of antibiotics. ACC is a multifunctional enzyme composed of three separate proteins. The biotin carboxylase component catalyzes the ATP-dependent carboxylation of biotin. The biotin carboxyl carrier protein features a biotin molecule covalently attached at Lys122 of the Escherichia coli enzyme. The carboxyltransferase subunit catalyzes the transfer of a carboxyl group from biotin to acetyl-coenzyme A (acetyl-CoA) to form malonyl-CoA. The objective of this study was to develop an assay for high-throughput screening for inhibitors of the carboxyltransferase subunit. The carboxyltransferase reaction was assayed in the reverse direction in which malonyl-CoA reacts with biocytin (an analog of the biotin carboxyl carrier protein) to form acetyl-CoA and carboxybiotin. The production of acetyl-CoA was coupled to citrate synthase, which produced citrate and coenzyme A. The amount of coenzyme A formed was detected using 5,5'-dithiobis(2-nitrobenzoic acid) (Ellman's reagent). The assay has been developed for use in both 96- and 384-well microplate formats and was validated using a known bisubstrate analog inhibitor of carboxyltransferase. The spectrophotometric readout in the visible absorbance range used in this assay does not generate the number of false negatives associated with frequently used NAD/NADH assay systems that rely on detection of NADH using UV absorbance.     

A sensitive colorimetric high-throughput screening method for lipase synthetic activity assay

A sensitive and practical high-throughput screening method for assaying lipase synthetic activity is described. Lipase-catalyzed transesterification between vinyl acetate and n-butanol in n-hexane was chosen as a model reaction. The released acetaldehyde was determined by the colorimetric method using 3-methyl-2-benzothialinone (MBTH) derivatization. In comparison with other methods, the major advantages of this process include high sensitivity, simple detection, inexpensive reagents, and low requirements for instruments.     

Development of a high-throughput screening assay for cytoprotective agents in rotenone-induced cell death

Parkinson’s disease (PD) is a neurodegenerative disease featured by selective loss of substantia nigra neurons. Rotenone administration in animals induces neurodegeneration accompanied by α-synuclein-positive Lewy body-like inclusions, recapturing typical histopathological features of PD. In an effort to screen for small-molecule agents to reverse rotenone-induced cytotoxicity, we developed and validated a sensitive and robust assay with neuroblastoma SK-N-SH cells. This assay was amenable to a high-throughput screening format with Z′ factor of 0.56. Robotic screening of a bioactive compound library led to the identification of carnosic acid that can effectively protect cells from rotenone treatment. Using a high-content image-based assay and Western blot analysis, we demonstrated that carnosic acid protects cells from rotenone stress by significant induction of HSP70 expression. Therefore, the assay reported here can be used to identify novel cytoprotective agents for clinical therapeutics of PD.