Utilization of fluorescence polarization and time resolved fluorescence resonance energy transfer assay formats for SAR studies: Src kinase as a model system

High-throughput screening (HTS), a major component of lead identification, often utilizes fluorescence-based assay technologies. For example, HTS kinase assays are formatted using a variety of fluorescence-based assay technologies including, but not limited to, dissociation enhanced lanthanide fluoroimmunoassay (DELFIA), time-resolved fluorescence resonance energy transfer (TR-FRET), and fluorescence polarization (FP). These assays offer tremendous advantages such as a nonradioactive format, ease of automation, and excellent reproducibility. Fluorescence-based assays frequently used for lead identification can also be useful for structure activity relationship (SAR) studies during lead optimization. An important issue when assessing an assay to be used for SAR is the ability of the assay to discriminate high-affinity small molecule inhibitors (pM-nM) from low-affinity inhibitors (microM-mM). The purpose of this study was to utilize HTS-friendly assay formats for SAR by developing TR-FRET, FP, and DELFIAassays measuring Src kinase activity and to define the theoretical lower limit of small molecule inhibitor detection achievable with these assay formats. The authors show that 2 homogeneous assay formats, TR-FRET and FP, allowed for the development of Src kinase assays with a lower limit of detection of K(i) = 0.01 nM. This study indicates that assay technologies typically used for HTS can be used during lead optimization by providing quantitative measurements of compound activity critical to driving SAR studies.     

Development, validation and implementation of immobilized metal affinity for phosphochemicals (IMAP)-based high-throughput screening assays for low-molecular-weight compound libraries

This protocol describes assay development, validation and implementation of automated immobilized metal affinity for phosphochemicals (IMAP)-based fluorescence polarization (FP) and time-resolved fluorescence resonance energy transfer (TR-FRET) high-throughput screening (HTS) assays for identification of low-molecular-weight kinase inhibitors. Both procedures are performed in miniaturized kinase reaction volumes and involve the stepwise addition of test or control compounds, enzyme and substrate/ATP. Kinase reactions are stopped by subsequent addition of IMAP-binding buffer. Assay attributes of the IMAP FP and TR-FRET methodologies are described. HTS assays developed using these procedures should result in Z-factors and low assay variability necessary for robust HTS assays. Providing that the required reagents and equipment are available, one scientist should be able to develop a 384-well, miniaturized HTS assay in approximately 6-8 weeks. Specific automated HTS assay conditions will determine the number of assay plates processed in a screening session, but two scientists should expect to process between 100 and 150 assay plates in one 8-h screening day.     

An improved zinc cocktail-mediated fluorescence polarization-based kinase assay for high-throughput screening of kinase inhibitors

During the past few years, high-throughput screening (HTS) has provided a useful resource to researchers involved in the development of kinase inhibitors as a novel therapeutic modality. However, with all the choices among kinase assays, there is not yet a one-size-fits-all assay. Therefore, selection of a specific kinase assay is a daunting task. HTS assays should be homogeneous, cost effective, use nonradioactive reagents, generic and not time consuming. Here, we report an improved method of assaying protein kinase activity using a zinc cocktail in a fluorescence polarization-(FP) based format. Assay conditions were standardized manually and validated in a HTS format using a liquid handler. We validated this assay for both serine/threonine and tyrosine (receptor/nonreceptor) kinases. The results obtained in the HTS assay system were comparable to the commercially available fluorescence-based assay. We suggest that the reported assay is a cost-effective alternative to the IMAP-based generic kinase assay.     

Assay concordance between SPA and TR-FRET in high-throughput screening

High-throughput screening (HTS) of large chemical libraries has become the main source of new lead compounds for drug development. Several specialized detection technologies have been developed to facilitate the cost- and time-efficient screening of millions of compounds. However, concerns have been raised, claiming that different HTS technologies may produce different hits, thus limiting trust in the reliability of HTS data. This study was aimed to investigate the reliability of the authors most frequently used assay techniques: scintillation proximity assay (SPA) and homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET). To investigate the data concordance between these 2 detection technologies, the authors screened a large subset of the Schering compound library consisting of 300,000 compounds for inhibitors of a nonreceptor tyrosine kinase. They chose to set up this study in realistic HTS scale to ensure statistical significance of the results. The findings clearly demonstrate that the choice of detection technology has no significant impact on hit finding, provided that assays are biochemically equivalent. Data concordance is up to 90%. The little differences in hit findings are caused by threshold setting but not by systematic differences between the technologies. The most significant difference between the compared techniques is that in the SPA format, more false-positive primary hits were obtained.     

Fluorescence polarization assay for inhibitors of the kinase domain of receptor interacting protein 1

Necrotic cell death is prevalent in many different pathological disease states and in traumatic injury. Necroptosis is a form of necrosis that stems from specific signaling pathways, with the key regulator being receptor interacting protein 1 (RIP1), a serine/threonine kinase. Specific inhibitors of RIP1, termed necrostatins, are potent inhibitors of necroptosis. Necrostatins are structurally distinct from one another yet still possess the ability to inhibit RIP1 kinase activity. To further understand the differences in the binding of the various necrostatins to RIP1 and to develop a robust high-throughput screening (HTS) assay, which can be used to identify new classes of RIP1 inhibitors, we synthesized fluorescein derivatives of Necrostatin-1 (Nec-1) and Nec-3. These compounds were used to establish a fluorescence polarization (FP) assay to directly measure the binding of necrostatins to RIP1 kinase. The fluorescein-labeled compounds are well suited for HTS because the assays have a dimethyl sulfoxide (DMSO) tolerance up to 5% and Z' scores of 0.62 (fluorescein-Nec-1) and 0.57 (fluorescein-Nec-3). In addition, results obtained from the FP assays and ligand docking studies provide insights into the putative binding sites of Nec-1, Nec-3, and Nec-4.     

A homogeneous assay of kinase activity that detects phosphopeptide using fluorescence polarization and zinc

Homogeneous antibody-free assays of protein kinase activity have great utility in high-throughput screening in support of drug discovery. In an effort to develop such an assay, we have used a pair of fluorescein-labeled peptides of identical amino acid sequence with and without phosphorylation on serine to mimic the substrate and product, respectively, of a kinase. Using fluorescence polarization (FP), we have demonstrated that a mixture of zinc sulfate, phosphate-buffered saline, and bovine serum albumin added to the peptides dramatically and differentially increased the fluorescence polarization of the phosphorylated peptide over its nonphosphorylated derivative. A similar FP differential was observed using different peptide pairs, though the magnitude varied. The FP values obtained using this method were directly proportional to the fraction of phosphopeptide present. Therefore, an FP assay was developed using a proprietary kinase. Using this FP method, linear reaction kinetics were obtained in enzyme titration and reaction time course experiments. The IC(50) values for a panel of inhibitors of kinase activity were determined using this FP method and a scintillation proximity assay. The IC(50) values were comparable between the two methods, suggesting that the zinc FP assay may be useful as an inexpensive high-throughput assay for identifying inhibitors of kinase activity.     

Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II)

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.     

Evaluation of fluorescent compound interference in 4 fluorescence polarization assays: 2 kinases, 1 protease,and 1 phosphatase

With the increasing use of fluorescence-based assays in high-throughput screening (HTS), the possibility of interference by fluorescent compounds needs to be considered. To investigate compound interference, a well-defined sample set of biologically active compounds, LOPAC, was evaluated using 4 fluorescein-based fluorescence polarization (FP) assays. Two kinase assays, a protease assay, and a phosphatase assay were studied. Fluorescent compound interference and light scattering were observed in both mixture- and single-compound testing under certain circumstances. In the kinase assays, which used low levels (1-3 nM) of fluorophore, an increase in total fluorescence, an abnormal decrease in mP readings, and negative inhibition values were attributed to compound fluorescence. Light scattering was observed by an increase in total fluorescence and minimal reduction in mP, leading to false positives. The protease and phosphatase assays, which used a higher concentration of fluorophore (20-1200 nM) than the kinase assays, showed minimal interference from fluorescent compounds, demonstrating that an increase in the concentration of the fluorophore minimized potential fluorescent compound interference. The data also suggests that mixtures containing fluorescent compounds can result in either false negatives that can mask a potential "hit" or false positives, depending on the assay format. Cy dyes (e.g., Cy3B and Cy5 ) excite and emit further into the red region than fluorescein and, when used in place of fluorescein in kinase 1, eliminate fluorescence interference and light scattering by LOPAC compounds. This work demonstrates that fluorescent compound and light scattering interferences can be overcome by increasing the fluorophore concentration in an assay or by using longer wavelength dyes.     

Readout technologies for highly miniaturized kinase assays applicable to high-throughput screening in a 1536-well format

This article discusses the development of homogeneous, miniaturized assays for the identification of novel kinase inhibitors from very large compound collections. In particular, the suitability of time-resolved fluorescence resonance energy transfer (TR-RET) based on phospho-specific antibodies, an antibody-independent fluorescence polarization (FP) approach using metal-coated beads (IMAP technology), and the determination of adenosine triphosphate consumption through chemiluminescence is evaluated. These readouts are compared with regard to assay sensitivity, compound interference, reagent consumption, and performance in a 1536-well format, and practical considerations for their application in primary screening or in the identification of kinase substrates are discussed. All of the tested technologies were found to be suitable for miniaturized high-throughput screening (HTS) in principle, but each of them has distinct limitations and advantages. Therefore, the target-specific selection of the most appropriate readout technology is recommended to ensure maximal relevance of HTS campaigns.     

Comparison of assay technologies for a nuclear receptor assay screen reveals differences in the sets of identified functional antagonists

Many assay technologies currently exist to develop high-throughput screening assays, and the number of choices continues to increase. Results from a previous study comparing assay technologies in our laboratory do not support the common assumption that the same hits would be found regardless of which assay technology is used. To extend this investigation, a nuclear receptor antagonist assay was developed using 3 assay formats: AlphaScreen, time-resolved fluorescence (TRF), and time-resolved fluorescence resonance energy transfer (TR-FRET). Compounds ( approximately 42000) from the Novartis library were evaluated in all 3 assay formats. A total of 128 compounds were evaluated in dose-response experiments, and 109 compounds were confirmed active from all 3 formats. The AlphaScreen, TRF, and TR-FRET assay technologies identified 104, 23, and 57 active compounds, respectively, with only 18 compounds active in all 3 assay formats. A total of 128 compounds were evaluated in a cell-based functional assay, and 35 compounds demonstrated activity in this cellular assay. Furthermore, 34, 11, and 16 hits that were originally identified in the dose-response experiment by AlphaScreen, TRF, and TR-FRET assay technologies, respectively, were functionally active. The results of the study indicated that AlphaScreen identified the greatest number of functional antagonists.     

A high-throughput, nonisotopic, competitive binding assay for kinases using nonselective inhibitor probes (ED-NSIP)

A novel competitive binding assay for protein kinase inhibitors has been developed for high-throughput screening (HTS). Unlike functional kinase assays, which are based on detection of substrate phosphorylation by the enzyme, this novel method directly measures the binding potency of compounds to the kinase ATP binding site through competition with a conjugated binding probe. The binding interaction is coupled to a signal amplification system based on complementation of beta-galactosidase enzyme fragments, a homogeneous, nonisotopic assay technology platform developed by DiscoveRx Corp. In the present study, staurosporine, a potent, nonselective kinase inhibitor, was chemically conjugated to a small fragment of beta-galactosidase (termed ED-SS). This was used as the binding probe to the kinase ATP binding pocket. The binding potencies of several inhibitors with diverse structures were assessed by displacement of ED-SS from the kinase. The assay format was specifically evaluated with GSK3alpha, an enzyme previously screened in a radioactive kinase assay (i.e., measurement of [(33)P]-gamma-ATP incorporation into the kinase peptide substrate). Under optimized assay conditions, nonconjugated staurosporine inhibited ED-SS binding in a concentration-dependent manner with an apparent potency (IC(50)) of 11 nM, which was similar to the IC(50) value determined in a radioactive assay. Furthermore, 9 kinase inhibitors with diverse structures, previously identified from chemical compound library screening, were screened using the competitive binding assay. The potencies in the binding assay were in very good agreement with those obtained previously in the isotopic functional activity assay. The binding assay was adapted for automated HTS using selected compound libraries in a 384-well microtiter plate format. The HTS assay was observed to be highly robust and reproducible (Z' factors > 0.7) with high interassay precision (R(2) > 0.96). Interference of compounds with the beta-galactosidase signal readout was negligible. In conclusion, the DiscoveRx competitive kinase binding assay, termed ED-NSIP trade mark, provides a novel method for screening kinase inhibitors. The format is homogeneous, robust, and amenable to automation. Because there is no requirement for substrate-specific antibodies, the assay is particularly applicable to Ser/Thr kinase assay, in which difficulties in identifying a suitable substrate and antibody preclude development of nonisotopic assays. Although the nonselective kinase inhibitor, staurosporine, was used here, chemically conjugating the ED fragment to other small molecule enzyme inhibitors is also feasible, suggesting that the format is generally applicable to other enzyme systems.     

Use of mixture distributions to deconvolute the behavior of "hits" and controls in high-throughput screening data

The stochastic nature of high-throughput screening (HTS) data indicates that information may be gleaned by applying statistical methods to HTS data. A foundation of parametric statistics is the study and elucidation of population distributions, which can be modeled using modern spreadsheet software. The methods and results described here use fundamental concepts of statistical population distributions analyzed using a spreadsheet to provide tools in a developing armamentarium for extracting information from HTS data. Specific examples using two HTS kinase assays are analyzed. The analyses use normal and gamma distributions, which combine to form mixture distributions. HTS data were found to be described well using such mixture distributions, and deconvolution of the mixtures to the constituent gamma and normal parts provided insight into how the assays performed. In particular, the proportion of hits confirmed was predicted from the original HTS data and used to assess screening assay performance. The analyses also provide a method for determining how hit thresholds--values used to separate active from inactive compounds--affect the proportion of compounds verified as active and how the threshold can be chosen to optimize the selection process.     

Further comparison of primary hit identification by different assay technologies and effects of assay measurement variability

High-throughput screening (HTS) has grown rapidly in the past decade, with many advances in new assay formats, detection technologies, and laboratory automation. Recently, several studies have shown that the choice of assay technology used for the screening process is particularly important and can yield quite different primary screening outcomes. However, because the screening assays in these previous studies were performed in a single-point determination, it is not clear to what extent the difference observed in the screening results between different assay technologies is attributable to inherent assay variability and day-to-day measurement variation. To address this question, a nuclear receptor coactivator recruitment assay was carried out in 2 different assay formats, namely, AlphaScreen and time-resolved fluorescence resonance energy transfer, which probed the same biochemical binding events but with different detection technologies. For each assay format, 4 independent screening runs in a typical HTS setting were completed to evaluate the run-to-run screening variability. These multiple tests with 2 assay formats allow an unambiguous comparison between the discrepancies of different assay formats and the effects of the variability of assay and screening measurements on the screening outcomes. The results provide further support that the choice of assay format or technology is a critical factor in HTS assay development.     

Development of a Transcreener kinase assay for protein kinase A and demonstration of concordance of data with a filter-binding assay format

A Transcreener kinase fluorescence polarization (FP) assay has been developed for the serine/threonine kinase protein kinase A (PKA). The PKA Transcreener kinase assay is an homogenous, competitive antibody-based FP assay that uses Far Red Alexa Fluor 633-labeled adenosine 5' disphosphate (ADP) tracer and mouse monoclonal anti-ADP antibody. The Transcreener PKA assay was validated with both known PKA inhibitors and library compounds. The Transcreener PKA assay is resistant to low-wavelength (or common) fluorescent interference from small-molecule library compounds and generates IC50 results comparable with current radioactive filter-binding assay.     

A widely applicable, high-throughput TR-FRET assay for the measurement of kinase autophosphorylation: VEGFR-2 as a prototype

Homogeneous time-resolved fluorescence resonance energy transfer (TR-FRET) assays represent a highly sensitive and robust high-throughput screening (HTS) method for the quantification of kinase activity. Traditional TR-FRET kinase assays detect the phosphorylation of an exogenous substrate. The authors describe the development and optimization of a TR-FRET technique that measures the autophosphorylation of vascular endothelial growth factor receptor 2 (VEGFR-2) kinase and extend its applicability to a variety of other kinases. The VEGFR-2 assay demonstrated dose-dependent inhibition by compounds known to modulate the catalytic activity of this receptor. In addition, kinetic analysis of a previously characterized VEGFR-2 inhibitor was performed using the method, and results were consistent with those obtained using a different assay format. Because of the known involvement of VEGFR-2 in angiogenesis, this assay should facilitate HTS for antiangiogenic agents. In addition, this general technique should have utility for the screening for inhibitors of kinases as potential therapeutic agents for many other disease indications.     

Data concordance from a comparison between filter binding and fluorescence polarization assay formats for identification of ROCK-II inhibitors

The Rho-associated coiled-coil-containing protein serine/threonine kinases ROCK-I and ROCK-II are thought to play a major role in cytoskeletal dynamics by serving as downstream effectors of the Rho/Rac family of cytokine- and growth factor-activated small GTPases. As such, the ROCK family members are attractive intervention targets for a variety of pathologies, including cancer and cardiovascular disease. The authors developed a high-throughput screen to identify ROCK-II inhibitors and report results from a direct comparison of 2 screening campaigns for ROCK-II inhibitors using fluorescence polarization (FP) and filter binding (FB). Screening protocols to identify inhibitors of ROCK-II were developed in FB and FP formats under similar assay and kinetic conditions. A 30,000-member compound library was screened using FB ((33)P) and FP detection systems, and compounds that were active in either assay were retested in 5-point curve confirmation assays. Analysis of these data showed an approximate 95% agreement of compounds identified as active in both assay formats. Also, compound potency determinations from FB and FP had a high degree of correlation and were considered equivalent. These data suggest that the assay methodology has little impact on the quality and productivity of the screen, provided that the assays are developed to standardize kinetic conditions.     

Scintillation proximity assay of arginine methylation

Methylation of arginine residues, catalyzed by protein arginine methyltransferases (PRMTs), is one important protein posttranslational modification involved in epigenetic regulation of gene expression. A fast and effective assay for PRMT can provide valuable information for dissecting the biological functions of PRMTs, as well as for screening small-molecule inhibitors of arginine methylation. Currently, among the methods used for PRMT activity measurement, many contain laborious separation procedures, which restrict the applications of these assays for high-throughput screening (HTS) in drug discovery. The authors report here a mix-and-measure method to measure PRMT activity based on the principle of scintillation proximity assay (SPA). In this assay, (3)H-AdoMet was used as methyl donor, and biotin-modified histone H4 peptide served as a methylation substrate. Following the methylation reaction catalyzed by PRMTs, streptavidin-coated SPA beads were added to the reaction solution, and SPA signals were detected by a MicroBeta scintillation counter. No separation step is needed, which simplifies the assay procedure and greatly enhances the assay speed. Particularly, the miniaturization and robustness suggest that this method is suited for HTS of PRMT inhibitors.     

An affinity-based fluorescence polarization assay for protein tyrosine phosphatases

Protein tyrosine phosphatases (PTPs) are important signaling enzymes that control such fundamental processes as proliferation, differentiation, survival/apoptosis, as well as adhesion and motility. Potent and selective PTP inhibitors serve not only as powerful research tools, but also as potential therapeutics against a variety illness including cancer and diabetes. PTP activity-based assays are widely used in high throughput screening (HTS) campaigns for PTP inhibitor discovery. These assays suffer from a major weakness, in that the reactivity of the active site Cys can cause serious problems as highly reactive oxidizing and alkylating agents may surface as hits. We describe the development of a fluorescence polarization (FP)-based displacement assay that makes the use of an active site Cys to Ser mutant PTP (e.g., PTP1B/C215S) that retains the wild-type binding affinity. The potency of library compounds is assessed by their ability to compete with the fluorescently labeled active site ligand for binding to the Cys to Ser PTP mutant. Finally, the substitution of the active site Cys by a Ser renders the mutant PTP insensitive to oxidation and alkylation and thus will likely eliminate "false" positives due to modification of the active site Cys that destroy the phosphatase activity.     

High-throughput identification of inhibitors of human mitochondrial peptide deformylase

The human mitochondrial peptide deformylase (HsPDF) provides a potential new target for broadly acting antiproliferative agents. To identify novel nonpeptidomimetic and nonhydroxamic acid-based inhibitors of HsPDF, the authors have developed a high-throughput screening (HTS) strategy using a fluorescence polarization (FP)-based binding assay as the primary assay for screening chemical libraries, followed by an enzymatic-based assay to confirm hits, prior to characterization of their antiproliferative activity against established tumor cell lines. The authors present the results and performance of the established strategy tested in a pilot screen of 2880 compounds and the identification of the 1st inhibitors. Two common scaffolds were identified within the hits. Furthermore, cytotoxicity studies revealed that most of the confirmed hits have antiproliferative activity. These findings demonstrate that the designed strategy can identify novel functional inhibitors and provide a powerful alternative to the use of functional assays in HTS and support the hypothesis that HsPDF inhibitors may constitute a new class of antiproliferative agent.