Development of a High-Throughput Fluorescence Polarization Assay to Identify Novel Ligands of Glutamate Carboxypeptidase II

Glutamate carboxypeptidase II (GCPII) is an important target for therapeutic and diagnostic interventions aimed at prostate cancer and neurologic disorders. Here we describe the development and optimization of a high-throughput screening (HTS) assay based on fluorescence polarization (FP) that facilitates the identification of novel scaffolds inhibiting GCPII. First, we designed and synthesized a fluorescence probe based on a urea-based inhibitory scaffold covalently linked to a Bodipy TMR fluorophore (TMRGlu). Next, we established and optimized conditions suitable for HTS and evaluated the assay robustness by testing the influence of a variety of physicochemical parameters (e.g., pH, temperature, time) and additives. Using known GCPII inhibitors, the FP assay was shown to be comparable to benchmark assays established in the field. Finally, we evaluated the FP assay by HTS of a 20 000-compound library. The novel assay presented here is robust, highly reproducible (Z' = 0.82), inexpensive, and suitable for automation, thus providing an excellent platform for HTS of small-molecule libraries targeting GCPII.     

A robust screen for inhibitors and enhancers of phosphoinositide-3 kinase (PI3K) activities by ratiometric fluorescence superquenching

Aberrant regulation of phosphoinositide 3-kinase (PI3K) activity is implicated in various diseases such as cancer and diabetes. Thus, high-throughput screening (HTS) of small-molecule inhibitors for PI3 kinases is an appealing strategy for drug development. Despite the attractiveness of lipid kinases as drug targets, screening for inhibitors for PI3K activities has been hampered by limited assay formats adaptable for HTS. The authors describe a homogeneous, direct, and nonradioactive assay for highly sensitive detection of PI3Kalpha, beta, delta, and gamma activities, which is suitable for HTS. The assay is based on fluorescence superquenching of a conjugated polymer upon metal-ion-mediated association of phosphorylated and dye-labeled substrates. As a result of phosphorylation, quencher and polymer are brought into proximity, and fluorescent energy transfer occurs. This event can be monitored as either fluorescence quench of the polymer or as enhanced emission from the quencher. Ratiometric analysis of the wavelengths eliminates interferences from autofluorescing compounds, which are present in HTS libraries. The platform has been adapted for the 384-well microplate format and delivers Z factors of > 0.6 at substrate conversions as low as 7%. Using this assay platform, several unreported inhibitors and activators of PI3Ks were identified in an 84- compound screen.     

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.     

Identification of novel drug scaffolds for inhibition of SARS-CoV 3-Chymotrypsin-like protease using virtual and high-throughput screenings

We have used a combination of virtual screening (VS) and high-throughput screening (HTS) techniques to identify novel, non-peptidic small molecule inhibitors against human SARS-CoV 3CLpro. A structure-based VS approach integrating docking and pharmacophore based methods was employed to computationally screen 621,000 compounds from the ZINC library. The screening protocol was validated using known 3CLpro inhibitors and was optimized for speed, improved selectivity, and for accommodating receptor flexibility. Subsequently, a fluorescence-based enzymatic HTS assay was developed and optimized to experimentally screen approximately 41,000 compounds from four structurally diverse libraries chosen mainly based on the VS results. False positives from initial HTS hits were eliminated by a secondary orthogonal binding analysis using surface plasmon resonance (SPR). The campaign identified a reversible small molecule inhibitor exhibiting mixed-type inhibition with a K_i value of 11.1 μM. Together, these results validate our protocols as suitable approaches to screen virtual and chemical libraries, and the newly identified compound reported in our study represents a promising structural scaffold to pursue for further SARS-CoV 3CLpro inhibitor development.     

High-throughput fluorescence assay for small-molecule inhibitors of autophagins/Atg4

Autophagy is an evolutionarily conserved process for catabolizing damaged proteins and organelles in a lysosome-dependent manner. Dysregulation of autophagy may cause various diseases, such as cancer and neurodegeneration. However, the relevance of autophagy to diseases remains controversial because of the limited availability of chemical modulators. Herein, the authors developed a fluorescence-based assay for measuring activity of the autophagy protease, autophagin-1(Atg4B). The assay employs a novel reporter substrate of Atg4B composed of a natural substrate (LC3B) fused to an assayable enzyme (PLA(2)) that becomes active upon cleavage by this cysteine protease. A high-throughput screening (HTS) assay was validated with excellent Z' factor (>0.7), remaining robust for more than 5 h and suitable for screening of large chemical libraries. The HTS assay was validated by performing pilot screens with 2 small collections of compounds enriched in bioactive molecules (n = 1280 for Lopac? and 2000 for Spectrum? library), yielding confirmed hit rates of 0.23% and 0.70%, respectively. As counterscreens, PLA(2) and caspase-3 assays were employed to eliminate nonspecific inhibitors. In conclusion, the LC3B-PLA(2) reporter assay provides a platform for compound library screening for identification and characterization of Atg4B-specific inhibitors that may be useful as tools for interrogating the role of autophagy in disease models.     

A novel flow cytometric HTS assay reveals functional modulators of ATP binding cassette transporter ABCB6

ABCB6 is a member of the adenosine triphosphate (ATP)-binding cassette family of transporter proteins that is increasingly recognized as a relevant physiological and therapeutic target. Evaluation of modulators of ABCB6 activity would pave the way toward a more complete understanding of the significance of this transport process in tumor cell growth, proliferation and therapy-related drug resistance. In addition, this effort would improve our understanding of the function of ABCB6 in normal physiology with respect to heme biosynthesis, and cellular adaptation to metabolic demand and stress responses. To search for modulators of ABCB6, we developed a novel cell-based approach that, in combination with flow cytometric high-throughput screening (HTS), can be used to identify functional modulators of ABCB6. Accumulation of protoporphyrin, a fluorescent molecule, in wild-type ABCB6 expressing K562 cells, forms the basis of the HTS assay. Screening the Prestwick Chemical Library employing the HTS assay identified four compounds, benzethonium chloride, verteporfin, tomatine hydrochloride and piperlongumine, that reduced ABCB6 mediated cellular porphyrin levels. Validation of the identified compounds employing the hemin-agarose affinity chromatography and mitochondrial transport assays demonstrated that three out of the four compounds were capable of inhibiting ABCB6 mediated hemin transport into isolated mitochondria. However, only verteporfin and tomatine hydrochloride inhibited ABCB6's ability to compete with hemin as an ABCB6 substrate. This assay is therefore sensitive, robust, and suitable for automation in a high-throughput environment as demonstrated by our identification of selective functional modulators of ABCB6. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel modulators of ABCB6 activity.     

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.     

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.     

TR-FRET-based high-throughput screening assay for identification of UBC13 inhibitors

UBC13 is a noncanonical ubiquitin conjugating enzyme (E2) that has been implicated in a variety of cellular signaling processes due to its ability to catalyze formation of lysine 63-linked polyubiquitin chains on various substrates. In particular, UBC13 is required for signaling by a variety of receptors important in immune regulation, making it a candidate target for inflammatory diseases. UBC13 is also critical for double-strand DNA repair and thus a potential radiosensitizer and chemosensitizer target for oncology. The authors developed a high-throughput screening (HTS) assay for UBC13 based on the method of time-resolved fluorescence resonance energy transfer (TR-FRET). The TR-FRET assay combines fluorochrome (Fl)-conjugated ubiquitin (fluorescence acceptor) with terbium (Tb)-conjugated ubiquitin (fluorescence donor), such that the assembly of mixed chains of Fl- and Tb-ubiquitin creates a robust TR-FRET signal. The authors defined conditions for optimized performance of the TR-FRET assay in both 384- and 1536-well formats. Chemical library screens (total 456 865 compounds) were conducted in high-throughput mode using various compound collections, affording superb Z' scores (typically >0.7) and thus validating the performance of the assays. Altogether, the HTS assays described here are suitable for large-scale, automated screening of chemical libraries in search of compounds with inhibitory activity against UBC13.     

Development of FRET-based high-throughput screening for viral RNase III inhibitors

The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose–response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.     

Development of a microplate-based, electrophoretic fluorescent protein kinase a assay: comparison with filter-binding and fluorescence polarization assay formats

A microplate-based electrophoretic assay has been developed for the serine/threonine kinase protein kinase A (PKA). The ElectroCapture PKA assay developed uses a positively charged, lissamine-rhodamine-labeled kemptide peptide substrate for the kinase reaction and Nanogen's ElectroCapture HTS Workstation and 384-well laminated membrane plates to electrophoretically separate the negatively charged phosphorylated peptide product from the kinase reaction mix. After the electrophoretic separation, the amount of rhodamine-labeled phosphopeptide product was quantified using a Tecan Ultra384 fluorescence reader. The ElectroCapture PKA assay was validated with both known PKA inhibitors and library compounds. The pK(iapp) results obtained in the ElectroCapture PKA assay were comparable to those generated with current radioactive filter-binding assay and antibody-based competitive fluorescence polarization PKA assay formats.     

A time-resolved fluorescence resonance energy transfer assay suitable for high-throughput screening for inhibitors of immunoglobulin E–receptor interactions

The interaction of immunoglobulin E (IgE) antibodies with the high-affinity receptor, FcεRI, plays a central role in initiating most allergic reactions. The IgE–receptor interaction has been targeted for treatment of allergic diseases, and many high-affinity macromolecular inhibitors have been identified. Small molecule inhibitors would offer significant advantages over current anti-IgE treatment, but no candidate compounds have been identified and fully validated. Here, we report the development of a time-resolved fluorescence resonance energy transfer (TR–FRET) assay for monitoring the IgE–receptor interaction. The TR–FRET assay measures an increase in fluorescence intensity as a donor lanthanide fluorophore is recruited into complexes of site-specific Alexa Fluor 488-labeled IgE-Fc and His-tagged FcεRIα proteins. The assay can readily monitor classic competitive inhibitors that bind either IgE-Fc or FcεRIα in equilibrium competition binding experiments. Furthermore, the TR–FRET assay can also be used to follow the kinetics of IgE-Fc–FcεRIα dissociation and identify inhibitory ligands that accelerate the dissociation of preformed complexes, as demonstrated for an engineered DARPin (designed ankyrin repeat protein) inhibitor. The TR–FRET assay is suitable for high-throughput screening (HTS), as shown by performing a pilot screen of the National Institutes of Health (NIH) Clinical Collection Library in a 384-well plate format.     

Development and validation of a high-throughput screen for inhibitors of SARS CoV and its application in screening of a 100,000-compound library

The authors have developed a high-throughput screen (HTS) that allows for the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries. The luminescent-based assay measures the inhibition of SARS CoV-induced cytopathic effect (CPE) in Vero E6 cells. The assay was validated in 96-well plates in a BSL3 containment facility. The assay is sensitive and robust, with Z values > 0.6, signal to background (S/B) > 16, and signal to noise (S/N) > 3. The assay was further validated with 2 different diversity sets of compounds against the SARS CoV. The "hit" rate for both libraries was approximately 0.01%. The validated HTS assay was then employed to screen a 100,000-compound library against SARS CoV. The hit rate for the library in a single-dose format was determined to be approximately 0.8%. Screening of the 3 libraries resulted in the identification of several novel compounds that effectively inhibited the CPE of SARS CoV in vitro-compounds which will serve as excellent lead candidates for further evaluation. At a 10-microM concentration, 3 compounds with selective indexes (SI50) of > 53 were discovered.     

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.     

A high-content biosensor-based screen identifies cell-permeable activators and inhibitors of EGFR function: implications in drug discovery

Early success of kinase inhibitors has validated their use as drugs. However, discovery efforts have also suffered from high attrition rates due to lack of cellular activity. We reasoned that screening for such candidates in live cells would identify novel cell-permeable modulators for development. For this purpose, we have used our recently optimized epidermal growth factor receptor (EGFR) biosensor assay to screen for modulators of EGFR activity. Here, we report on its validation under high-throughput screening (HTS) conditions displaying a signal-to-noise ratio of 21 and a Z' value of 0.56-attributes of a robust cell-based assay. We performed a pilot screen against a library of 6912 compounds demonstrating good reproducibility and identifying 82 inhibitors and 66 activators with initial hit rates of 1.2% and 0.95%, respectively. Follow-up dose-response studies revealed that 12 of the 13 known EGFR inhibitors in the library were confirmed as hits. ZM-306416, a vascular endothelial growth factor receptor (VEGFR) antagonist, was identified as a potent inhibitor of EGFR function. Flurandrenolide, beclomethasone, and ebastine were confirmed as activators of EGFR function. Taken together, our results validate this novel approach and demonstrate its utility in the discovery of novel kinase modulators with potential use in the clinic.     

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.     

High-throughput fluorescence polarization assay for chemical library screening against anti-apoptotic Bcl-2 family member Bfl-1

Overexpression of the anti-apoptotic Bcl-2 family proteins occurs commonly in human cancers. Bfl-1 is highly expressed in some types of malignant cells, contributing significantly to tumor cell survival and chemoresistance. Therefore, it would be desirable to have chemical antagonists of Bfl-1. To this end, we devised a fluorescence polarization assay (FPA) using Bfl-1 protein and fluorescein-conjugated Bid BH3 peptide, which was employed for high-throughput screening of chemical libraries. Approximately 66 000 compounds were screened for the ability to inhibit BH3 peptide binding to Bfl-1, yielding 14 reproducible hits with ≥50% displacement. After dose-response analysis and confirmation using a secondary assay based on time-resolved fluorescence resonance energy transfer (TR-FRET), two groups of Bfl-1-specific inhibitors were identified, including chloromaleimide and sulfonylpyrimidine series compounds. FPAs generated for each of the six anti-apoptotic Bcl-2 proteins demonstrated selective binding of both classes of compounds to Bfl-1. Analogs of the sulfonylpyrimidine series were synthesized and compared with the original hit for Bfl-1 binding by both FPAs and TR-FRET assays. The resulting structure-activity relation analysis led to the chemical probe compound CID-2980973 (ML042). Collectively, these findings demonstrate the feasibility of using the HTS assay for discovery of selective chemical inhibitors of Bfl-1.