The endoplasmic reticulum (ER), a multifunctional organelle, plays a central role in cellular signaling, development, and stress response. Dysregulation of ER homeostasis has been associated with human diseases, such as cancer, inflammation, and diabetes. A broad spectrum of stressful stimuli including hypoxia as well as a variety of pharmacological agents can lead to the ER stress response. In this study, we have developed a stable ER stress reporter cell line that stably expresses a β-lactamase reporter gene under the control of the ER stress response element (ESRE) present in the glucose-regulated protein, 78 kDa (GRP78) gene promoter. This assay has been optimized and miniaturized into a 1536-well plate format. In order to identify clinically used drugs that induce ER stress response, we screened approximately 2800 drugs from the NIH Chemical Genomics Center Pharmaceutical Collection (NPC library) using a quantitative high-throughput screening (qHTS) platform. From this study, we have identified several known ER stress inducers, such as 17-AAG (via HSP90 inhibition), as well as several novel ER stress inducers such as AMI-193 and spiperone. The confirmed drugs were further studied for their effects on the phosphorylation of eukaryotic initiation factor 2α (eIF2α), the X-box-binding protein (XBP1) splicing, and GRP78 gene expression. These results suggest that the ER stress inducers identified from the NPC library using the qHTS approach could shed new lights on the potential therapeutic targets of these drugs.

The cover picture shows a TR-FRET-based functional assay for screening small-molecule modulators of co-activator-associated arginine methyltransferase 1 (CARM1), based on its methylation of PABP1. The formation of the ternary complex (Me-GFP-PABP1, Me-PABP1 Ab, and Tb-2nd Ab) enables time-resolved detection of the long-lifetime terbium (Tb)-to-GFP FRET signal. Light at 340 nm is used to excite Tb, which emits at 495 nm. The proximity of the donor Tb to the acceptor GFP results in energy transfer from Tb to GFP, and this induces the specific FRET signal at 520 nm. On p. 827 ff., W. Xu et al. describe the development and optimization of the homogenous Me-GFP-PABP1-based TR-FRET assay for monitoring the expression and enzymatic activity of CARM1 in MCF7 breast cancer cells. The assay has been optimized for high-throughput screening to identify CARM1 allosteric activators. More importantly, this methodology has the potential to be extended to other protein arginine methyltransferases through the development of appropriate substrate–GFP fusion proteins and methylation-site-specific antibodies.

Epigenetics is an emerging field that demands selective cell‐permeable chemical probes to perturb, especially in vivo, the activity of specific enzymes involved in modulating the epigenetic codes. Coactivator‐associated arginine methyltransferase 1 (CARM1) is a coactivator of estrogen receptor α (ERα), the main target in human breast cancer. We previously showed that twofold overexpression of CARM1 in MCF7 breast cancer cells increased the expression of ERα‐target genes involved in differentiation and reduced cell proliferation, thus leading to the hypothesis that activating CARM1 by chemical activators might be therapeutically effective in breast cancer. Selective, potent, cell‐permeable CARM1 activators will be essential to test this hypothesis. Here we report the development of a cell‐based, time‐resolved (TR) FRET assay that uses poly(A) binding protein 1 (PABP1) methylation to monitor cellular activity of CARM1. The LanthaScreen TR‐FRET assay uses MCF7 cells expressing GFP‐PABP1 fusion protein through BacMam gene delivery system, methyl‐PABP1 specific antibody, and terbium‐labeled secondary antibody. This assay has been validated as reflecting the expression and/or activity of CARM1 and optimized for high throughput screening to identify CARM1 allosteric activators. This TR‐FRET platform serves as a generic tool for functional screening of cell‐permeable, chemical modulators of CARM1 for elucidation of its in vivo functions.

Mutations in LRRK2 (leucine-rich repeat kinase 2) have been linked to inherited forms of PD (Parkinson's disease). Substantial pre-clinical research and drug discovery efforts have focused on LRRK2 with the hope that small-molecule inhibitors of the enzyme may be valuable for the treatment or prevention of the onset of PD. The pathway to develop therapeutic or neuroprotective agents based on LRRK2 function (i.e. kinase activity) has been facilitated by the development of both biochemical and cell-based assays for LRRK2. LRRK2 is phosphorylated on Ser910, Ser935, Ser955 and Ser973 in the N-terminal domain of the enzyme, and these sites of phosphorylation are likely to be regulated by upstream enzymes in an LRRK2 kinase-activity-dependent manner. Knowledge of these phosphorylation sites and their regulation can be adapted to high-throughput-screening-amenable platforms. The present review describes the utilization of LRRK2 phosphorylation as indicators of enzyme inhibition, as well as how such assays can be used to deconvolute the pathways in which LRRK2 plays a role.

Implementing functional cell-based screens in early antibody discovery has become increasingly important to select antibodies with the desired profile. However, this is limited by assay tolerance to crude antibody preparations and assay sensitivity. The current study aims to address this challenge and identify routes forward. Two common types of high-throughput screening (HTS) antibody sample, derived from either phage display or hybridoma techniques, have been screened across a wide range of CellSensor beta-lactamase reporter assays in a variety of cell backgrounds to more extensively characterize assay tolerance. Pathway-, sample-, and cell background-specific effects were observed. Reporter assays for agonism were less affected by crude antibody preparations, with 8 of 21 sample tolerant, and the potential to implement an additional 8 assays by choosing the best-tolerated sample type. Antagonist mode assays exhibited more complexity, with potentiating as well as inhibitory effects. However, 5 of 24 antagonist assays were fully tolerant, with the potential to implement an additional 11 assays. Different subsets of assays were affected in agonist versus antagonist mode, and hybridoma sample sets were better tolerated overall. The study clearly demonstrates the potential to use cell-based reporter assays in biologics HTS, particularly if the method of antibody production is considered in the context of the required assay mode (agonist/antagonist).

Abstract Receptor tyrosine kinases (RTKs) are important pharmacological targets in oncology. Current biochemical assays designed to assess compound efficacy utilize truncated forms of the kinase lacking the transmembrane or extracellular domains and fail to address how these domains might affect the observed pharmacology. Purification of the full-length recombinant receptor can be both costly and difficult to scale for large screening campaigns. Additionally, because of the generic nature of most substrates used in activity assays for RTKs, small amounts of contaminating kinases can interfere with assay results. Numerous cell-based assays for pathway analysis can also serve as readouts for these targets, but they are not a direct measurement of kinase activity. To address these limitations, we have employed a BacMam-mediated gene delivery system to express full-length receptor tyrosine kinases with C-terminal green fluorescent protein (GFP) fusions in a variety of cellular backgrounds. Expression of these full-length RTKs has enabled the development of two complimentary assay platforms. First, we present a competitive displacement assay that uses a europium (Eu)-labeled anti-GFP antibody and an AlexaFluor® 647-labeled active site probe to characterize compound binding to the RTK in a lysate-based format. Here, the GFP moiety serves as an epitope tag to allow for very specific and sensitive detection of compound binding, discriminating compounds with sub-nanomolar affinity. Using the same BacMam RTK-GFP constructs, we have also developed a simple, addition-only, cell-based method to detect auto-phosphorylation of the RTK via TR-FRET between a terbium (Tb)-labeled anti-phospho-tyrosine antibody and the GFP moiety. Together, these tools can be used in a high-throughput screening format with a fluorescence-based readout to more fully characterize compound efficacy against full-length RTKs expressed in native cellular contexts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3881. doi:1538-7445.AM2012-3881

Background Mutations in the leucine-rich repeat kinase-2 (LRRK2) have been linked to Parkinson’s disease. Recent studies show that inhibition of LRRK2 kinase activity decreased the level of phosphorylation at its own Ser910 and Ser935, indicating that these sites are prime targets for cellular readouts of LRRK2 inhibition. Methodology/Principal Findings Using Time-Resolved Förster Resonance Energy Transfer (TR-FRET) technology, we developed a high-throughput cellular assay for monitoring LRRK2 phosphorylation at Ser935. LRRK2-Green Fluorescence Protein (GFP) fusions were expressed in cells via BacMam. Phosphorylation at Ser935 in these cells is detected using a terbium labeled anti-phospho-Ser935 antibody that generates a TR-FRET signal between terbium and GFP. LRRK2 wild-type and G2019S are constitutively phosphorylated at Ser935 in cells as measured by TR-FRET. The phosphorylation level is reduced for the R1441C mutant and little could be detected for the kinase-dead mutant D1994A. The TR-FRET cellular assay was further validated using reported LRRK2 inhibitors including LRRK2-IN-1 and our results confirmed that inhibition of LRRK2 can reduce the phosphorylation level at Ser935. To demonstrate the utility of this assay for screening, we profiled a small library of 1120 compounds. Three known LRRK2 inhibitors were identified and 16 hits were followed up in the TR-FRET and a cytotoxicity assay. Interestingly, out of the top 16 hits, five are known inhibitors of IκB phosphorylation, two CHK1 and two CDC25 inhibitors. Thirteen hits were further tested in a biochemical LRRK2 kinase activity assay and Western blot analysis for their effects on the phosphorylation of Ser910, Ser935, Ser955 and Ser973. Conclusions/Significance We developed a TR-FRET cellular assay for LRRK2 Ser935 phosphorylation that can be applied to the screening for LRRK2 inhibitors. We report for the first time that several compounds such as IKK16, CHK1 inhibitors and GW441756 can inhibit LRRK2 Ser935 phosphorylation in cells and LRRK2 kinase activity in vitro.

Abstract Post-translational modifications such as phosphorylation, acetylation and methylation play important roles in regulating the structures and functions of histones, which in turn regulate gene expression and DNA repair and replication. Histone modifying enzymes, such as deacetylases, methyltransferases and demethylases, have been pursued as therapeutic targets for various diseases. However, detection of the activities of these enzymes in high-throughput cell-based formats has remained challenging. We have developed high-throughput LanthaScreen® cellular assays for histone H3 site-specific modifications. These assays utilize cells expressing green fluorescence protein (GFP) tagged-histone H3 transiently delivered via BacMam and terbium labeled-anti-histone H3 modification-specific antibodies. Robust time-resolved Foerster resonance energy transfer signals were detected for H3 lysine-9 di-methylation (K9me2), lysine-4 di- (K4me2) and tri-methylation (K4me3) and lysine-27 tri-methylation (K27me3). Consistent with previous reports, hypoxic stress increased K4 methylation levels and methyltransferase G9a inhibitor UNC-0638 decreased K9me2 levels significantly with little effects on other modifications. Further validation of these assays using methyltransferases-specific RNAi oligos demonstrated that EZH2-specific RNAi reduced the level of K27me3 with little effect on the other three modifications, whereas G9a RNAi and SMYD3 RNAi reduced K9me2 and K4me2/3, respectively. The use of BacMam gene delivery system enables the measurement of these histone methylations in a variety of cell backgrounds including primary cells. In conclusion, we have developed homogenous cellular assays that can enable the high-throughput screening for modulators of histone H3 methylation at various lysine sites. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5006. doi:1538-7445.AM2012-5006

Upon genomic insult, the tumor suppressor p53 is phosphorylated and acetylated at specific serine and lysine residues, increasing its stability and transactivation function. Deacetylases, including the type III histone deacetylase SIRT1, remove acetyl groups from p53 and counterbalance acetyltransferase activity during a DNA damage response. This report describes a series of high-throughput LanthaScreen (R) time-resolved Forster resonance energy transfer (TR-FRET) immunoassays for detection of intracellular p53 phosphorylation of Ser15 and acetylation of Lys382 upon treatment with DNA damage agents, such as etoposide. These assays were used to measure the deacetylase activity of SIRT1 and/or Type I/II Histone deacetylases (HDACs). First, BacMam-mediated overexpression of SIRT1 resulted in dose-dependent deacetylation of GFP-p53 following etoposide treatment of U-2 OS cells, confirming that GFP-p53 serves as a SIRT1 substrate in this assay format. Further, overexpression of the acetyltransferase p300 via BacMam increased the acetylation of GFP-p53 at Lys382. Next, siRNA-mediated knockdown of SIRT1 resulted in increased GFP-p53 acetylation, indicating that endogenous SIRT1 activity can also be measured in U-2 OS cells. Consistent with these results, GFP-p53 acetylation was also increased upon treatment of cells with a small-molecule inhibitor of SIRT1, EX-527. The effect of this compound was dramatically increased when used in combination with chemotherapeutic drug and/or the HDAC inhibitor Trichostatin A, confirming a proposed synergistic mechanism of p53 deacetylation by SIRT1 and Type I/II HDACs. Taken together, the cellular assays described here can be used as high-throughput alternatives to traditional immunoassays such as western blotting for identifying pharmacological modulators of specific p53-modifying enzymes.

The nuclear receptor retinoid-related orphan receptor gamma (ROR gamma) has become an attractive target for drug discovery due to its important role in the development and differentiation of Th17 cells, a subset of T cells that produce interleukin-17 and are involved in the pathogenesis of human inflammatory and autoimmune diseases. To facilitate the drug discovery efforts in this area, we have developed a cellular assay for screening for ROR gamma inverse agonists. We stably engineered a tetracycline-inducible Gal4 DNA-binding domain/ROR gamma ligand-binding domain fusion protein into an upstream activation sequence driven-beta-lactamase reporter gene cell line. Due to its constitutive activity, the induced Gal4-ROR gamma expression leads to increased reporter activity, which can be knocked down using ROR gamma ligand-binding domain-specific RNA interference oligos. Using this assay, we tested several recently reported ligands for ROR gamma and observed varying levels of partial inverse agonist activity at mu M concentrations. Additionally, we screened a small library of biologically active compounds with this assay and demonstrated its robustness and usefulness in high-throughput screening and follow-up studies for this emerging drug target.

INTRODUCTION:Over the past decade, there has been an increased number of FDA approved small molecule kinase inhibitors for the treatment of cancer. This is due, in part, to an increased understanding of the fundamental aspects of kinase biology, coupled with advances in the methods used to study the inhibitory effects of small molecules on kinase activity. Underlying the development of these inhibitors are profiling methods that are used to assess the effect of potential compounds against their desired and undesired targets. The advancement of kinase profiling has stemmed from the development of basic assay technology that allows compounds to be tested against ever larger panels of kinases in a robust, cost-effective manner. Methods have also been developed that rapidly assess compound activity against specific activation states of kinases. There has also been a development of newer methods that move beyond traditional biochemical formats, which take a 'whole cell' approach to compound profiling.AREAS COVERED:This review provides an overview of traditional biochemical-based kinase profiling as well as an introduction to advances that have been made by moving compound profiling into a cell-based format.EXPERT OPINION:While central to the appropriate prioritization and optimization of compounds during the hit to lead phase of early-stage pharmaceutical development, every compound profiling format must be critically assessed so that one can make informed decisions through an understanding of their strengths and limitations. These decisions will ultimately be balanced against cost, complexity and its biological relevance.

Abstract Epigenetic histone modifications such as, methylation, acetylation, and phosphorylation play important roles in regulating the structure and functions of histones which in turn regulate essential gene expression. Dysregulation of the enzymes responsible for these modifications has been linked to cancer and many other diseases. In particular, histone deacetylases (HDACs) and histone methyl transferase (HMTs) have been a recent focus for drug discovery. However, detection of these enzymatic activities in an HTS cell-based format has remained challenging, especially since many of these enzymes may require the formation of protein complexes for relevant activity. To help overcome these challenges, we have developed and validated high-throughput compatible LanthaScreen® cellular assays for the analysis of histone methylation, acetylation, and phoshorylation, in cell backgrounds of interest. The acetylation, methylation and phosphorylation of H3 at specific residues can be detected with terbium-labeled antibodies against the specific site and modification. The expression of GFP-H3 substrate allows for detection of antibody binding in cell lysates via TR-FRET between the terbium and GFP. Since the actual modification takes place in the intact cell, native protein complexes that may be important for enzyme function are preserved. Together, these assays enable interrogation of multiple enzymatic activities responsible for epigenetic modifications of challenging histone targets Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 67. doi:10.1158/1538-7445.AM2011-67

Posttranslational modifications such as phosphorylation, acetylation, and methylation play important roles in regulating the structures and functions of histones, which in turn regulate gene expression and DNA repair and replication. Histone-modifying enzymes, such as deacetylases, methyltransferases and demethylases, have been pursued as therapeutic targets for various diseases. However, detection of the activities of these enzymes in high-throughput cell-based formats has remained challenging. The authors have developed high-throughput LanthaScreen cellular assays for Histone H3 site-specific modifications. These assays use cells expressing green fluorescence protein-tagged Histone H3 transiently delivered via BacMam and terbium-labeled anti-Histone H3 modification-specific antibodies. Robust time-resolved Förster resonance energy transfer signals were detected for H3 lysine-9 acetylation and dimethylation (H3K9me2), serine-10 phosphorylation, K4 di- and trimethylation, and K27 trimethylation. Consistent with previous reports, hypoxic stress increased K4 methylation levels, and methyltransferase G9a inhibitor UNC-0638 decreased K9me2 levels significantly, with little effects on other modifications. To demonstrate the utility of this assay platform in screening, the K9 acetylation assay was used to profile the Enzo Epigenetics Library. Twelve known HDAC inhibitors were identified as hits and followed up in a dose-response format. In conclusion, this assay platform enables high-throughput cell-based analysis of diverse types of posttranslational modifications of Histone H3.

Abstract Kinase activity-based assays are cost-effective and widely used to drive early drug discovery and lead identification, however, they typically require purified, active kinase preparations be produced that are shown to phosphorylate a known substrate. While it is possible to satisfy these requirements for the well-studied kinases, there are many kinases which lack one or more of these requirements, such as CDK8 and STK33, making drug discovery efforts difficult. To solve for these assay development problems, we have developed a TR-FRET-based binding assay platform which allows for characterization of compounds with kinases that are not sufficiently purified or do not demonstrate measurable activity against putative or potential substrates. This assay addresses kinase assay requirements early in the drug discovery process, and should enable more targets to be screened sooner, resulting in compounds being made available for lead optimization faster. The ability to detect and characterize binding of compounds to non-activated kinases is also a benefit to the lead optimization process, as compounds that bind preferentially to kinases in their non-activated state, such as Imatinib, are sometimes preferred due to their selectivity. In this poster, we present solutions for kinases that are difficult to address with activity assays and provide a comparison of compound affinities for active and non-activated forms of a diverse set of kinases to enable faster lead optimization. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C95.

The authors recently reported the development and application of multiple LanthaScreen cellular assays to interrogate specific steps within the PI3K/Akt pathway. The importance of this signaling cascade in regulating fundamental aspects of cell growth and survival, as well as in the progression of cancer, underscores the need for portable cell-based assays for compound profiling in multiple disease-relevant cell backgrounds. To meet this need, the authors have now expanded their LanthaScreen assay platform across a variety of cell types using a gene delivery technology known as BacMam. Here, they have demonstrated the successful detection of Akt-dependent phosphorylation of PRAS40 at Thr246 in 10 different cell lines harboring mutations known to activate the PI3K/Akt pathway. In addition, they generated inhibitory profiles of 17 known pathway inhibitors in these same cells to validate the approach of using the BacMam-enabled LanthaScreen cellular assay format to rapidly profile compounds in disease-relevant cell types. Importantly, their results provide a broad illustration of how the genetic alterations that affect PI3K/Akt signaling can also influence the inhibitory profile of a given compound.

Abstract Post-translational modifications such as phosphorylation, acetylation and ubquitination play important roles in regulating the structure and functions of histones and transcription factors such as p53, which in turn regulate essential gene expression. Dysregulation of these post-translational modifications has been linked to cancer and metabolic diseases. In particular, histone deacetylases (HDACs) have been pursued as valuable targets for various therapeutic interventions. However, detection of these enzymatic activities in a cell-based format has remained intractable using HTS-compatible technologies. To enable the drug discovery for these post-translational modification enzymes, we have developed and validated high-throughput compatible LanthaScreen® cellular assays for the analysis of histone and/or p53-specific acetylation, ubquitination and phoshorylation in cell backgrounds of interest. The cell cycle-dependent acetylation of histone H3 at Lys9 and the phosphorylation of histone H3 at Ser10 can be detected with terbium-labeled anti-Histone H3 acetyl-lys 9 and anti-phospho-Ser10 specific antibodies, respectively. In addition, poly-ubiquitination of Histone H2B can be measured with terbium-labeled anti-poly-ubiquitin antibody. We have also applied this technology and developed cellular assays for measuring DNA-damage induced phosphorylation at Ser15 and acetylation at Lys382 of p53. These assays were further validated with both small molecule inhibitor and siRNA against specific HDAC family members. Our results suggest that the deacetylation of Histone H3 Lys 9 is mediated by type I/II HDACs, whereas the deacetylation of p53 at Lys382 is mediated synergistically by both SIRT1 and type I/II HDAC activities. Together, these assays enable interrogation of multiple enzymatic activities responsible for post-translational modifications of challenging targets such as histones and p53. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4867.

Signaling pathways and their protein target constituents (e.g. kinases) have become important therapeutic targets in many disease areas. Traditional selectivity profiling for kinase inhibitors has relied upon screening panels of recombinant enzymes in biochemical assay formats. Recent studies have highlighted the importance of using cellular assays to better approximate true biological selectivity. We have developed a portfolio of CellSensor beta-lactamase transcriptional reporter gene assays that can be used to screen for perturbagens of various endogenous signaling pathways. Here we describe a multi-pathway profiling approach for generating compound-pathway selectivity maps. To demonstrate the utility of this approach, we have screened 32 known compounds across a diverse panel of 12 key signaling pathways and generated the first comprehensive cellular pathway selectivity profiles of several clinically approved kinase and other well-known bioactive inhibitors. Selectivity score comparisons identified several kinase inhibitors that were more promiscuous than predicted by traditional biochemical profiling methods. For example, we identified effects of sorafenib on the JAK/STAT pathway and demonstrated the potential therapeutic indication of sorafenib in treating leukemia/myeloproliferative disorder patients harboring TEL-JAK2 or JAK2V617F mutations. Our results indicate that multi-pathway profiling can efficiently characterize both on and off-pathway compound activities, revealing potential novel pathways and opportunities for drug repositioning purposes and/or safety liabilities in one profiling campaign.

Moderate environmental and physiological stressors are known to initiate protective heat shock response (HSR) leading to cell survival. HSR is largely mediated by the activation of heat shock factor (HSF), resulting in increased heat shock protein expression. Dysregulation of the HSR signaling has been associated with various diseases including cancer, inflammation and neurodegenerative disorders. Compounds that can modulate HSR have been pursued for the treatment of these diseases. To facilitate the discovery of HSR modulators, we developed a high-throughput amenable betalactamase transcriptional reporter gene assay for monitoring the function of HSF. HeLa cells were engineered to express the beta-lactamase reporter under the control of HSF response elements (HSE) present in the HSP70 gene promoter. The HSE-beta lactamase (HSE-bla) reporter gene assay was validated by using HSF-specific siRNAs and known small molecule modulators. Taking the advantage of fluorescence resonance energy transfer (FRET)-based cell permeable betalactamase substrate, this assay can be miniaturized into 1536-well format. Our results demonstrate that the assay is robust and can be applied to high-throughput screening (HTS) for modulators of HSR.