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The epidermal growth factor receptor (EGFR) pathway is one of the most deregulated molecular pathways in human epithelial cancers. Many approved drugs were optimized to directly target EGFR but yielded only modest clinical improvement in cancer patients due to low efficacy and drug resistance. Transactivation of EGFR by other cell surface receptors such as G-protein-coupled receptors (GPCRs) was proposed to explain this lack of efficacy. Even if direct EGFR activation and transactivation by GPCR contribute to the activation of the same signaling pathways, they are often studied as independent events resulting in partial investigation of a drug's mechanism of action. We present a novel high-throughput approach that integrates interrogation of direct activation of EGFR and its transactivation via GPCR activation. Using distinct technology platforms, three readouts were used to measure (1) direct activation of GPCR via cyclic adenosine monophosphate (cAMP) detection, (2) direct activation of EGFR through the release of intracellular Ca(2+), and (3) EGFR transactivation by GPCR using the detection of p-extracellular-signal-regulated kinases 1/2 (p-ERK1/2). In addition to being simple, quick, and homogenous, our methods were shown to be more sensitive than those in current use. These enabling tools should improve the knowledge pertaining to GPCRs and receptor tyrosine kinases trans-regulation and facilitate the design of more potent and better targeted new therapeutic strategies.

Assay technologies that were originally developed for high-throughput screening (HTS) have recently proven useful in drug discovery for activities located upstream (target identification and validation) and downstream (ADMET) of HTS. Here the authors investigated and characterized the biological properties of a novel target, IRE1alpha, a bifunctional kinase/RNase stress sensor of the endoplasmic reticulum (ER). They have developed a novel assay platform using the HTS technology AlphaScreen to monitor the dimerization/oligomerization and phosphorylation properties of the cytosolic domain of IRE1alpha. They show in vitro that dimerization/oligomerization of the cytosolic domain of IRE1 correlated with the autophosphorylation ability of this domain and its endoribonuclease activity toward XBP1 mRNA. Using orthogonal in vitro and cell-based approaches, the authors show that the results obtained using AlphaScreen were biologically relevant. Preliminary characterization of assay robustness indicates that both AlphaScreen assays should be useful in HTS for the identification of IRE1 activity modulators.

AlphaScreen (Amplified Luminescent Proximity Homogeneous Assay Screen) is versatile assay technology developed to measuring analytes using a homogenous protocol. This technology is an example of a bead-based proximity assay and was developed from a diagnostic assay technology known as LOCI (Luminescent Oxygen Channeling Assay). Here, singlet oxygen molecules, generated by high energy irradiation of Donor beads, travel over a constrained distance (approx. 200 nm) to Acceptor beads. This results in excitation of a cascading series of chemical reactions, ultimately causing generation of a chemiluminescent signal. In the past decade, a wide variety of applications has been reported, ranging from detection of analytes involved in cell signaling, including protein:protein, protein:peptide, protein:small molecule or peptide:peptide interactions. Numerous homogeneous HTS-optimized assays have been reported using the approach, including generation of second messengers (such as accumulation of cyclic AMP, cyclic GMP, inositol [1, 4, 5] trisphosphate or phosphorylated ERK) from liganded GPCRs or tyrosine kinase receptors, post-translational modification of proteins (such as proteolytic cleavage, phosphorylation, ubiquination and sumoylation) as well as protein-protein and protein-nucleic acid interactions. Recently, the basic AlphaScreen technology was extended in that the chemistry of the Acceptor bead was modified such that emitted light is more intense and spectrally defined, thereby markedly reducing interference from biological fluid matrices (such as trace hemolysis in serum and plasma). In this format, referred to as AlphaLISA, it provides an alternative technology to classical ELISA assays and is suitable for high throughput automated fluid dispensing and detection systems. Collectively, AlphaScreen and AlphaLISA technologies provide a facile assay platform with which one can quantitate complex cellular processes using simple no-wash microtiter plate based assays. They provide the means by which large compound libraries can be screened in a high throughput fashion at a diverse range of therapeutically important targets, often not readily undertaken using other homogeneous assay technologies. This review assesses the current status of the technology in drug discovery, in general, and high throughput screening (HTS), in particular.

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.

The interaction between nuclear receptors (NRs) and their coactivators, a key step in transcription regulation, requires a short consensus sequence called the LXXLL motif found in the coactivators' structure. Using the AlphaScreen technology, the authors have taken advantage of this receptor-coactivator interaction to develop a highly sensitive assay to identify and characterize compounds modulating NR activity. Estrogen and retinoic acid receptors were chosen as models to demonstrate the versatility of the AlphaScreen technology: (1) the assay can be designed using different antibodies to capture either full-length receptors or receptor domains that have been tagged, (2) the assay can differentiate between ligands that act as agonists or antagonists because only agonists will allow recruitment of the coactivator sequence-derived peptide, and (3) the assay gives the opportunity to screen for antagonists targeting the ligand-binding site or the dimerization interface between the receptor and the coactivator. Titration of the receptor and biotinylated peptide indicates that AlphaScreen is highly sensitive, requiring nanomolar concentration of reagents. Competition isotherms performed with known receptor antagonists demonstrate that the assay is a useful tool to rank the antagonists according to their order of potency. Overall, the results presented here indicate that the versatility, sensitivity, robustness, and ease of execution of the AlphaScreen NR assay will allow for efficient screening of NR modulators.

Nuclear receptors, including the androgen receptor (AR), regulate target cell transcription through interaction with auxiliary proteins to modify chromatin structure. We describe herein a novel AR-interacting protein, termed ARIP4, that has structural features typical of the SNF2-like protein family. With regard to the Snf2 domain, the closest homolog of ARIP4 is the ATRX protein. ARIP4 is a nuclear protein and comprises 1466 amino acids. It interacts with AR in vitro and in cultured yeast and mammalian cells. ARIP4 can be labeled with 8-azido-[γ-32P]ATP and exhibits DNA-dependent ATPase activity. Like several ATP-dependent chromatin remodeling proteins, ARIP4 generates superhelical torsion within linear DNA fragments in an ATP-dependent manner. With a stably integrated target promoter, ARIP4 elicits a modest enhancement of AR-dependent transactivation. In transient cotransfection assays, ARIP4 modulates AR function in a promoter-dependent manner; it enhances receptor activity on minimal promoters, but does not activate more complex promoters. ARIP4 mutants devoid of ATPase activity fail to alter DNA topology and behave as trans-dominant negative regulators of AR function in transient assays.

Small ubiquitin-like proteins (SUMO) are recently discovered post-translational modifiers that regulate protein functions and intracellular trafficking. In this study, we are describing two chemoluminescence-based assays, one for SUMOylation and another one for SUMO-mediated protein-protein interactions. These assays can be used to characterize the activity and kinetics of the enzymes that catalyze SUMOylation, and in high-throughput screening for inhibitors of SUMOylation and SUMO-dependent protein-protein interactions. These novel assays represent the most sensitive assays for ubiquitin-like systems published to date. Similar strategies can be used to develop assays for other ubiquitin-like modification systems.

The 21st Century Bioanalytical Laboratory Platforms initiative in the BIOTEC section of the American Association of Pharmaceutical Scientists (AAPS) began with a pre-conference workshop held in Seattle, WA in June of 2009. This workshop brought together members of the pharmaceutical and biotechnology industries, with instrument and reagent manufacturers to discuss the current and potential future state of the bioanalytical laboratories supporting biologics development. At the conclusion of the workshop, four sub-teams were formed to further develop the ideas and concepts raised during the 2-day workshop. The sub-teams are reagents, automation, e-solutions, and platforms. This paper discusses the critical attributes of a research and development ligand binding assay (LBA) platform and the desired characteristics new platforms should strive to offer in the future. This paper is not intended to be a review and comparison of the current platforms on the market, as this has been done and published elsewhere (1–9). The platforms team consists of a balanced cross-section of the industry with representatives from pharmaceutical, biotechnology, contract research organizations, and instrument manufacturers. The Platforms team have collaborated to discuss and arrive at a consensus regarding the most useful characteristics of a bioanalytical platform for biologics. We present here the results of these discussions. A platform is the technology employed in an analytical method to transduce a biochemical event into a measureable output or signal. This signal allows the bioanalytical scientist to accurately and reproducibly make measurements to analyze different aspects of a specific biologic target (therapeutic, biomarker, and anti-drug antibody) such as its pharmacokinetics, immunogenicity, potency, or effect of biomarkers. An instrument is the tool utilized minimally to measure a platform’s output and convert the resultant signal into interpretable information the analytical scientist can use but can incorporate other aspects such as liquid handling. Many platforms employ optical signals including the absorbance of light through a medium (10) or the emission of fluorescence (10) or luminescence (11). A variety of light detectors are used to measure these optical signals including photo diodes, charge-coupled device cameras, and photo-multiplier tubes (Table I). Sections in this paper provide details on the desirable analytical characteristics, multiplexing, platform flexibility and throughput, desirable instrument characteristics, and finally, life cycle management of the ideal LBA platform. Table I Commonly Used Platforms The analytical characteristics of today’s ligand binding assays are primarily influenced by three major factors—the quality of the reagents, assay format, and the choice of the analytical platform. This paper describes only those aspects derived from the analytical platform.

Histone posttranslational modifications are among the epigenetic mechanisms that modulate chromatin structure and gene transcription. Histone methylation and demethylation are dynamic processes controlled respectively by histone methyltransferases (HMTs) and demethylases (HDMs). Several HMTs and HDMs have been implicated in cancer, inflammation, and diabetes, making them attractive targets for drug therapy. Hence, the discovery of small-molecule modulators for these two enzyme classes has drawn significant attention from the pharmaceutical industry. Herein, the authors describe the development and optimization of homogeneous LANCE Ultra and AlphaLISA antibody-based assays for measuring the catalytic activity of two epigenetic enzymes acting on lysine 4 of histone H3: SET7/9 methyltransferase and LSD1 demethylase. Both the SET7/9 and LSD1 assays were designed as signal-increase assays using biotinylated peptides derived from the N-terminus of histone H3. In addition, the SET7/9 assay was demonstrated using full-length histone H3 protein as substrate in the AlphaLISA format. Optimized assays in 384-well plates are robust (Z' factors ≥0.7) and sensitive, requiring only nanomolar concentrations of enzyme and substrate. All assays allowed profiling of known SET7/9 and LSD1 inhibitors. The results demonstrate that the optimized LANCE Ultra and AlphaLISA assay formats provide a relevant biochemical screening approach toward the identification of small-molecule inhibitors of HMTs and HDMs that could lead to novel epigenetic therapies.

Histone proteins are an integral part of DNA packaging into chromatin, a dynamic process partly regulated by post-translation modification of histone N-terminal tails. Aberrant histone acetylation (ac) or methylation (me) levels is associated with a number of diseases. For example, histone deacetylases (HDACs and sirtuins) regulate the expression of many genes involved in neurodegeneration and can be aberrantly expressed in different tumors. In addition, deregulation of the methyl marks H3K4me3 and H3K27me3 are found associated with the development of several types of cancer. This work presents the development of high-throughput AlphaLISA® in cyto assays to monitor four specific histone marks: H3K9ac, H3K4me2, H3K27ac and H3K27me3. These assays were performed using an all-in-one well histone extraction protocol requiring no acid extraction or centrifugation steps. The level of each histone mark was first measured in cell titration experiments, seeding cells from 500 to 10 000 in 384 well plates. The chemical modulation of H3K4me2, H3K9ac and H3K27ac was monitored following overnight treatment of HeLa, HEK293, and Jurkat cells with the non-selective histone deacetylase inhibitors sodium butyrate and trichostatin A. Signal increases were all corroborated by Western blot analysis using the same antibodies as in the AlphaLISA detection assay. In the absence of chemical modulation, measurement of different H3K27me3 mark levels was performed in two B cell lymphoma cell lines: OCI-LY-19 and SU-DHL-6. OCI-LY-19 cells express wild-type EZH2 methyltransferase while SU-DHL-6 cells bear a heterozygous EZH2 mutation (Y641N) that alters its substrate selectivity, resulting in increased H3K27me3 levels. These novel cell-based assays showed suitability for high-throughput screening (HTS) protocols as demonstrated by Z’ factors superior to 0.6 for all four marks. In summary, these homogeneous cell-based AlphaLISA assays allow the simple and rapid monitoring of cellular histone H3 mark levels. 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 3879. doi:1538-7445.AM2012-3879