Time-resolved Förster Resonance Energy Transfer Assays for Measurement of Endogenous Phosphorylated STAT Proteins in Human Cells.

The Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling pathway plays a crucial role in mediating cellular responses to cytokines and growth factors. STAT proteins are activated by tyrosine phosphorylation mediated mainly by JAKs. The abnormal activation of STAT signaling pathways is implicated in many human diseases, especially cancer and immune-related conditions. Therefore, the ability to monitor STAT protein phosphorylation within the native cell signaling environment is important for both academic and drug discovery research. The traditional assay formats available to quantify phosphorylated STAT proteins include western blotting and the enzyme-linked immunosorbent assay (ELISA). These heterogeneous methods are labor-intensive, low-throughput, and often not reliable (specific) in the case of western blotting. Homogeneous (no-wash) methods are available but remain expensive. Here, detailed protocols are provided for the sensitive, robust, and cost-effective measurement in a 384-well format of endogenous levels of phosphorylated STAT1 (Y701), STAT3 (Y705), STAT4 (Y693), STAT5 (Y694/Y699), and STAT6 (Y641) in cell lysates from adherent or suspension cells using the novel THUNDER time-resolved Förster resonance energy transfer (TR-FRET) platform. The workflow for the cellular assay is simple, fast, and designed for high-throughput screening (HTS). The assay protocol is flexible, uses a low-volume sample (15 µL), requires only one reagent addition step, and can be adapted to low-throughput and high-throughput applications. Each phospho-STAT sandwich immunoassay is validated under optimized conditions with known agonists and inhibitors and generates the expected pharmacology and Z'-factor values. As TR-FRET assays are ratiometric and require no washing steps, they provide much better reproducibility than traditional approaches. Together, this suite of assays provides new cost-effective tools for a more comprehensive analysis of specific phosphorylated STAT proteins following cell treatment and the screening and characterization of specific and selective modulators of the JAK/STAT signaling pathway.

Development of homogeneous nonradioactive methyltransferase and demethylase assays targeting histone H3 lysine 4.

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.

A sensitive, homogeneous, and high-throughput assay for lysine-specific histone demethylases at the H3K4 site.

Histone methylation is a regulated feature of nucleosomes that can have an impact on gene expression. The methylation state of histone residues has also been found in recent years to be associated with various disorders. Tools for detecting methylation state changes are very useful for dissecting the function of these epigenetic marks. In this work, a sensitive homogeneous assay for histone demethylase activity at the H3K4 site has been developed in a time-resolved fluorescent resonance energy transfer assay format. The assay is based on the detection of the unmethylated H3 peptide by a fluorescent europium-chelate labeled monoclonal antibody binding specifically to the H3K4 site. The assay was validated for histone lysine-specific demethylase 1 and was demonstrated to be a suitable assay for inhibitor profiling and high-throughput screening.

Development of a high-throughput AlphaScreen assay measuring full-length LRRK2(G2019S) kinase activity using moesin protein substrate.

Mutations within the LRRK2 (leucine-rich repeat kinase 2) gene predispose humans to develop late-onset Parkinson's disease (PD). The most prevalent of these mutations, G2019S, has been shown to increase LRRK2 kinase activity. Therefore, the discovery of small molecule inhibitors of LRRK2(G2019S) through high-throughput screening (HTS) may provide a novel therapeutic strategy for treating PD. Current biochemical assays monitoring the activity of LRRK2(G2019S) either are radioactive or use short peptidic substrates. Here we describe the development and optimization of a novel HTS AlphaScreen assay for measuring the catalytic activity of full-length LRRK2(G2019S) using its putative physiological protein substrate moesin. The high sensitivity of this optimized 384-well assay allowed the use of enzyme concentrations as low as 0.75nM. The estimated apparent K(m) value for adenosine triphosphate (6 microM) using the glutathione S-transferase-moesin substrate was much lower than the one previously reported using LRRKtide, a synthetic peptide derived from moesin. Testing of nonselective kinase inhibitors (staurosporine, H-1152, and Y-27632) generated potencies consistent with published data. Finally, robotic validation of the assay yielded an average Z' factor of 0.80. Overall, these results indicate that the present HTS AlphaScreen assay might provide a more relevant biochemical approach for the discovery of novel LRRK2(G2019S) inhibitors.

Single-well monitoring of protein-protein interaction and phosphorylation-dephosphorylation events.

We combined oxygen channeling assays with two distinct chemiluminescent beads to detect simultaneously protein phosphorylation and interaction events that are usually monitored separately. This novel method was tested in the ERK1/2 MAP kinase pathway. It was first used to directly monitor dissociation of MAP kinase ERK2 from MEK1 upon phosphorylation and to evaluate MAP kinase phosphatase (MKP) selectivity and mechanism of action. In addition, MEK1 and ERK2 were probed with an ATP competitor and an allosteric MEK1 inhibitor, which generated distinct phosphorylation-interaction patterns. Simultaneous monitoring of protein-protein interactions and substrate phosphorylation can provide significant mechanistic insight into enzyme activity and small molecule action.

Metabolic interactions between low doses of benzo[a]pyrene and tributyltin in arctic charr (Salvelinus alpinus): a long-term in vivo study.

We have previously reported that short-term, single exposure to a high dose of tributyltin (TBT), a widely used antifouling biocide, inhibited both the in vivo metabolism and metabolic activation of the carcinogenic polycyclic aromatic hydrocarbon benzo[a]pyrene (BaP) in fish; (BaP), in turn, stimulated TBT metabolism. Here, we provide further mechanistic evidence of mutual metabolic interactions between BaP and TBT in response to long-term, repeated exposures to low doses. Juvenile Arctic charr (Salvelinus alpinus) received 10 separate i.p. injections (a single injection every 6 days) of BaP (3 mg/kg), TBT (0.3 mg/kg), or both in combination; control fish received corn oil vehicle only. Two days after the 2nd (Day 8), 6th (Day 32), and 10th dose (Day 56), blood, bile, and liver samples were collected and analyzed for a suite of biomarkers. HPLC/fluorescence analysis indicated that TBT cotreatment inhibited the formation of (+)-anti-BaP diol-epoxide adducts with plasma albumin (53%, Day 8), hepatic DNA (27%, Day 32), or both albumin and globin (50 and 58%, Day 56) compared to BaP alone. This antagonistic interaction was associated with a time-dependent modulation (inhibition at Day 8, enhancement at Day 32) of both cytochrome P450 (P450)1A-mediated ethoxyresorufin O-deethylase (EROD) activity and biliary BaP metabolite formation. TBT cotreatment also inhibited (39%) the BaP-mediated induction of hepatic glutathione S-transferase (GST) activity observed at Day 8. Treatment with TBT alone increased EROD activity (60%) at Day 32, but decreased both GST activity (70 and 37%) and glutathione content (24% and 16%) at Days 32 and 56, respectively. GC/MS analysis revealed that, at Day 56, BaP cotreatment increased (200%) the levels of biliary butyltin compounds, including mono- and dibutyltin metabolites. This potentiative interaction was associated with a protective effect of BaP cotreatment against the TBT-mediated decreases in GST activity and glutathione content. The current results demonstrate that, whereas TBT inhibited BaP bioactivation via a time-dependent modulation of P4501A induction, BaP stimulated the Phase II metabolism of TBT and/or its biliary excretion. The mutual metabolic interactions between these two widespread aquatic pollutants reinforce the need for long-term in vivo interactive studies at low doses.