Stéphane Parent

THE BRET2/ARRESTIN ASSAY IN STABLE RECOMBINANT CELLS: A PLATFORM TO SCREEN FOR COMPOUNDS THAT INTERACT WITH G PROTEIN-COUPLED RECEPTORS (GPCRS)*

ABSTRACT In BRET2 (Bioluminescence Resonance Energy Transfer), a Renilla luciferase (RLuc) is used as the donor protein, while a Green Fluorescent Protein (GFP2) is used as the acceptor protein. In the presence of the cell permeable substrate DeepBlueC™, RLuc emits blue light at 395 nm. If the GFP2 is brought into close proximity to RLuc via a specific biomolecular interaction, the GFP2 will absorb the blue light energy and reemit green light at 510 nm. BRET2 signals are therefore easily determined by measuring the ratio of green over blue light (510/395 nm) using appropriate dual channel luminometry instruments (e.g., Fusion™ Universal Microplate Analyzer, Packard BioScience). Since no light source is required for BRET2 assays, the technology does not suffer from high fluorescent background or photobleaching, the common problems associated with standard FRET-based assays. Using BRET2, we developed a generic G Protein–Coupled Receptor (GPCR) assay based on the observation that activation of the majority of GPCRs by agonists leads to the interaction of β-arrestin (a protein that is involved in receptor desensitization and sequestration) with the receptor. We established a cell line stably expressing the GFP2 : β-arrestin 2 fusion protein, and showed that it can be used to monitor the activation of various transiently expressed GPCRs, in BRET2/arrestin assays. In addition, using the HEK 293/GFP2 : β-arrestin 2 cell line as a recipient, we generated a double-stable line co-expressing the vasopressin 2 receptor (V2R) fused to RLuc (V2R : RLuc) and used it for the pharmacological characterization of compounds in BRET2/arrestin assays. This approach yields genuine pharmacology and supports the BRET2/arrestin assay as a tool that can be used with recombinant cell lines to characterize ligand–GPCR interactions which can be applied to ligand identification for orphan receptors.

Soluble amyloid precursor protein alpha and beta in CSF in Alzheimer's disease

OBJECTIVE Cerebral accumulation of amyloid β (Aβ) is a pathological hallmark of Alzheimers disease (AD). Proteolytic processing of amyloid precursor protein (APP) by α- or β-secretase results in two soluble metabolites, sAPPα and sAPPβ, respectively. However, previous data have shown that both α- and β-secretase have multiple cleavage sites. The aim of this study was to characterize the C-termini of sAPPα and sAPPβ in cerebrospinal fluid (CSF) by mass spectrometry (MS) and to evaluate whether different combinations of these fragments better separate between AD patients and controls by comparing two different sAPP immunoassays. METHODS Using immunoprecipitation and high resolution MS, the APP species present in CSF were investigated. CSF levels of sAPPα and sAPPβ from patients with AD (n=43) and from non-demented controls (n=44) were measured using AlphaLISA and MSD immunoassays that employ different antibodies for C-terminal recognition of sAPPα. RESULTS Four different C-terminal forms of sAPP were identified, sAPPβ-M671, sAPPβ-Y681, sAPPα-Q686, and sAPPα-K687 (APP770 numbering). Neither immunoassay for the sAPP species could separate the two patient groups. The correlation (R(2)) between the two immunoassays was 0.41 for sAPPα and 0.45 for sAPPβ. CONCLUSION Using high resolution MS, we show here for the first time that sAPPα in CSF ends at Q686 and K687. The findings also support the conclusion from several previous studies that sAPPα and sAPPβ levels are unaltered in AD.

AlphaScreen®-Based Characterization of the Bifunctional Kinase/RNase IRE1α A Novel and Atypical Drug Target

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, IRE1α, 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 IRE1α. 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.

Integrated Cell-Based Platform to Study EGFR Activation and Transactivation

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 drugs 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.

The use of AlphaLISA in measurement of Aβ1-15/16 as a potential biomarker in neurodegenerative diseases

Magdalena Nutu, Philippe Bourgeois, Henrik Zetterberg, Erik Portelius, Francesco Lipari, St ephane Parent, Oskar Hansson, Kaj Blennow, The Sahlgrenska Academy, Gothenburg, Sweden; PerkinElmer Biosignal, Inc, Montreal, Quebec, Canada; University of Gothenburg, M€olndal, Sweden; Institute of Neuroscience and Physiology, M€olndal, Sweden; PerkinElmer Inc, Montreal, Quebec, Canada; Depatment of Neurology, Sk ane University Hospital, Malm€o, Sweden; Institute of Neuroscience and Physiology, The Sahlgrenska Academy, University of Gothenburg, M€olndal, Sweden.