CUSTOMIZED RECOMBINANT CELL LINES EXPRESSING YOUR TARGET OF INVEST
Euroscreen FAST is offering fully customized development for cell lines in short turnaround time including:
- Molecular biology: receptor cloning (human or ortholog) and mutagenesis
- Stable Calcium optimized cell lines (forced or unforced) using Aequorin assay for GPCR or Calcium channels.
- Stable cell lines for tailored purpose (functional and/or radioligand binding)
Euroscreen FAST is bringing 20 years of expertise and a successful track record for your GPCR needs such as:
- Residence time experiments for Kon/Koff determination
Direct kinetics of ligand-receptor interactions are not usually taken into account in the optimization of a drug’s pharmacokinetics properties. However, several studies have proposed that slow dissociation rates in vitro can be associated to long-acting effects of a compound in vivo.. This important parameter can be measured as the rate at which the complex dissociates (expressed as koff or dissociative half-life) to the free ligand and its target protein. The dissociative half-life has been connected to improved clinical efficacy in a large number of marketed drugs.
Below we exemplify the determination of the kon/Koff for the potent and selective D2L antagonist Risperidone by looking at the association of [3H]-Spiperone in the presence of increasing concentrations of the antagonist
-Cell migration assay (recombinant and primary cells)
Cell migration is a fundamental function of normal cellular processes, including embryonic development, angiogenesis, wound healing, immune response, and inflammation. One such process, leukocyte extravasation, is crucial for appropriate and effective immune response. Leukocytes normally exist in a resting state as they circulate through the body. However, upon interaction with small molecules known as chemoattractants, they rapidly respond with endothelial adhesion followed by emigration from the vasculature and chemotaxis to the site of inflammation.
Below we show the quantitative determination of the chemotaxis induced by chemokine MIP-1b as well as potent CCR5 antagonist TAK-779 using recombinant CCR5 CHO-K1 cell line.
-Cytokine Release from LPS stimulated human PBMC
The human LPS-stimulated PBMC model is a good preliminary profiling assay to evaluate a compound's effect on inflammatory cytokine production.
Below we show the inhibitory effect of dexamethasone on pro-inflammatory TNFa cytokine release from LPS-stimulated human Peripheral Blood Mononuclear Cells.
- Rat Isolated b-islet Glucose Stimulated Insulin Secretion (GSIS) assay
Type 2 Diabetes (T2D) is a devastating worldwide public health problem. Drug therapies are available to address this disease, but the common appearance of adverse effects, or lack of efficacy after long-term use, causes concern. Moreover, the growing patient population suffering from T2D and associated metabolic diseases creates a demand for new entrants into this therapeutic market. GPCRs are among the target for the largest number of marketed drug in this area. Taking advantage of our expertise in the field and our extensive catalogue of naturally-coupled GPCR cell-based assays, we are now offering the possibility to support the pre-clinical development and build confidence in the selection of compounds that have desired effects in man.
Below we show the stimulatory effect of GLP-1(7-36) peptide on glucose-induced insulin release from freshly isolated rat pancreatic islets.
- Glucose Stimulated Insulin Secretion (GSIS)
Freshly isolated Wistar rat islets were incubated with increasing concentrations of glucose with or without GLP-1 for 90 min at 37°C in a 96-well plate. Cell supernatants were harvested and insulin content was measured with an Insulin HTRFTM quantitation kit.
- Off-the-shelf and custom-based enzymatic assays
Enzymes represent a fast-growing class of targets in the drug discovery industry, as principal targets for screening like kinases or enzymes implicated in epigenetic modulation, or as potential safety issues when assessing off-target effects. Consequently, we have recently put our scientific expertise to the development and validation of several enzymatic assays including kinases, monoamine oxidases, catechol-O-methyltransferase, phosphodiesterases, cyclooxygenase, or acetylcholine-esterase.
Below we show the Steroid 5-a-Reductase (SRD5a) assay that was developed for a specific application. CHO-K1 cells expressing recombinant SRD5a are used to induce testosterone conversion to androstanolone. This enzymatic activity is dose-dependently inhibited by Linolenic Acid and intact testosterone is quantified using an HTRFTM kit.
GPCR BIASED INTRACELLULAR SIGNALING EVALUATION
Small molecules modulating GPCR are able to selectively affect one signaling pathway over other for specific GPCR thereby “biasing” the signaling. The current explanation for this biased agonism is that GPCRs can adopt multiple active conformations stabilized by different molecules affecting intracellular signaling in different ways. This divergent behavior brings further complexity in a Drug Discovery program and thus requires a methodology improvement for providing in vitro pharmacology and SAR optimization in the fastest way. Such improvement can be achieved by a multiplexing approach enabling simultaneous analysis of several intracellular signaling pathways.GPR43 (also known as FFA2), a G-protein coupled receptor, is activated by short-chain fatty acids (SCFA) including propionate and acetate and is coupled to both Gai and Gaq proteins. GPR43 has been investigated in models pertaining to the treatment of type II diabetes and dyslipidemia. A synthetic small-molecule, Phenylacetamide (Φacetamide) has recently been shown to activate GPR43 in an allosteric manner and to induce an inhibition of lipolysis through a Gai dependent coupling. These data indicate that GPR43 agonists may have an important therapeutic role in the management of type II diabetes, dyslipidemia and aspects of the metabolic syndrome.
We present here the development of a 384-well plate multiplexed assay using both Aequorin and HTRF™ cAMP for the evaluation of Gai- and Gaq -coupling in response to SCFA and Φacetamide as well as identification of biased small molecule agonists.