A homogeneous enzyme fragment complementation cyclic AMP screen for GPCR agonists.
In the new high-throughput screening (HTS) campaign, receptor functional assays, 3',5'-cyclic adenosine monophosphate (cAMP), intracellular [Ca(2)+](i), phosphatidylinositol turnover, and reporter-based assays are being used as primary screens as they are now developed as homogeneous and automation-friendly assays. FlashPlate assay and scintillation proximity assay using radiolabeled cAMP have been used for measuring cAMP. A nonradioactive homogeneous HTS assay using HitHunter trade mark enzyme fragment complementation (EFC) technology was evaluated for measuring cAMP in adherent and suspension cells overexpressing a Galpha(s)-coupled receptor. In the EFC-cAMP assay, the beta-galactosidase (beta-gal) donor fragment-cAMP (ED-cAMP) conjugate complements with the beta-gal enzyme acceptor (EA) fragment to form an active beta-gal enzyme. Binding of ED-cAMP conjugate to the anti-cAMP antibody prevents its complementation with the EA fragment to form an active enzyme. Cyclic AMP in the samples compete with ED-cAMP to bind to the anti-cAMP antibody, thus increasing the free ED-cAMP that can complement with the EA fragment to form an active enzyme that is assayed with a luminescent substrate. Thus, this assay results in a positive signal unlike other technologies, wherein the signal is completed by cAMP in the sample. Glucagon-like peptide (GLP)-1 binds to GLP-1 receptor (with a Kd of 0.2 nM) signals through Galpha(s) to activate adenylate cyclase, which results in an increase of intracellular cAMP (EC(50) of 0.3 nM). GLP-1 stimulation of cAMP levels measured by the EFC method was similar in both adherent and suspension cell formats (EC(50)~0.3 nM) at different cell numbers. The assay was further validated with forskolin, exendin, and several active GLP-1 peptide analogues. The stimulation of cAMP by GLP-1 and forskolin was effectively inhibited by the adenylate cyclase inhibitors MDL-12330A and SQ-22536, confirming that the increased cAMP is through the AC pathway. The assay tolerates dimethyl sulfoxide (DMSO) up to 10%, and tartrazine does not interfere with the assay with the adherent cells up to 1 mM and affects minimally up to 10 microM in suspension cells. The assay is very robust, with a Z' value of 0.7 to 0.8. The assay was validated with several plates of low molecular weight nonpeptide compounds and peptide agonists with different potencies. The suspension cell protocol is a robust homogeneous assay that involves fewer steps than the adherent cell protocol and is suitable for HTS. The cAMP assay using EFC technology is advantageous in that it has a greater dynamic range of detection; is nonradioactive, very sensitive, robust; has minimal interference from DMSO and colored compounds; and is amenable for automation. An added advantage of this assay is that the cAMP is measured as a positive signal, thereby reducing the incidence of false positives.
Drug Discovery, Pharmaceutical Research Institute, Bristol-Myers Squibb, Princeton, NJ, USA.
Rajasree
R
Golla
Ramakrishna
R
Seethala
D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002448:Cell Adhesion; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D004396:Coloring Agents / Q000737:chemistry; D006224:Cricetinae; D000242:Cyclic AMP / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000737:chemistry; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004798:Enzymes / Q000235:genetics / Q000378:metabolism*; D000067757:Glucagon-Like Peptide-1 Receptor; D006801:Humans; D008163:Luminescent Measurements; D008967:Molecular Biology / Q000379:methods* / Q000592:standards; D025941:Protein Interaction Mapping / Q000379:methods; D043562:Receptors, G-Protein-Coupled / Q000819:agonists* / Q000187:drug effects / Q000378:metabolism; D018027:Receptors, Glucagon / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D013645:Tartrazine / Q000737:chemistry; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D004396:Coloring Agents; D004798:Enzymes; C000593833:GLP1R protein, human; D000067757:Glucagon-Like Peptide-1 Receptor; D043562:Receptors, G-Protein-Coupled; D018027:Receptors, Glucagon; D011994:Recombinant Proteins; D005576:Colforsin; D000242:Cyclic AMP; D001616:beta-Galactosidase; D013645:Tartrazine; D004121:Dimethyl Sulfoxide