High-throughput screening using beta-lactamase reporter-gene technology for identification of low-molecular-weight antagonists of the human gonadotropin releasing hormone receptor.
G-protein coupled receptors (GPCRs) signal via G-proteins to intracellular second messengers. Assays that link transcription of a detectable reporter to promoters that are activated by such signaling cascades are highly sensitive and allow screening for compounds that either activate or inactivate a GPCR of interest. This study describes the development and performance of an antagonistic screen on the human gonadotropin releasing hormone receptor (GnRH-R). Compounds (245,000) were tested in a high-throughput screen using a Chinese hamster ovary cell line stably expressing the human GnRH-R and the Ca2+ sensitive reporter nuclear factor activated in T-cells/ activator protein-1-beta-lactamase. In total, 4,160 active compounds were identified. Colored and toxic compounds, as well as dust and compound aggregates, have been depicted as artifacts. To deselect non-target hits, several follow-up assays, including luminescent and fluorescent Ca2+ mobilization assays and radioligand binding, were developed for the GnRH-R. These assays were validated using peptide and low-molecular-weight GnRH-R reference compounds before hits from screening were also profiled in these assays. For several reference compounds the use of different assay technologies resulted in a poor correlation of potency values. In conclusion, beta-lactamase as a primary high-throughput screening assay is a powerful complementation to other screening technologies. The beta-lactamase technology has several advantages, including lack of cell lysis and ratiometric read-out, which augments assay robustness. Based on technology comparison, it is not adequate to assume that the same hits would be found regardless of which assay technology is used.
Oosterom
Julia
J
Molecular Pharmacology, N.V. Organon, Oss, The Netherlands. julia.oosterom@organon.com
van Doornmalen
Els J P
EJ
Lobregt
Sendy
S
Blomenröhr
Marion
M
Zaman
Guido J R
GJ
D000818:Animals; D001681:Biological Assay / Q000379:methods*; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D015195:Drug Design; D004353:Drug Evaluation, Preclinical / Q000379:methods; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008163:Luminescent Measurements; D008970:Molecular Weight; D025941:Protein Interaction Mapping / Q000379:methods*; D011966:Receptors, LHRH / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism*
D011966:Receptors, LHRH; D011994:Recombinant Proteins; D001618:beta-Lactamases