Assessment of a recombinant androgen receptor binding assay: initial steps towards validation.
Despite more than a decade of research in the field of endocrine active compounds with affinity for the androgen receptor (AR), still no validated recombinant AR binding assay is available, although recombinant AR can be obtained from several sources. With funding from the European Union (EU)-sponsored 6th framework project, ReProTect, we developed a model protocol for such an assay based on a simple AR binding assay recently developed at our institution. Important features of the protocol were the use of a rat recombinant fusion protein to thioredoxin containing both the hinge region and ligand binding domain (LBD) of the rat AR (which is identical to the human AR-LBD) and performance in a 96-well plate format. Besides two reference compounds [dihydrotestosterone (DHT), androstenedione] ten test compounds with different affinities for the AR [levonorgestrel, progesterone, prochloraz, 17alpha-methyltestosterone, flutamide, norethynodrel, o,p'-DDT, dibutylphthalate, vinclozolin, linuron] were used to explore the performance of the assay. At least three independent experiments per compound were performed. The AR binding properties of reference and test compounds were well detected, in terms of the relative ranking of binding affinities, there was good agreement with published data obtained from experiments using recombinant AR preparations. Irrespective of the chemical nature of the compound, individual IC(50)-values for a given compound varied by not more than a factor of 2.6. Our data demonstrate that the assay reliably ranked compounds with strong, weak, and no/marginal affinity for the AR with high accuracy. It avoids the manipulation and use of animals, as a recombinant protein is used and thus contributes to the 3R concept. On the whole, this assay is a promising candidate for further validation.
Freyberger
Alexius
A
Bayer Schering Pharma AG, BSP-GDD-GED-GTOX Special Toxicology, Wuppertal, Germany. alexius.freyberger@bayerhealthcare.com
Weimer
Marc
M
Tran
Hoai-Son
HS
Ahr
Hans-Jürgen
HJ
D059002:Androgen Receptor Antagonists; D000728:Androgens / Q000737:chemistry / Q000494:pharmacology*; D000826:Animal Testing Alternatives; D000818:Animals; D001667:Binding, Competitive; D001681:Biological Assay / Q000379:methods* / Q000592:standards; D004305:Dose-Response Relationship, Drug; D052244:Endocrine Disruptors / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D020128:Inhibitory Concentration 50; D008024:Ligands; D011485:Protein Binding; D051381:Rats; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins / Q000819:agonists / Q000037:antagonists & inhibitors; D015203:Reproducibility of Results
D059002:Androgen Receptor Antagonists; D000728:Androgens; D052244:Endocrine Disruptors; D008024:Ligands; D011944:Receptors, Androgen; D011993:Recombinant Fusion Proteins