Transactivation assays

Zhu ZR. 2007. Use of cryopreserved transiently transfected cells in high-throughput pregnane X receptor transactivation assay. J. Biomol. Screen. 12, 248-254. 

Transactivation assays in stably transfected cell lines involve cells stably transfected with both human ER and ER-responsive reporter genes, the latter of which have easily detected activity [30]. Transiently transfected cells have been used successfully... 

Organon disclosed A510 steroids as ERa-selective agonists [50]. Compound 10 showed 290-fold ERa selectivity in a receptor-binding assay and over 50-fold ERa selectivity in a CHO cell-based transactivation assay. 

Benzothiophenes 2-Phenyl-benzothiophenes were described as ERP-selective agonists by Organon [71]. It was found that dibutyryl analog 42 exhibits 15-fold ERP selectivity in a CHO cell-based transactivation assay. Similar observations were made by Novartis [72]. Receptor binding studies showed 12-fold ERP selectivity for 43. Introduction of substituents at C3 or C4 reduced ERP selectivity. 

The binding assays as well as the transactivation assays can be adapted to high-throughput screening formats, and allow a suitable determination of relative affinities and efficacies even for large libraries. 

The compound class was further optimized towards antagonistic activity. A ring contraction to the pyrazoline scaffold and decoration of this ring with several substituents resulted in a series of compounds exemplified by 23. This displayed weak antagonistic activity in the MMTV reporter gene transactivation assay in CV-1 cells compared to mifepristone (RU-486) (Scheme 6.11). 

PRE-Luc transactivation assay in CV1 cells it was antagonistic with an IC50 of 6.7 nM. This antagonistic effect was confirmed in an in vivo decidualization assay. 

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