The co-transfection assay is a novel functional assay using cells transiently transfected with plasmids encoding intracellular receptors and corresponding reporter genes. Using this assay,
natural product extracts were tested to identify compounds that modulate intracellular receptor activity, measured as changes in reporter gene activity. A
crude extract of the marine alga Cymopolia barbata was found to inhibit
progesterone-stimulated reporter gene expression in cells transfected with the human
progesterone receptor (hPR) and an appropriate reporter construct. Purification of the active constituents of the extract, guided by the co-transfection assay, yielded two diastereomers of
cyclocymopol monomethyl ether, possessing opposing pharmacological activities with the hPR. The antagonist (3R)-cyclocymopol monomethyl
ether (
LG100127) blocked 1 nM
progesterone-stimulated reporter gene expression with an IC50 value of 549 +/- 55 nM in the co-transfection assay. The agonist (3S)-cyclocymopol monomethyl
ether (
LG100128) had efficacy similar to that of
progesterone and an EC50 value of 35 +/- 2 nM. Stimulation by
progesterone of the hPR in the human
breast cancer cell line T-47D results in enhanced expression of
alkaline phosphatase;
LG100127 blocked
alkaline phosphatase expression stimulated either by
progesterone or by
LG100128, and
LG100128 mimicked
progesterone in this assay. Both diastereomers displaced [3H]
progesterone from baculovirus-expressed hPR.
LG100127 and
LG100128 each interacted with the human
androgen receptor but did not interact with the human
glucocorticoid receptor,
estrogen receptor,
vitamin D receptor, or
retinoid receptors. In summary, these in vitro studies describe the first nonsteroidal pharmacophores for the hPR and demonstrate the use of the co-transfection assay in their discovery.