The Development And Application Of Functional Assays For Sreening Of Progesterone Receptor Antagonist

This thesis intends to develop functional drug screening assays targeting humanprogesterone receptor (hPR) through means of molecular biology, cell biology andpharmacoly. They were applied in vitro and in vivo to evaluate novel non-steroidalprogesterone receptor antagonists.First, a repoter gene vector MMTV-pGL3 was constructed by molecular cloningbased on progesterone response element, MMTV-LTR. A reporter gene assay wasestablished subsequently by cotransfecting hPR and MMTV-pGL3 plasmids to CV-1cells. Two non-steroidal hits identified by the hPR competitive binding method wereclassified in this assay as PR antagonists. One of which, namely, SH00019581, wasused as a template for structure and activity relationship studies. As a result, a seriesof non-steroidal structurally related analogs were made and their pharmacologicalproperties assessed in vitro. One such, DYL-(6)-14 with an IC50 of 79.8 nM,displayed no cross-reactivity on other steroid receptors, except for human androgenreceptor. Its inhibitory effect on mouse uterine decidualization was preliminarilyconfirmed in vivo.In addition, calcium mobilization assays for α4β2-nAChR and α3β4-nAChRwere set up and used for confirmation screening of the initial hits (17 in total)discovered by the binding assay (α4β2-nACh receptor).