Comparison between steroid binding to membrane progesterone receptor alpha (mPRalpha) and to nuclear progesterone receptor: correlation with physicochemical properties assessed by comparative molecular field analysis and identification of mPRalpha-specific agonists

Abstract

Recent results showing that the binding characteristics of 33 steroids for human membrane progesterone receptor alpha (hu-mPRalpha) differ from those for the nuclear progesterone receptor (nPR) suggest that hu-mPRalpha-specific agonists can be identified for investigating its physiological functions. The binding affinities of an additional 21 steroids for hu-mPRalpha were determined to explore the structure-activity relationships in more detail and to identify potent, specific mPRalpha agonists. Four synthetic progesterone derivatives with methyl or methylene groups on positions 18 or 19, 18a-methylprogesterone (18-CH(3)P4, Org OE 64-0), 13-ethenyl-18-norprogesterone (18-CH(2)P4, Org 33663-0), 19a-methylprogesterone (19-CH(3)P4, Org OD 13-0) and 10-ethenyl-19-norprogesterone (19-CH(2)P4, Org OD 02-0), showed similar or higher affinities than progesterone for hu-mPRalpha and displayed mPRalpha agonist activities in G-protein and MAP kinase activation assays. All four steroids also bound to the nPR in cytosolic fractions of MCF-7 cells. However, two compounds, 19-CH(2)P4 and 19-CH(3)P4, showed no nPR agonist activity in a nPR reporter assay and therefore are selective mPRalpha agonists suitable for physiological investigations. The structure-binding relationships of the combined series of 54 steroids for hu-mPRalpha deviated strikingly from those of a published set of 60 3-keto or 3-desoxy steroids for nPR. Close correlations were observed between the receptor binding affinities of the steroids and their physicochemical properties calculated by comparative molecular field analysis (CoMFA) for both hu-mPRalpha and nPR. A comparison of the CoMFA field graphs for the two receptors revealed several differences in the structural features required for binding to hu-mPRalpha and nPR which could be exploited to develop additional mPR-specific ligands.

Figure 1

A and C, Competition curves of ligand binding to recombinant human mPRα on plasma membranes of transfected MDA-231 cells expressed as a percentage of maximum specific [³H]P4 binding. P4, progesterone; 18-CNP4, 3,20-dioxopregn-4-ene-18-carbonitrile; 19-OHP4, 19-hydroxyprogesterone; 1,4-pregnad, pregna-1,4-diene-3,20-dione; 4,6-pregnad, 4,6-pregna-4,6-diene-3,20-dione. For key to other steroid abbreviations see Table 1. Assays were repeated 3 times and similar results were obtained in each assay. B, Molecular structures of four synthetic progestins with high affinities for mPRα.

Figure 2

G protein (A) and MAPK (B) activation by various progestins in MDA-231 cells transfected with human mPRα. A, Specific [³⁵S]GTPγS binding to plasma membranes was measured after 100 nM progestin treatment for 20 min. CTL, vehicle control; P4, progesterone; 02-0, 19-CH₂P4 (Org OD 02-0); 13-0, 19-CH₃P4 (Org OD 13-0); 64-0, 18-CH₃P4 (Org OE 64-0); 33663, 18-CH₂P4 (Org 33663-0); ***: P<0.0001, **: P<0.001 compared to Veh. B, MAPK activation in human mPRα-transfected MDA-231 cells (231-mPRα cells) compared to untransfected controls (231-CTL cells) after 100 nM progestin treatment for 10 min. sV, steroid vehicle control; eV, EGF vehicle control; EGF, epidermal growth factor positive control; R5020, promegestone; ERK, total ERK; P-ERK, active phosphorylated ERK. Both assays were repeated 3 times and similar results were obtained in each assay.

Figure 3

Concentration-response relationship of MAPK activation by 19-CH₂P4 (Org OD 02-0) and progesterone in MDA-231 cells transfected with human mPRα over the range of 1nM to 100 nM.. A, Representative results from a single MAPK activation assay. B, Combined results of response to 19-CH₂P4 from four separate experiments. * P < 0.05, ** P<0.001 compared to Veh.

Figure 4

Competition curves of binding by progesterone and various progestins to human nPR in cytosolic fractions of MCF-7 cells expressed as a percentage of maximum specific [³H]P4 binding. Cells were pretreated with estradiol-17β for 72 hrs prior to the assay to upregulate nPR expression. For key to steroid abbreviations see legend for Figure 2. Assays were repeated 3 times and similar results were obtained in each assay.

Figure 5

Effects of treatment with various progestins on transactivation of human PR-B using a PRE luciferase reporter system in PR-B –transfected MCF-7 cells. A, Agonist activities of the progestins in the PR transactivation assay at a concentration of 50 nM or 50 nM P4 + 50 nM 64 (or 50 nM 02). Veh, vehicle control; P4, progesterone; 64, 18-CH₃P4 (Org OE 64-0); 02, 19-CH₂P4 (Org OD 02-0); 13, 19-CH₃P4 (Org OD 13-0); 33663, 18-CH₂P4 (Org 33663-0).***: P<0.0001 compared to vehicle (Veh), #: P<0.05 compared to P4; B, Agonist and antagonist activities of 19-CH₂P4 and 19-CH₃P4 in the PR transactivation assay at concentrations of 20 and 100 nM in response to treatment with 5 nM progesterone. ***: P<0.0001 compared to Veh, +: P<0.05 compared to P4. C Agonist activities of 19-CH₂P4 (OD-02-0) over the concentration range 1–50 nM.**: P<0.001 compared to Veh. Assays were repeated 3 times and similar results were obtained each time.

Figure 6

Physicochemical properties of steroid binding to mPRα (on right) and nPR (on left) assessed by comparative molecular field analyses (CoMFA) A. CoMFA steric and electrostatic field contour plots for the mPRα and nPR models. Progesterone is shown as the reference compound in the top view and the side view. The green volumes indicate favourable steric interactions and the yellow volumes represent unfavourable steric interactions. The red volumes indicate regions where the presence of negative potential contributes to the affinity. The blue volumes indicate regions where the presence of positive potential increases binding affinity. B. Plots of CoMFA predicted vs measured pIC50 values of 48 mPRα ligands and 60 nPR ligands. C. Details of the two CoMFA analyses using the pIC50 values of 48 mPRα ligands and of 60 nPR ligands. The structures of all the ligands are shown in Supplementary Table 1.

Figure 6

Physicochemical properties of steroid binding to mPRα (on right) and nPR (on left) assessed by comparative molecular field analyses (CoMFA) A. CoMFA steric and electrostatic field contour plots for the mPRα and nPR models. Progesterone is shown as the reference compound in the top view and the side view. The green volumes indicate favourable steric interactions and the yellow volumes represent unfavourable steric interactions. The red volumes indicate regions where the presence of negative potential contributes to the affinity. The blue volumes indicate regions where the presence of positive potential increases binding affinity. B. Plots of CoMFA predicted vs measured pIC50 values of 48 mPRα ligands and 60 nPR ligands. C. Details of the two CoMFA analyses using the pIC50 values of 48 mPRα ligands and of 60 nPR ligands. The structures of all the ligands are shown in Supplementary Table 1.

Figure 7

A, Receptor binding profiles of nine steroids for nPR and mPRα expressed as pIC50 values. Org 2058 and P4 were used as reference compounds for the nPR and mPRα receptors, respectively. P4, progesterone; 2058, Org 2058; R5020, promegestone; NP4, 19-norprogesterone; Test, testosterone; Nan, nandrolone; Ethi, ethisterone; Net, norethisterone, Norg, norgestrel. B, Receptor binding profiles of five steroids for nPR and mPRα expressed as pIC50. P4 was used as reference compound for both the nPR and mPRα. P4, progesterone; OE-64, 18-CH₃P4; OD-02, 19-CH₂P4; 33663, 18-CH₂P4; OD-13, 19-CH₃P4.