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Department of Pharmacology and Cancer Biology (B.L.W., G.P., P.G., D.P.M.), Duke University Medical Center, Durham, North Carolina 27710; Molecular Endocrinology Group (J.C.W., J.A.C.), NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709; Department of Biochemistry and Molecular Biology (M.B., W.V.V.), Louisiana State University Medical School, New Orleans, Louisiana 70112; Ligand Pharmaceuticals, Inc. (D.E.M.), San Diego, California 92121; Research Triangle Institute (C.E.C.), Chemistry and Life Sciences, Research Triangle Park, North Carolina 27709
Address all correspondence and requests for reprints to: D. P. McDonnell, Department of Pharmacology and Cancer Biology, Duke University Medical School, Box 3813, Durham, North Carolina 27710. E-mail: McDon016{at}acpub.duke.edu
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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All of the currently available antiprogestins are steroidal in nature and are derived from a 19-nor testosterone backbone (10, 13, 14). It is likely that nonsteroidal antiprogestins with improved selectivity will be developed. In their absence, efforts to dissociate antiprogestational from antiglucocorticoid activity have been limited to modifications of existing steroidal antiprogestins. Unfortunately, a selective steroidal antiprogestin has not yet emerged. We believe that progress in this area has been limited by the approach that has been used in the past to screen for dissociated antiprogestins. Typically, in vitro receptor binding assays, assessing PR/GR selectivity, have been used to guide medicinal chemistry. This approach has not yet yielded a dissociated antiprogestin as it has been found that most compounds that display a reduced GR binding activity exhibit a commensurate decrease in affinity for PR (10). This observation suggested that a more predictive screen for novel antiprogestins was needed, one that did not discriminate based on receptor binding affinity, but rather on the ability of a compound to differentially affect PR or GR signaling.
Much of the justification for a mechanism-based approach to develop dissociated antiprogestins has come from our previous studies on the mechanism of action of PR agonists and antagonists (15, 16, 17, 18). In these earlier studies, we identified two classes of antiprogestins that interact with similar, though distinct, regions within the PR ligand binding domain, resulting in unique alterations in PR structure (18). Subsequently, it was determined that members of one class of antiprogestins identified exhibited pure antiprogestenic activity in all contexts examined, whereas members of the second class functioned as antiprogestins in most contexts but had the ability to function as partial agonists in others (18). A potential molecular explanation for the differential activity of these two classes of antagonists was revealed when it was determined that the pure antiprogestins permitted the formation of high affinity interactions of PR with the nuclear receptor corepressors SMRT and NCoR, whereas the tissue selective antiprogestins (mixed agonists) formed weak associations with the same proteins. Importantly, overexpression of either corepressor had a pronounced effect on the activity of the PR mixed agonists where complete suppression of the partial agonist activity of these compounds was achieved. Cumulatively, these findings indicated that although the two classes of antiprogestins displayed similar PR binding affinities, they were mechanistically different. Based on this observation, which established a link between PR structure and biological activity, we considered that it may be possible to identify compounds that interact with both PR and GR but may not affect the transcriptional activity of these receptors in a similar manner. Therefore, in this study we used a series of mechanism based approaches to screen libraries of high affinity steroidal antiprogestins for compounds with reduced antiglucocorticoid activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Mammalian transfections and luciferase assays
HeLa and T47D cells were maintained in MEM and RPMI
supplemented with 10% FCS, respectively. Cells were plated in 24-well
plates, 24–48 h before transfection. HeLa cells were transiently
transfected for 3 h with a total of 3 µg of DNA per triplicate
using Lipofectin. T47D cells were similarly transfected with Lipofectin
for 2 h. After transfection, the cells were immediately washed and
incubated with the designated ligands for 24 or 48 h. The
cells were then lysed and analyzed for luciferase and ß-galactosidase
activity as previously reported (20).
Cell viability
CEM-C7 cells were maintained in RPMI 1640 media containing 10%
dialyzed, heat-inactivated FBS. Cells were seeded at 1 x
105 to 3 x 105 cells per ml in 6-well
plates and incubated with the designated ligands for 72 h.
Following the incubation, 500 µl of cells were removed and the number
of viable cells was assayed using trypan blue exclusion.
Relative binding affinities
All procedures were performed using a Biomek 1000 automated
workstation (Beckman Coulter Instruments, Inc., Fullerton,
CA). Ten-fold serial dilutions (10-6–10-10) of
the compound to be tested were prepared in a 10 mM Tris (pH
7.6) 0.3 MKCl, 5 mM DTT solution. A 100 µl
aliquot of each dilution was transferred to a polystyrene tube
containing 5 nM [3H] progesterone or
[3H] dexamethasone (Amersham, Arlington
Heights, IL). To each tube either PR containing extracts from
baculovirus (20 µg total protein) or GR containing extracts from
MDA-231 cells (250 µg total protein) were added and incubated
overnight at 4 C. Hydroxylapatite slurry (100 µl) in 10
mM Tris (pH 7.6) and 2 mM DTT were added and
the tubes were incubated for an additional 30 min at 4 C, after which
they were centrifuged to recover the pellets. Hydroxylapatite pellets
were washed four times with 1% Triton X-100, 10 mM Tris
(pH 7.6), 5 mM DTT after which they were resuspended in 800
µl Ecoscint A scintillation fluid (National Diagnostic, Manville,
NJ), and the activity was measured on a LS60001C scintillation counter
(Beckman Coulter Instruments, Inc., Fullerton, CA).
Immunohistochemistry
The subcellular distribution of human GR transiently transfected
into COS-1 cells has been previously described (21). Briefly, COS-1
cells (African Green Monkey Kidney, ATCC) were grown in
DMEM (Gibco BRL, Gaithersburg, MD) containing 9 mg/ml
glucose, 100 IU/ml penicillin, 100 µg/ml streptomycin, and
supplemented with 2 mM glutamine and 10% of a 1:1 mixture
of FCS/calf serum (FCS:CS) (Irvine Scientific, Santa Ana,
CA). Cultures were maintained at 37 C in a humidified atmosphere of 5%
CO2. The cells were passed every 3–4 days and were
maintained in culture for no longer than 15 passages. Cells were
transfected by the commercial agent DMRIE C (Gibco BRL) as
per manufacturer’s instructions. Cells were incubated with the
appropriate DNA/DMRIE C mixture for 4 h and placed in DMEM
supplemented with steroid-stripped FCS:CS and further incubated at 37 C
for 24 h. Transfected cells were then placed in two-chamber glass
slides and incubated for an additional 24 h and then treated with
100 nM hormone or vehicle for 1 h. Cells were fixed
and processed for immunohistochemical staining as previously described
(21).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Previous studies with these compounds
indicated that they could be separated into one of three groups based
on how they interacted with PR (18). In this study, we evaluated
whether the biological activity of these compounds reflected these
mechanistic classifications. This was accomplished by evaluating each
compound for agonist and antagonist activity in PR-containing T47D
cells on the endogenous progesterone-responsive alkaline phosphatase
gene (19). Although the alkaline phosphatase gene is regulated by PR,
it is not clear if this activity occurs in a direct or an indirect
manner. As observed in Fig. 1A, progesterone administration induced significant alkaline phosphatase
activity in this cell system. Compounds that, based on their effect on
PR structure, were predicted to function as antagonists [RTI 3021–002
(RTI-002), RTI 3021–003 (RTI-003), and RTI 3021–012 (RTI-012)],
exhibited no measurable agonist activity. Conversely, compounds that
interacted with PR in a manner similar to progesterone [RTI 2207–222
(RTI-222), RTI 2207–225 (RTI-225), and RTI 2207–226 (RTI-226)]
functioned as agonists. The PR ligands, RTI 3021–020 (RTI-020), RTI
3021–021 (RTI-021), and RTI 3021–022 (RTI-022), which induce unique
structural alterations within the receptor, exhibited partial agonist
activity in this assay, a result that distinguished them from agonists
and antagonists. The classification of these compounds as partial
agonists, as distinct from weak agonists, was confirmed by examining
their ability to inhibit progesterone induced expression of alkaline
phosphatase activity. As shown in Fig. 1B, the pure antagonists all
functioned as potent PR antagonists and quantitative inhibition was
achieved at concentrations as low as 100 nM. The partial
agonist activity of RTI-020, -021, and -022 was confirmed by
demonstrating that they inhibit progesterone activated PR
transcriptional activity to a level equivalent to their maximal agonist
activity. Although the direct measurement of alkaline phosphatase
activity indicated that like progesterone, RTI-222, -225, and -226
function as PR agonists they may not function in an identical manner to
progesterone in this assay. Specifically, it is noted that the maximal
efficacy of the RTI agonists is significantly less than progesterone
(Fig. 1A). Paradoxically, these compounds do not inhibit progesterone
agonist activity when tested in the antagonist mode. As yet, we have
been unable to explain this result. As shown below, however, additional
experiments indicate that this particular activity of the RTI agonists
may be unique to the alkaline phosphatase
promoter.
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, the pure antagonists and agonists
functioned predictably. However, in this environment RTI-020, -021, and
-022 do not exhibit measurable agonist activity (Fig. 2A) and at 100
nM all members of this class functioned as efficient
antagonists of progesterone agonist activity (> 95% efficacy; Fig. 2B). Similar results were obtained in transfected CV-1 cells using the
same experimental paradigm (data not shown). Taken together, these
results confirmed that PR ligands could be separated into at least
three functionally distinct classes. We proceeded, therefore, to assess
the antiglucocorticoid activity of these newly identified PR
antagonists and partial antagonists.
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). When compared with
dexamethasone, it was observed that RTI-022, RTI-012, and RU486 (the
standard used in our assays) had similar GR binding affinities (RBAs
5.7, 5.2, and 13.9, respectively) to the pure agonist dexamethasone. A
similar analysis comparing the affinities of these compounds for PR
indicated that, compared with progesterone, the RBAs for RTI-012,
RTI-022, and RU486 were 12.7, 11.9, and 6.8, respectively. Thus, if
ligand binding is the primary determinant of antagonist efficacy, then
these compounds should display equivalent antiprogestenic and
antiglucocorticoid activities. To test this hypothesis, we compared the
ability of RU486, RTI-012, and RTI-022 to inhibit PR and GR
transcriptional activity in transfected cells.
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, demonstrate
that all three compounds are effective PR antagonists. In accord with
the observed affinity differences, we noticed that the antagonist
potency of RU486 was slightly greater than either of the two RTI
compounds, which themselves behaved quite similarly in this assay.
Thus, in this cell and promoter context, the in vitro PR
binding affinity of these compounds and their PR antagonist efficacy
match closely. Similar results were observed in HeLa cells in which PR
and a PR-responsive promoter were cotransfected (data not shown).
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. As expected from its GR-binding
affinity, RU486 functioned as an effective GR antagonist. Quite
surprisingly, however, RTI-012 and RTI-022, whose affinities for GR
were similar to each other and to that displayed by RU486, did not
function as potent GR antagonists. Specifically, under the conditions
of this assay the antagonist potencies of RU486 and RTI-012 differed by
over 100-fold, whereas a greater than 1000-fold difference in potency
was observed between RU486 and RTI-022. The differences between RTI-012
and RTI-022 may relate to subtle mechanistic differences between these
compounds. Alternatively, it is possible that RTI-012 is converted to
its 17
-OH metabolite, a transformation that would not express itself
in the in vitro binding assays and may enhance its receptor
binding affinity. These informative results indicated that, with
respect to GR antagonism, there was a large discrepancy between GR
antagonist efficacy and binding affinity. It must also be mentioned
that neither RTI-012, nor RTI-022 exhibited any GR agonist activity
when assayed on a GR-responsive promoter in transfected mammalian cells
(data not shown).
RTI-012 and RTI-022 efficiently promote the interaction of PR, but
not GR, with target gene promoters in vivo
We considered that one reason for the difference in GR antagonist
efficacy manifest by RU486, RTI-022, and RTI-012 was that they were not
equally effective at delivering GR to DNA. This possibility was tested
by assessing the ability of these compounds to activate transcription
of a GR-VP16 fusion plasmid. In this assay, GR/ligand complexes that
bind DNA permit the activation of transcription by the VP16 activation
domain contained within the chimeric GR. This approach was chosen as we
and others have shown that the VP16 activation function, when used in
the context of a receptor chimera, permits both agonists and
antagonists to activate transcription upon DNA binding (22, 26). Thus,
antagonists will function as agonists if they can deliver the chimera
receptor to DNA. For this analysis, HeLa cells were transiently
transfected with an expression vector encoding the GR-VP16 chimera
together with one of two different GRE containing luciferase reporter
vectors, MMTV-LUC or PRE-TK-LUC. As shown in Fig. 4A, both dexamethasone and RU486
efficiently delivered GR-VP16 to DNA. Interestingly, this is not the
case when the assay is performed in the presence of either RTI-012 or
RTI-022. Under the conditions of this assay, using saturating
concentrations of test compounds, we observed that the GR/DNA binding
activity of RTI-012 and RTI-022 was only 35% and 6%, respectively, of
that observed in the presence of RU486 when assayed on the MMTV-LUC
promoter. A similar result was observed when the assay was performed on
the PRE-TK-LUC promoter. For comparative purposes, we performed the
same type of assay using PR-VP16. The results of this analysis shown in
Fig. 4 indicate that both RTI-022 and RTI-012 are capable of inducing
high affinity PR-DNA interactions in a manner that is indistinguishable
from RU486. Thus, we conclude from these results that the inability of
RTI-012 and RTI-022 to efficiently deliver GR to DNA may explain their
relatively weak GR-antagonist activity.
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) demonstrated that there were
differences in the ability of antagonists to promote GR target promoter
associations (RU486[tmt]RTI-012 > RTI-022). One explanation for
this activity is that there were differences in the ability of these
compounds to promote nuclear translocation. GR is unique among the
nuclear receptors in that it resides in the cytoplasm of target cells
in the absence of ligand (21). Upon binding an agonist such as
dexamethasone, the receptor translocates to the nucleus where it exerts
its regulatory activities (21). To test the effect of the RTI compounds
on GR nuclear translocation we transfected COS-1 cells with an
expression vector for GR and examined the cellular localization of the
recombinant receptor using immunohistochemical techniques following
treatment of the cells with selected agonists and antagonists. The
results of this analysis are shown in Fig. 5. Both dexamethasone and RU486 promoted
an efficient translocation of GR to the nuclear compartment of these
cells. However, under the conditions of this assay both RTI-012 and
RTI-022 were only partially active in this regard. We therefore
concluded that RTI-012 or RTI-022 function predominantly as competitive
antagonists on GR because the resulting receptor-ligand complexes
cannot translocate efficiently to the nucleus and compete for DNA
binding with agonist activated receptor.
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). As previously
reported, RU486 completely prevented dexamethasone-induced apoptosis
when these compounds were added in equimolar concentrations and gave
50% protection when added at a concentration 1/10th that of
dexamethasone. This is the expected result given that RU486 has nearly
a 13-fold higher affinity for GR than does dexamethasone.
Interestingly, when assayed under the same conditions RTI-022, whose
affinity for GR is only 2.5-fold less than RU486, required 50–100
times more compound to evoke the same response. Cumulatively,
therefore, our data, emanating from both cotransfection and cell based
assays, indicate that PR antagonists can be developed that do not
significantly impact GR signaling.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this study, we undertook a molecular approach to understand the mechanism by which antiprogestins manifest antagonist activity on PR and GR. The currently available antiprogestins also function as effective antiglucocorticoids (13, 14). Thus, for applications that require chronic administration there is a medical need to develop dissociated antiprogestins; compounds that display no or reduced antiglucocorticoid activity (9). However, there has been little success in identifying antiprogestins that do not function as antiglucocorticoids (10, 13, 14). This may relate to the fact that the currently available antiprogestins are steroidal, derived from the same chemical backbone, and so may function by very similar mechanisms (13). The recent identification of a new class of PR mixed agonists, which interact with the PR hormone binding domain in a distinct manner, prompted us to reexamine the issue of GR cross-reactivity of PR antagonists (18). In this study, we profiled this new series of PR ligands and determined that the compounds RTI-022 and RTI-012 that functioned as potent PR antagonists in vitro exhibited significantly less GR antagonist activity than their receptor binding affinities would predict. To understand the discrepancy between binding affinity and biological potency, we compared the ability of RU486, RTI-022, and RTI-012 to facilitate the interaction of GR with target gene promoters. These studies revealed that neither RTI-012 or RTI-022 were as effective as RU486 at inducing nuclear translocation of GR. In contrast, however, RTI-012, RTI-022 and RU486 efficiently facilitated PR/DNA interactions and demonstrated comparable progesterone antagonist activities. Thus, although we previously had shown that RTI-012 and RTI-022 interact with different regions of the PR-ligand binding domain and do not inhibit PR-transcriptional activity in the same manner, they both efficiently delivered PR to DNA indicating that it was post DNA binding events that distinguished these compounds. When assayed on GR, we were surprised to find that RTI-012 and RTI-022, that displayed high affinity GR binding, were not potent antagonists. This was in great distinction to RU486, an affinity matched ligand, which functioned as a potent PR and GR antagonist. Thus, although we can classify compounds as PR agonists, antagonists or partial agonists based on how they interact with PR, these classifications do not predict the likely GR cross-reactivity of specific compounds. Thus, at this point, we believe that the unique chemical structures of RTI-012 and RTI-022 have some effect on GR that distinguishes them from RU486. This interesting possibility will be followed up in subsequent studies. Regardless, these data strongly support our hypothesis that binding affinity alone is not sufficient to predict the biological activity of a receptor antagonist.
The studies presented here, and those of others, are compatible with
the existence of two distinct types of antagonists, competitive and
active antagonists (Fig. 7). Using GR
antagonism as an example, we propose that the RTI compounds function
only as competitive antagonists; a one-step process in which agonists
and antagonists only compete for receptor binding. Possibly because of
a specific conformational change, the resultant GR ligand complex does
not enter the nucleus and therefore does not directly oppose the
actions of residual agonist activated receptor. Because competitive
inhibitors do not prevent agonist occupied receptors from binding DNA
and activating transcription, their antagonist activity is governed
mainly by affinity. In contrast to RTI-022 and RTI-012, RU486 functions
as an active antagonist of GR transcriptional activity. Thus, RU486 not
only competitively inhibits agonist binding to GR but permits the
formation of a ligand-GR complex that can participate actively in the
inhibition process. Specifically, these complexes can bind with high
affinity to target gene promoters and block agonist activated receptor
from interacting with its DNA-target site. In some contexts, members of
this class of active antagonists can function as partial agonists; an
event that can only occur when the receptor binds DNA. Indeed, Nordeen
et al. (34) have demonstrated that RU486 can in fact
manifest partial GR agonist activity in some contexts. Although the
mechanism of this partial agonism remains to be determined, it is
likely that differences in the expression of specific receptor
associated proteins contribute significantly to the degree of agonist
activity manifest by the RU486/GR complex. Therefore, in those contexts
in which RU486 exhibits partial agonist activity, competitive
inhibitors, at saturating doses, are more likely to function as pure
antagonists. As a final note on this topic, we believe that because
RU486, RTI-012, and RTI-022 efficiently deliver PR to DNA, they are
functioning as active antagonists of PR transcriptional activity. This
highlights the need to qualify the classification of a given compound
with respect to a specific receptor.
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In summary, this work has led to the identification of RTI-022 and RTI-012, compounds that function as competitive antagonists of GR function and active antagonists of PR transcriptional activity. These mechanistic differences manifest themselves as a 1- to 400-fold discrepancy between binding and antagonist efficacy with respect to GR activity and comparable binding and antagonist potency on PR. Thus, a separation between GR and PR antagonism is afforded by virtue of differences in the mechanism of action of these compounds on the two different receptors. This result validates using a mechanism-based approach to develop dissociated antiprogestins that, when used in combination with traditional direct binding approaches, is likely to be a powerful combination in the discovery of dissociated antiprogestins. In addition to providing useful insights into the pharmacology of PR and GR, we believe that RTI-012 and RTI-022 will find use in vivo, both as research tools and hopefully as drugs where it is important to separate antiprogestenic from antiglucocticoid activities.
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Acknowledgments |
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Footnotes |
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2 Supported by an Advanced Predoctoral Fellowship from the
Pharmaceutical Research and Manufacturers of America Foundation.
3 Supported by a predoctoral fellowship from U.S. Army Medical
Research and Materiel Command.
Received February 6, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-substituted analogs of the antiprogestin RU486 induce a unique
conformation in the human progesterone receptor resulting in mixed
agonist activity. Proc Natl Acad Sci USA 93:8739–8744This article has been cited by other articles:
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