The Novel Progesterone Receptor Antagonists RTI 3021–012 and RTI3021–022 Exhibit Complex Glucocorticoid Receptor Antagonist Activities: Implications for the Development of Dissociated Antiprogestins

0013-7227/99/$03.00/0 Endocrinology Copyright © 1999 by The Endocrine Society Vol. 140, No. 3 Printed in U.S.A. The Novel Progesterone Receptor Antagonists RTI 3021– 012 and RTI 3021–022 Exhibit Complex Glucocorticoid Receptor Antagonist Activities: Implications for the Development of Dissociated Antiprogestins* B. L. WAGNER†, G. POLLIO, P. GIANGRANDE‡, J. C. WEBSTER, M. BRESLIN, D. E. MAIS, C. E. COOK, W. V. VEDECKIS, J. A. CIDLOWSKI, AND D. P. MCDONNELL 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 ABSTRACT We have identified two novel compounds (RTI 3021– 012 and RTI 3021– 022) that demonstrate similar affinities for human progesterone receptor (PR) and display equivalent antiprogestenic activity. As with most antiprogestins, such as RU486, RTI 3021– 012, and RTI 3021– 022 also bind to the glucocorticoid receptor (GR) with high affinity. Unexpectedly, when compared with RU486, the RTI antagonists manifest significantly less GR antagonist activity. This finding indicates that, with respect to antiglucocorticoid function, receptor binding affinity is not a good predictor of biological activity. We have determined that the lack of a clear correlation between the GR binding affinity of the RTI compounds and their antagonist activity reflects the unique manner in which they modulate GR signaling. Previously, we proposed a two step “active inhibition” model to explain steroid receptor antagonism: 1) competitive inhibition of agonist binding; and 2) competition of the antagonist bound receptor with that activated T HE STEROID HORMONE progesterone is a key regulator of the processes involved in the development and maintenance of reproductive function (1). However, the efficacy of antiprogestins as treatments for brain meningiomas, breast cancer, uterine fibroids, and endometriosis have implicated progesterone in the pathology of these diseases (2– 9). Consequently, although a relatively new class of molecules, the antiprogestins are likely to have a wide range of clinical applications. The most widely used antiprogestin, RU486 (mifepristone), was originally developed as an antiglucocorticoid but was subsequently shown to be a potent Received February 6, 1998. 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: . * This work was supported by NIH Grants DK-50494 (D.P.M.) and DK-47211 (W.V.V.). † Supported by an Advanced Predoctoral Fellowship from the Pharmaceutical Research and Manufacturers of America Foundation. ‡ Supported by a predoctoral fellowship from U.S. Army Medical Research and Materiel Command. by agonists for DNA response elements within target gene promoters. Accordingly, we observed that RU486, RTI 3021– 012, and RTI 3021– 022, when assayed for PR antagonist activity, accomplished both of these steps. Thus, all three compounds are “active antagonists” of PR function. When assayed on GR, however, RU486 alone functioned as an active antagonist. RTI 3021– 012 and RTI 3021– 022, on the other hand, functioned solely as “competitive antagonists” since they were capable of high affinity GR binding, but the resulting ligand receptor complex was unable to bind DNA. These results have important pharmaceutical implications supporting the use of mechanism based approaches to identify nuclear receptor modulators. Of equal importance, RTI 3021– 012 and RTI 3021– 022 are two new antiprogestins that may have clinical utility and are likely to be useful as research reagents with which to separate the effects of antiprogestins and antiglucocorticoids in physiological systems. (Endocrinology 140: 1449 –1458, 1999) and effective antiprogestin (10). As an antiprogestin, RU486 is used to induce medical abortions and as a missed menses inducer (11, 12). For these applications, the drug is given acutely and, consequently, the antiglucorticoid activity is unlikely to cause any lasting side effects. For chronic administration, however, such as would be required for most endocrinopathies, it is likely that the antiglucocorticoid activity of these compounds would not be desirable. Therefore, there has been a great deal of interest in developing compounds that will inhibit progesterone receptor (PR) transcriptional activity but do not interfere with the biological actions of glucocorticoids. 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 1449 1450 DEVELOPMENT OF DISSOCIATED ANTIPROGESTINS 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 (15–18). 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 r (...truncated)