Oestrogens are involved in the growth, development and homeostasis of a number of tissues. The physiological effects of these steroids are mediated by a ligand-inducible nuclear transcription factor, the oestrogen receptor (ER). Hormone binding to the ligand-binding domain (LBD) of the ER initiates a series of molecular events culminating in the activation or repression of target genes. Transcriptional regulation arises from the direct interaction of the ER with components of the cellular transcription machinery. Here we report the crystal structures of the LBD of ER in complex with the endogenous oestrogen, 17beta-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 A, respectively. The structures provide a molecular basis for the distinctive pharmacophore of the ER and its catholic binding properties. Agonist and antagonist bind at the same site within the core of the LBD but demonstrate different binding modes. In addition, each class of ligand induces a distinct conformation in the transactivation domain of the LBD, providing structural evidence of the mechanism of antagonism.

Molecular basis of agonism and antagonism in the oestrogen receptor.

Methods are provided for inhibiting stenosis following vascular trauma or disease in a mammalian host, comprising administering to the host a therapeutically effective dosage of a therapeutic conjugate containing a vascular smooth muscle binding protein that associates in a specific manner with a cell surface of the vascular smooth muscle cell, coupled to a therapeutic agent dosage form that inhibits a cellular activity of the muscle cell. Methods are also provided for the direct and/or targeted delivery of therapeutic agents to vascular smooth muscle cells that cause a dilation and fixation of the vascular lumen by inhibiting smooth muscle cell contraction, thereby constituting a biological stent.

Therapeutic inhibitor of vascular smooth muscle cells

We have found that certain tetrasubstituted pyrazoles are high-affinity ligands for the estrogen receptor (ER) (Fink et al Chem Biol 1999, 6, 205-219) and that one pyrazole is considerably more potent as an agonist on the ERalpha than on the ERbeta subtype (Sun et al Endocrinology 1999, 140, 800-804) To investigate what substituent pattern provides optimal ER binding affinity and the greatest enhancement of potency as an ERalpha-selective agonist, we prepared a number of tetrasubstituted pyrazole analogues with defined variations at certain substituent positions Analysis of their binding affinity pattern shows that a C(4)-propyl substituent is optimal and that a p-hydroxyl group on the N(1)-phenyl group also enhances affinity and selectivity for ERalpha The best compound in this series, a propylpyrazole triol (PPT, compound 4g), binds to ERalpha with high affinity (ca 50% that of estradiol), and it has a 410-fold binding affinity preference for ERalpha It also activates gene transcription only through ERalpha Thus, this compound represents the first ERalpha-specific agonist We investigated the molecular basis for the exceptional ERalpha binding affinity and potency selectivity of pyrazole 4g by a further study of structure-affinity relationships in this series and by molecular modeling These investigations suggest that the pyrazole triols prefer to bind to ERalpha with their C(3)-phenol in the estradiol A-ring binding pocket and that binding selectivity results from differences in the interaction of the pyrazole core and C(4)-propyl group with portions of the receptor where ERalpha has a smaller residue than ERbeta These ER subtype-specific interactions and the ER subtype-selective ligands that can be derived from them should prove useful in defining those biological activities in estrogen target cells that can be selectively activated through ERalpha

Pyrazole Ligands: Structure−Affinity/Activity Relationships and Estrogen Receptor-α-Selective Agonists

Forty years ago, Lerner and coworkers (1958) discovered the first nonsteroidal antiestrogen and Jensen (Jensen and Jacobson, 1960) identified a target for drug action, the ER. This knowledge opened the door for the clinical development of tamoxifen which we now know provides a survival advantage in both node-positive and node-negative patients with ER-positive disease (Early Breast Cancer Trialists Collaborative Group, 1992, 1998). The drug has been studied extensively, and the results have provided an invaluable insight into possible ancillary advantages of "antiestrogens", i.e., maintenance of bone density and the prevention of coronary heart disease, and possible disadvantages, i.e., rat liver carcinogenesis and an increased risk of endometrial cancer. Most importantly, the identification of the target site-specific actions of tamoxifen caused a paradigm shift in the prospective uses of antiestrogens from a direct exploitation of the antitumor properties to the broader application as a preventative for osteoporosis, but with the beneficial side effects of preventing breast and endometrial cancer. Raloxifene, a second-generation SERM, has all the properties in the laboratory that would encourage development as a safe preventative for osteoporosis (Jordan et al., 1997). As a result, raloxifene has been evaluated in more than 11,000 postmenopausal women and found to maintain bone density with significant decreases in breast cancer incidence and no increase in endometrial thickness. Raloxifene is now available as a preventative for osteoporosis in postmenopausal women. There is every reason to believe that a multifaceted agent like raloxifene will find widespread use, and there will be continuing interest by the pharmaceutical industry in the development of new agents with even broader applications. The extensive clinical effort is augmented by past molecular innovations in the laboratory and the future promise of new discoveries. The cloning and sequencing of the ER (Green et al., 1986; Greene et al., 1986) has allowed the development of an ER knock-out mouse (Lubahn et al., 1993) that compliments Jensen's pioneering work (Jensen and Jacobson, 1962) and describes the consequences of the loss of ER alpha. However, ER beta (Kuiper et al., 1996), the second ER, has provided an additional dimension to the description of estrogen and antiestrogen action. For the future, the development of ER beta monoclonal antibodies, the classification of target sites for the protein around the body, and the creation of ER beta and ER alpha, beta knock-out mice will identify new therapeutic targets to modulate physiological functions. Clearly, the successful crystallization of ER alpha with raloxifene (Brzozowski et al., 1997) must act as a stimulus for the crystallization of ER beta. The central issue for research on antiestrogen pharmacology is the discovery of the mechanism (or mechanisms) of target site-specificity for the modulation of estrogenic and antiestrogenic response. The description of a stimulatory pathway for antiestrogens through an AP-1 ER beta signal transduction pathway (Paech et al., 1997), although interesting, may not entirely explain the estrogenicity of antiestrogens. The model must encompass the sum of pharmacological consequences of signal transduction through ER alpha and ER beta with the simultaneous competition from endogenous estrogens at both sites. This is complicated because estradiol is an antagonist at ER beta through AP-1 sites (Paech et al., 1997), so this is clearly not the pathway for estrogen-induced bone maintenance in women. Estrogen is stimulatory through ER alpha, but antiestrogens are usually partial agonists and may either block or stimulate genes. However, we suggest that the ER alpha stimulatory pathway could be amplified through selective increases in coactivators. The principle is illustrated with the MDA-MB-231 cells stably transfected with the cDNAs for the wild-type and the amino acid 351 mutan

Basic Guide to the Mechanisms of Antiestrogen Action

Context.—Raloxifene is a selective estrogen receptor modulator that has estrogen-agonistic
effects on bone and estrogen-antagonistic effects on breast and uterus.Objective.—To identify the effects of raloxifene on markers of cardiovascular risk
in postmenopausal women, and to compare them with those induced by hormone
replacement therapy (HRT).Design.—Double-blind, randomized, parallel trial.Setting.—Eight sites in the United States.Participants.—390 healthy postmenopausal women recruited by advertisement.Intervention.—Participants were randomized to receive 1 of 4 treatments: raloxifene,
60 mg/d; raloxifene, 120 mg/d; HRT (conjugated equine estrogen, 0.625 mg/d,
and medroxyprogesterone acetate, 2.5 mg/d); or placebo.Main Outcome Measures.—Change and percent change from baseline of lipid levels and coagulation
parameters after 3 months and 6 months of treatment.Results.—At the last visit completed, compared with placebo, both dosages of
raloxifene significantly lowered low-density lipoprotein cholesterol (LDL-C)
by 12% (P<.001), similar to the 14% reduction
with HRT (P<.001). Both dosages of raloxifene
significantly lowered lipoprotein(a) by 7% to 8% (P<.001),
less than the 19% decrease with HRT (P<.001).
Raloxifene increased high-density lipoprotein-2 cholesterol (HDL2-C)
by 15% to 17% (P<.05), less than the 33% increase
with HRT (P<.001). Raloxifene did not significantly
change high-density lipoprotein cholesterol (HDL-C), triglycerides, or plasminogen
activator inhibitor-1 (PAI-1); whereas HRT increased HDL-C by 11% and triglycerides
by 20%, and decreased PAI-1 by 29% (for all, P<
.001). Raloxifene significantly lowered fibrinogen by 12% to 14% (P<.001), unlike HRT, which had no effect. Neither treatment changed
fibrinopeptide A or prothrombin fragment 1 and 2.Conclusions.—Raloxifene favorably alters biochemical markers of cardiovascular risk
by decreasing LDL-C, fibrinogen, and lipoprotein(a), and by increasing HDL2-C without raising triglycerides. In contrast to HRT, raloxifene had
no effect on HDL-C and PAI-1, and a lesser effect on HDL2-C and
lipoprotein(a). Further clinical trials are necessary to determine whether
these favorable biochemical effects are associated with protection against
cardiovascular disease.

https://jamanetwork.com/journals/jama/articlepdf/187517/JOC72104.pdf

Effects of Raloxifene on Serum Lipids and Coagulation Factors in Healthy Postmenopausal Women

The 2-arylbenzothiophene raloxifene, 1, is a selective estrogen receptor modulator which is currently under clinical evaluation for the prevention and treatment of postmenopausal osteoporosis. A series of raloxifene analogs which contain modifications to the 2-arylbenzothiophene core have been prepared and evaluated for the ability to bind to the estrogen receptor and inhibit MCF-7 breast cancer cell proliferation in vitro. Their ability to function as tissue-selective estrogen agonists in vivo has been assayed in a short-term, ovariectomized (OVX) rat model with end points of serum cholesterol lowering, uterine weight gain, and uterine eosinophil peroxidase activity. These studies have demonstrated that (1) the 6-hydroxy and, to a lesser extent, the 4'-hydroxy substituents of raloxifene are important for receptor binding and in vitro activity, (2) small, highly electronegative 4'-substituents such as hydroxy, fluoro, and chloro are preferred both in vitro and in vivo, (3) increased steric bulk at the 4'-position leads to increased uterine stimulation in vivo, and (4) additional substitution of the 2-aryl moiety is tolerated while additional substitution at the 4-, 5-, or 7-position of the benzothiophene results in reduced biological activity. In addition, compounds in which the 2-aryl group is replaced by alkyl, cycloalkyl, and naphthyl substituents maintain a profile of in vitro and in vivo biological activity qualitatively similar to that of raloxifene. Several novel structural variants including 2-cyclohexyl, 2-naphthyl, and 6-carbomethoxy analogs also demonstrated efficacy in preventing bone loss in a chronic OVX rat model of postmenopausal osteopenia, at doses of 0.1-10 mg/kg.

Structure-activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene

This invention provides a novel series of non-peptidyl compounds which are useful in the treatment or prevention of a physiological disorder associated with an excess of tachykinins. This invention also provides methods for the treatment of such physiological disorders as well as pharmaceutical formulations which employ these novel compounds.

Non-peptide tachykinin receptor antagonists

This invention provides the novel compound of formula (I) having the chemical name (R)-3-(1H-indol-3-yl)-1-[N-(2-methoxybenzyl)acetylamino]-2-[N-2-(4-(piperidin-1-yl)piperidin-1-yl)acetyl)amino]propane dihydrochloride trihydrate.

Sung-Yong Stephen Cho

Papers

Structure-activity relationships of selective estrogen receptor modulators: modifications to the 2-arylbenzothiophene core of raloxifene

Non-peptide tachykinin receptor antagonists

Heterocyclic tachykinin receptor antagonist

Practical and Enantiospecific Synthesis of LY303870, a Novel NK‐1 Antagonist.

Antagonistes hétérocycliques du récepteur des tachykinines