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

Through an effort to develop novel ligands that have subtype selectivity for the estrogen receptors alpha (ERalpha) and beta (ERbeta), we have found that 2,3-bis(4-hydroxyphenyl)propionitrile (DPN) acts as an agonist on both ER subtypes, but has a 70-fold higher relative binding affinity and 170-fold higher relative potency in transcription assays with ERbeta than with ERalpha. To investigate the ERbeta affinity- and potency-selective character of this DPN further, we prepared a series of DPN analogues in which both the ligand core and the aromatic rings were modified by the repositioning of phenolic hydroxy groups and by the addition of alkyl substituents and nitrile groups. We also prepared other series of DPN analogues in which the nitrile functionality was replaced with acetylene groups or polar functions, to mimic the linear geometry or polarity of the nitrile, respectively. To varying degrees, all of the analogues show preferential binding affinity for ERbeta (i.e., they are ERbeta affinity-selective), and many, but not all of them, are also more potent in activating transcription through ERbeta than through ERalpha (i.e., they are ERbeta potency-selective). meso-2,3-Bis(4-hydroxyphenyl)succinonitrile and dl-2,3-bis(4-hydroxyphenyl)succinonitrile are among the highest ERbeta affinity-selective ligands, and they have an ERbeta potency selectivity that is equivalent to that of DPN. The acetylene analogues have higher binding affinities but somewhat lower selectivities than their nitrile counterparts. The polar analogues have lower affinities, and only the fluorinated polar analogues have substantial affinity selectivities. This study suggests that, in this series of ligands, the nitrile functionality is critical to ERbeta selectivity because it provides the optimal combination of linear geometry and polarity. Furthermore, the addition of a second nitrile group beta to the nitrile in DPN or the addition of a methyl substitutent at an ortho position on the beta-aromatic ring increases the affinity and selectivity of these compounds for ERbeta. These ERbeta-selective compounds may prove to be valuable tools in understanding the differences in structure and biological function of ERalpha and ERbeta.

Estrogen receptor-beta potency-selective ligands: structure-activity relationship studies of diarylpropionitriles and their acetylene and polar analogues.

In this review, we have traced the path of estradiol from its entry into the cell to the time of its release from the receptor. We feel that all of the current models are limited in one respect or another. We have examined most critically those currently most in vogue. The entry of estradiol into the cell is widely assumed to be simply a matter of diffusion. We have highlighted data that suggest the existence of a protein-mediated transport, but feel that the available data are too limited to make a definite conclusion.

Current Models of Steroid Hormone Action: A Critique

The estradiol pharmacophore: Ligand structure-estrogen receptor binding affinity relationships and a model for the receptor binding site

Publisher Summary This chapter discusses the photoaffinity labeling which could be used as a method that allows to unleash the reagent at a particular time and place, when the chemical affinity labeling restricts it. The chapter notes that the possibility that a labile group of appropriate reactivity cannot be incorporated into the ligand molecule without excessive disturbance of the recognition process, there are two limitations to the affinity labeling approach. The first challenge; the range of chemical reactivity of groups that can be incorporated into the ligand is limited by the fact that these groups must not react so rapidly with water that they are destroyed hydrolytically before the ligand that carries them can reach the binding site. And secondly, it is becoming clear that some biological problems require a reagent whose reactivity remains masked until the experimenter chooses to activate it. Both of the two limitations of classical chemical affinity labeling discussed above can in principle be circumvented by the use of a photogenerated reagent.

[8] Photoaffinity labeling

Studies on the uterine, cytoplasmic estrogen binding protein. Thermal stability and ligand dissociation rate. An assay of empty and filled sites by exchange.

Photoaffinity labels for estrogen binding proteins of rat uterus.

The photoreactivity of 20 estrogen derivatives (4 azides 4 diazos 4 triarylethylenes and 8 miscellaneous) was analyzed by a rat uterus cytosol exchange assay. The photosensitive derivative was incubated with rat uterus supernatant fraction with or without estradiol for 1-2 hours at 4 degrees C. then irradiated with 254 nm or 315 nm light at 2 degrees C. while samples were removed for assay of tritiated estrogen-binding sites. The reaction efficiency or percent of estrogen-binding capacity consumed by photolysis was computed. The reaction efficiency of azides and diazos ranged from 0%-21% and of triarylethylenes 20%-51%. Benzil o-iodophenol o-nitrophenol alpha-oximo ketone stilbene and styrene chromophores were unreactive. The kinetics of reaction was usually biphasic with a rapid initial rate due to photolysis of the photosensitive moiety and a slower rate interpreted as direct photolytic degradation of the estrogen-binding protein. 6-oxoestradiol had a first order reaction.

Photoreactivity of some light-sensitive estrogen derivatives. Use of an exchange assay to determine their photointeraction with the rat uterine estrogen binding protein.

Howard J. Johnson

Papers

Studies on the uterine, cytoplasmic estrogen binding protein. Thermal stability and ligand dissociation rate. An assay of empty and filled sites by exchange.

Photoaffinity labels for estrogen binding proteins of rat uterus.

Photoreactivity of some light-sensitive estrogen derivatives. Use of an exchange assay to determine their photointeraction with the rat uterine estrogen binding protein.

Reagents for photoaffinity labeling of estrogen binding proteins. Synthesis of some azide and diazo derivatives of estradiol, estrone, and hexestrol