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Title:Second-site inhibitors of the estrogen and androgen hormone receptors
Author(s):Parent, Alexander A.
Director of Research:Katzenellenbogen, John A.
Doctoral Committee Chair(s):Katzenellenbogen, John A.
Doctoral Committee Member(s):van der Donk, Wilfred A.; Hergenrother, Paul J.; Gennis, Robert B.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):estrogen receptor
androgen receptor
nuclear receptor
Abstract:The estrogen and androgen receptors are members of the nuclear hormone receptor protein superfamily and play an important role in the development of primary and secondary female and male sexual characteristics. Although necessary for proper development, in certain contexts activation of these receptors contributes to the formation and progression of hyperplastic diseases, namely breast and prostate cancers. Current treatment for metastatic hormone responsive breast and prostate tumors involves either chemical ‘castration’ techniques which target the synthesis of the endogenous estrogen and androgen agonists, or the administration of hormone antagonists which displace endogenous agonist from the ligand binding domain of the receptor causing a conformational change such that the transcriptional activity of the protein is inhibited. While efficacious for a duration ranging from a few months to years, breast and prostate tumors eventually cease to respond to these treatments, entering a hormone-refractory state for which there is no adequate treatment. Multiple new approaches to estrogen and androgen receptor inhibition have been reported in recent years. Among these are efforts to directly disrupt the nuclear receptor/coactivator and the nuclear receptor/DNA interactions, and to increase degradation of receptor through binding of small-molecules at non-traditional binding sites. Due to the essential nature of these interactions for estrogen and androgen receptor signaling, it has been proposed that such second-site inhibition may be less susceptible to the mutations and receptor and coregulator overexpression that are currently exhibited in hormone-refractory breast and prostate cancers. As described herein, we have investigated a number of chemical systems for their ability to disrupt the estrogen and androgen receptor/steroid receptor coactivator interaction. In chapters 2 and 3 we describe the structure-based design of pyrimidine-core small-molecules that mimic the three interacting leucine/phenylalanine residues of the estrogen and androgen receptor coactivators. Members of this library bind to both nuclear receptors and disrupt the nuclear receptor/coactivator complex with Ki’s in the low micromolar range. In our screening against the two subtypes of the estrogen receptor, we discovered a only few compounds that exhibited binding to the estrogen receptor beta, and the vast majority show strong preference for estrogen receptor alpha inhibition. Based on its ability to accommodate larger aromatic residues at the coactivator binding site, we anticipated that pyrimidine-core molecules with bulky aromatic substituents would selectively bind to the androgen receptor. Indeed, this approach produced multiple coactivator binding inhibitors that displayed potent and selective inhibition of the androgen receptor, without concomitant disruption of the estrogen receptor/coactivator complex. During the synthesis of the pyrimidine-core coactivator binding inhibitors, a tetra-aryl cyclobutane compound that showed potent estrogen receptor/coactivator disruption was unexpectedly isolated. This compound stemmed from the photocycloaddition of 2,4-dichloro-6-styrylpyrimidine, an intermediate in the synthesis for a number of the pyrimidine-core coactivator binding inhibitors, which occurred accidentally under ambient conditions. Based on this observation, six additional azastilbenes were synthesized and their photoreactivity in the solid and solution states explored as detailed in chapter 4. The results from these experiments demonstrated a quantitative link between the pi-stacking ability of the azastilbenes (as determined by high-level quantum mechanical calculations) and cyclobutane formation in solution. Conversely, regio and stereoselective cyclobutane production in the solid state is controlled by the three dimensional orientation of the monomers in the crystal lattice as predicted by the ‘topochemical postulate.’ The creation of an expanded cyclobutane-core coactivator binding inhibitor library is described in chapter 5. Specifically, the pyrimidine rings of the tetra-aryl cycobutanes were either symmetrically diaminated, or alterntatively, each was substituted with a single beta-aminoethanethiol followed by amination to produce compounds with improved aqueous solubility. The most potent compound in this series disrupts the estrogen receptor/coactivator interaction with a Ki of ca. 70 nM. Initial results from screens for androgen receptor inhibition with these compounds produced a promising tetramethoxy tetra-aryl cyclobutane hit. In cell-based assays this compound has an activity profile similar to MDV3100, an antiandrogen currently in phase III clinical trials, and demonstrates no toxicity at any of the assayed concentrations. Based on this initial success, a focused library containing cyclobutanes with one or two-atom substituents on the pyrimidine rings was synthesized, producing a number of additional active compounds. Interestingly, as shown by EC50 shift assays, these androgen receptor-inhibitory compounds appear to be acting through a traditional antagonist mechanism, displacing agonist and inducing an antagonist conformational change in the receptor. The final chapter in the thesis involves the synthesis of a library of hydrocarbon ‘stapled’ peptides that target the androgen receptor coactivator binding groove. This class of alpha-helix-stabilized peptides has shown efficacy in mouse models, and demonstrates decreased rates of proteolytic degradation and increased cellular permeability. Although the stapled peptides showed selective inhibition of the androgen receptor/coactivator interaction in protein-based assays, in cellular models the activity was either greatly decreased or completely anulled. In spite of this disappointment, one of these peptides, SPAR-5, exhibited the unique ability to upregulate androgen-receptor dependent transcription in the presence of an agonist ligand. Protein-recruitment profiling of the SPAR-5/androgen receptor/agonist complex demonstrated a unique coregulator profile, indicative of a super-agonist receptor structure. Furthermore, the ability of SPAR-5 to restore full agonist activity in signaling-deficient androgen receptors was established in a polyglutamine model of Kennedy’s disease, a type of androgen insensitivity syndrome.
Issue Date:2012-02-01
Rights Information:Copyright 2011 Alexander A. Parent
Date Available in IDEALS:2012-02-01
Date Deposited:2011-12

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