Nuclear receptors (NRs) complexed with agonist ligands activate transcription by recruiting coactivator protein complexes. In principle, one should be able to inhibit the transcriptional activity of the NRs by blocking this transcriptionally critical receptor-coactivator interaction directly, using an appropriately designed coactivator binding inhibitor (CBI). To guide our design of various classes of CBIs, we have used the crystal structure of an agonist-bound estrogen receptor (ER) ligand binding domain (LBD) complexed with a coactivator peptide containing the LXXLL signature motif bound to a hydrophobic groove on the surface of the LBD. One set of CBIs, based on an outside-in design approach, has various heterocyclic cores (triazenes, pyrimidines, trithianes, cyclohexanes) that mimic the tether sites of the three leucines on the peptide helix, onto which are appended leucine residue-like substituents. The other set, based on an inside-out approach, has a naphthalene core that mimics the two most deeply buried leucines, with substituents extending outward to mimic other features of the coactivator helical peptide. A fluorescence anisotropy-based coactivator competition assay was developed to measure the specific binding of these CBIs to the groove site on the ER-agonist complex with which coactivators interact; control ligand-binding assays assured that their interaction was not with the ligand binding pocket. The most effective CBIs were those from the pyrimidine family, the best binding with K(i) values of ca. 30 microM. The trithiane- and cyclohexane-based CBIs appear to be poor structural mimics, because of equatorial vs axial conformational constraints, and the triazene-based CBIs are also conformationally constrained by amine-substituent-to-ring resonance overlap, which is not the case with the higher affinity alkyl-substituted pyrimidines. The pyrimidine-based CBIs appear to be the first small molecule inhibitors of NR coactivator binding.

We report here on the design, synthesis, and evaluation of small molecule inhibitors of the interaction between a steroid receptor coactivator and estrogen receptor alpha. These inhibitors are based upon an amphipathic benzene scaffold whose hydrophobic face mimics the leucine-rich alpha-helical consensus sequence on the steroid receptor coactivators that interacts with a shallow groove on estrogen receptor alpha. Several of these molecules are among the most potent inhibitors of this interaction described to date and are active at low micromolar concentrations in both in vitro models of estrogen receptor action and in cell-based assays of estrogen receptor-mediated coactivator interaction and transcription.

Nuclear hormone receptor (NR) function relies on association of agonist-bound receptors with steroid receptor coactivator (SRC) proteins through a small pentapeptide motif (LXXLL) of the SRC that binds to a hydrophobic groove on the NR. We have synthesized a series of bicyclo[2.2.2]octanes that are close structural mimics of the two key lysine residues of this SRC sequence as bound in the hydrophobic groove of the estrogen receptor. These bicyclic systems block the NR-SRC interaction with modest potency.Nuclear receptors (NRs) are transcription factors that control many physiological and pathological processes by directly regulating the expression of select target genes. 1 Members of the NR gene superfamily have multi-domain structures, with a conserved DNAbinding domain and a ligand-binding domain (LBD). The transcriptional activity of NRs is initiated by hormone binding that stabilizes the conformation of the LBD. When an agonist ligand binds, conformational stabilization rigidifies surface features that function as docking sites for coactivator protein complexes that are recruited to modify chromatin structure and activate RNA polymerase II. The most notable of these are the p160 class of steroid receptor coactivators (SRCs). 2,3 X-ray crystallography has revealed details of these NR-SRC interactions, 4-9 which are largely mediated by a short, conserved, pentapeptide LXXLL (L=leucine, X=amino acid) motif of the SRC, termed the NR box. The interaction between the estrogen receptor α (ERα) LBD and a peptide corresponding to NR box 2 of SRC1 (RHKILHRLLQE) is shown in Fig. 1. 5 Selected residues of the peptide interact with the LBD through a two-turn amphipathic α-helical motif that places the first and third leucine residues (L690 and L694) in a deep, solvent-exposed hydrophobic groove made up of residues from various helices of the LBD; histidine and arginine residues of the peptide (H691 and R692, removed for clarity) are solvent exposed and appear unnecessary for the interaction. 5 The intrinsic dipole moment of the coactivator α-helix is aligned with charged residues on the surface of the ERα-LBD (E542 interacts with the peptide N-terminus and K362 with its C-terminus), together forming a "charge clamp". 5The traditional strategy to block agonist signaling of NRs involves hormone antagonists (antihormones) that bind to the LBD, displacing the agonist and stabilizing alternative © 2007 Elsevier Ltd. All rights reserved. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. An alternative approach to interrupting NR signaling is the use of compounds capable of blockin...

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