Tomas Bonn

Molecular basis of agonism and antagonism in the oestrogen receptor.

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, 17β-oestradiol, and the selective antagonist raloxifene, at resolutions of 3.1 and 2.6 Å, 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.

Structure of the Ligand-Binding Domain of Oestrogen Receptor Beta in the Presence of a Partial Agonist and a Full Antagonist

Oestrogens exert their physiological effects through two receptor subtypes. Here we report the three‐dimensional structure of the oestrogen receptor beta isoform (ERβ) ligand‐binding domain (LBD) in the presence of the phyto‐oestrogen genistein and the antagonist raloxifene. The overall structure of ERβ‐LBD is very similar to that previously reported for ERα. Each ligand interacts with a unique set of residues within the hormone‐binding cavity and induces a distinct orientation in the AF‐2 helix (H12). The bulky side chain of raloxifene protrudes from the cavity and physically prevents the alignment of H12 over the bound ligand. In contrast, genistein is completely buried within the hydrophobic core of the protein and binds in a manner similar to that observed for ERs endogenous hormone, 17β‐oestradiol. However, in the ERβ–genistein complex, H12 does not adopt the distinctive ’agonist‘ position but, instead, lies in a similar orientation to that induced by ER antagonists. Such a sub‐optimal alignment of the transactivation helix is consistent with genisteins partial agonist character in ERβ and demonstrates how ERs transcriptional response to certain bound ligands is attenuated.

The three-dimensional structure of the liver X receptor beta reveals a flexible ligand-binding pocket that can accommodate fundamentally different ligands.

The structures of the liver X receptor LXRβ (NR1H2) have been determined in complexes with two synthetic ligands, T0901317 and GW3965, to 2.1 and 2.4 Å, respectively. Together with its isoform LXRα (NR1H3) it regulates target genes involved in metabolism and transport of cholesterol and fatty acids. The two LXRβ structures reveal a flexible ligand-binding pocket that can adjust to accommodate fundamentally different ligands. The ligand-binding pocket is hydrophobic but with polar or charged residues at the two ends of the cavity. T0901317 takes advantage of this by binding to His-435 close to H12 while GW3965 orients itself with its charged group in the opposite direction. Both ligands induce a fixed “agonist conformation” of helix H12 (also called the AF-2 domain), resulting in a transcriptionally active receptor.

Indazole-based liver X receptor (LXR) modulators with maintained atherosclerotic lesion reduction activity but diminished stimulation of hepatic triglyceride synthesis.

A series of substituted 2-benzyl-3-aryl-7-trifluoromethylindazoles were prepared as LXR modulators. These compounds were partial agonists in transactivation assays when compared to 1 (T0901317) and were slightly weaker with respect to potency and efficacy on LXRalpha than on LXRbeta. Lead compounds in this series 12 (WAY-252623) and 13 (WAY-214950) showed less lipid accumulation in HepG2 cells than potent full agonists 1 and 3 (WAY-254011) but were comparable in efficacy to 1 and 3 with respect to cholesterol efflux in THP-1 foam cells, albeit weaker in potency. Compound 13 reduced aortic lesion area in LDLR knockout mice equivalently to 3 or positive control 2 (GW3965). In a 7-day hamster model, compound 13 showed a lesser propensity for plasma TG elevation than 3, when the compounds were compared at doses in which they elevated ABCA1 and ABCG1 gene expression in duodenum and liver at equal levels. In contrast to results previously published for 2, the lack of TG effect of 13 correlated with its inability to increase liver fatty acid synthase (FAS) gene expression, which was up-regulated 4-fold by 3. These results suggest indazoles such as 13 may have an improved profile for potential use as a therapeutic agent.

Characterization of bacterially expressed rat estrogen receptor β ligand binding domain by mass spectrometry: Structural comparison with estrogen receptor α

Abstract Functional rat estrogen receptor β ligand binding domain (rERβ LBD, aa 210–485) and human estrogen receptor α ligand binding domain (hERα LBD, aa 301–553) were expressed in Escherichia coli. Hormone binding assays revealed that both ERβ and ERα LBDs bound the natural ligand estradiol (E2) with similar affinity (Kd ∼ 100 pM). Competitive binding experiments were carried out with ICI 164384, 4-hydroxytamoxifen, 16α-bromo-estradiol, and genistein employing [3H]E2 as a tracer. No significant differences in responses of ERα and ERβ LBDs to ICI 164384 and 4-hydroxytamoxifen were observed. 16α-Bromo-estradiol and genistein discriminated between the ER subtypes and acted as ERα and ERβ selective ligands, respectively. Final purification of recombinant proteins was achieved on an E2 affinity column, where they were subjected to in situ carboxymethylation. The partially carboxymethylated proteins actively bound E2. The carboxymethylated rERβ LBD had a molecular mass of 32251.6 Da, equivalent to the calculated mass with the addition of three carboxymethyl groups. No other proteins (of lower or higher molecular mass) were detected, so the LBD was considered structurally authentic and pure. By using a combination of intact protein mass spectrometric fragmentation and trypsin proteolysis (98% sequence coverage), it was established that rERβ cysteine-289 and -354 were not carboxymethylated on the affinity column, suggesting that they were shielded from alkylation in the E2-bound conformation state. Concurrent analysis of hERα LBD showed that under the same experimental conditions, the two equivalent ERα cysteines were not alkylated (αC381 and αC447). These data support close structural relationship between the E2-bound ERα LBD and ERβ LBD proteins.

Structural insights into the mechanisms of agonism and antagonism in oestrogen receptor isoforms

Here we summarise the results that have emerged from our structural studies on the oestrogen receptor (ER) ligand-binding domain. We have investigated the conformational effects of a variety of ligands on the structures of both ER isoforms. Each class of ligand (agonists, partial agonists and selective oestrogen receptor modulators) induces a unique conformation in the receptors ligand-dependent transcriptional activation function. Together these studies have broadened our understanding of ER function by providing a unique insight into ERs ligand specificity and the structural changes that underlie receptor agonism and antagonism.