Nuclear receptor liver receptor homologue 1 (LRH-1) regulates pancreatic cancer cell growth and proliferation.

An essential regulator of gene transcription, nuclear receptor liver receptor homologue 1 (LRH-1) controls cell differentiation in the developing pancreas and maintains cholesterol homeostasis in adults. Recent genome-wide association studies linked mutations in the LRH-1 gene and its up-stream regulatory regions to development of pancreatic cancer. In this work, we show that LRH-1 transcription is activated up to 30-fold in human pancreatic cancer cells compared to normal pancreatic ductal epithelium. This activation correlates with markedly increased LRH-1 protein expression in human pancreatic ductal adenocarcinomas in vivo. Selective blocking of LRH-1 by receptor specific siRNA significantly inhibits pancreatic cancer cell proliferation in vitro. The inhibition is tracked in part to the attenuation of the receptor's transcriptional targets controlling cell growth, proliferation, and differentiation. Previously, LRH-1 was shown to contribute to formation of intestinal tumors. This study demonstrates the critical involvement of LRH-1 in development and progression of pancreatic cancer, suggesting the LRH-1 receptor as a plausible therapeutic target for treatment of pancreatic ductal adenocarcinomas.

Molecular basis for dimer formation of TRbeta variant D355R.

Protein quality and stability are critical during protein purification for X-ray crystallography. A target protein that is easy to manipulate and crystallize becomes a valuable product useful for high-throughput crystallography for drug design and discovery. In this work, a single surface mutation, D355R, was shown to be crucial for converting the modestly stable monomeric ligand binding domain of the human thyroid hormone receptor (TR LBD) into a stable dimer. The structure of D335R TR LBD mutant was solved using X-ray crystallography and refined to 2.2 A resolution with R(free)/R values of 24.5/21.7. The crystal asymmetric unit reveals the TR dimer with two molecules of the hormone-bound LBD related by twofold symmetry. The ionic interface between the two LBDs comprises residues within loop H10-H11 and loop H6-H7 as well as the C-terminal halves of helices 8 of both protomers. Direct intermolecular contacts formed between the introduced residue Arg 355 of one TR molecule and Glu 324 of the second molecule become a part of the extended dimerization interface of 1330 A(2) characteristic for a strong complex assembly that is additionally strengthened by buffer solutes.

Structural insight into the mode of action of a direct inhibitor of coregulator binding to the thyroid hormone receptor.

The development of nuclear hormone receptor antagonists that directly inhibit the association of the receptor with its essential coactivators would allow useful manipulation of nuclear hormone receptor signaling. We previously identified 3-(dibutylamino)-1-(4-hexylphenyl)-propan-1-one (DHPPA), an aromatic beta-amino ketone that inhibits coactivator recruitment to thyroid hormone receptor beta (TRbeta), in a high-throughput screen. Initial evidence suggested that the aromatic beta-enone 1-(4-hexylphenyl)-prop-2-en-1-one (HPPE), which alkylates a specific cysteine residue on the TRbeta surface, is liberated from DHPPA. Nevertheless, aspects of the mechanism and specificity of action of DHPPA remained unclear. Here, we report an x-ray structure of TRbeta with the inhibitor HPPE at 2.3-A resolution. Unreacted HPPE is located at the interface that normally mediates binding between TRbeta and its coactivator. Several lines of evidence, including experiments with TRbeta mutants and mass spectroscopic analysis, showed that HPPE specifically alkylates cysteine residue 298 of TRbeta, which is located near the activation function-2 pocket. We propose that this covalent adduct formation proceeds through a two-step mechanism: 1) beta-elimination to form HPPE; and 2) a covalent bond slowly forms between HPPE and TRbeta. DHPPA represents a novel class of potent TRbeta antagonist, and its crystal structure suggests new ways to design antagonists that target the assembly of nuclear hormone receptor gene-regulatory complexes and block transcription.

Perspectives on designs of antiandrogens for prostate cancer.

The androgen receptor (AR) regulates gene transcription in many tissues and is profoundly important in prostate cancer. Antiandrogens compete with the natural hormone and are front line therapeutics to treat prostate cancer. However, antiandrogens frequently become ineffective after prolonged treatment because of development of tumor resistance. This paper reviews design principles for new generations of antiandrogens: super antagonists and surface allosteric modulators. Super antiandrogens are compounds with higher binding affinity than natural agonists and that contain precisely engineered hydrophobic groups that disrupt AR function. AR surface is also an attractive alternative target. Surface inhibitors are small molecules that directly block the receptor-co-activator interface, preventing co-activator recruitment. The challenges to designing these compounds are significant but so is the potential for treatment of the disease.

Interaction between the androgen receptor and a segment of its corepressor SHP.

The mechanisms of functional repression of the androgen receptor (AR) are crucial for the regulation of genes involved in physiological development as well as for the progression of prostate cancer. To date, only two in vivo inhibitors of AR-mediated transcription have been identified: DAX-1 and SHP (small heterodimer partner). SHP is a regulatory nuclear receptor (NR) that lacks DNA-binding and activation domains. Using X-ray crystallography, the interaction between peptide segments of the SHP repressor containing LxxLL-like motifs and the ligand-binding domain of AR have been investigated. Under the crystallization conditions used, it was found that of the three NR Boxes present in the SHP protein sequence, only NR Box 2 (LKKIL motif) formed a complex with AR. Determination of the crystal structure revealed that ten amino acids of the SHP peptide (14-mer) are ordered through interactions with AR. Two side chains make unique interactions that were not reported for other AR-peptide complexes. The NR Box 2 of SHP binds to an adaptable hydrophobic groove on the surface of AR in a fashion observed for other NR-LxxLL-like complexes. Comparisons of AR structures bound to coactivator peptides and the SHP peptide revealed structural similarity of their binding sites, suggesting that transcriptional AR activity may be inhibited by SHP by competing with AR coactivators.

Discovery of small molecule inhibitors of the interaction of the thyroid hormone receptor with transcriptional coregulators.

Thyroid hormone (3,5,3'-triiodo-L-thyronine, T3) is an endocrine hormone that exerts homeostatic regulation of basal metabolic rate, heart rate and contractility, fat deposition, and other phenomena (1, 2). T3 binds to the thyroid hormone receptors (TRs) and controls their regulation of transcription of target genes. The binding of TRs to thyroid hormone induces a conformational change in TRs that regulates the composition of the transcriptional regulatory complex. Recruitment of the correct coregulators (CoR) is important for successful gene regulation. In principle, inhibition of the TR-CoR interaction can have a direct influence on gene transcription in the presence of thyroid hormones. Herein we report a high throughput screen for small molecules capable of inhibiting TR coactivator interactions. One class of inhibitors identified in this screen was aromatic beta-aminoketones, which exhibited IC50 values of approximately 2 microm. These compounds can undergo a deamination, generating unsaturated ketones capable of reacting with nucleophilic amino acids. Several experiments confirm the hypothesis that these inhibitors are covalently bound to TR. Optimization of these compounds produced leads that inhibited the TR-CoR interaction in vitro with potency of approximately 0.6 microm and thyroid signaling in cellular systems. These are the first small molecules irreversibly inhibiting the coactivator binding of a nuclear receptor and suppressing its transcriptional activity.