Inhibitors of UHRF1 base flipping activity showing cytotoxicity against cancer cells.

Ubiquitin-like containing PHD and RING finger domain 1 (UHRF1) is a nuclear multi-domain protein overexpressed in numerous human cancer types. We previously disclosed the anthraquinone derivative UM63 that inhibits UHRF1-SRA domain base-flipping activity, although having DNA intercalating properties. Herein, based on the UM63 structure, new UHRF1-SRA inhibitors were identified through a multidisciplinary approach, combining molecular modelling, biophysical assays, molecular and cell biology experiments. We identified AMSA2 and MPB7, that inhibit UHRF1-SRA mediated base flipping at low micromolar concentrations, but do not intercalate into DNA, which is a key advantage over UM63. These molecules prevent UHRF1/DNMT1 interaction at replication forks and decrease the overall DNA methylation in cells. Moreover, both compounds specifically induce cell death in numerous cancer cell lines, displaying marginal effect on non-cancer cells, as they preferentially affect cells with high level of UHRF1. Overall, these two compounds are promising leads for the development of anti-cancer drugs targeting UHRF1.

Structural basis for a molecular allosteric control mechanism of cofactor binding to nuclear receptors.

Transcription regulation by steroid hormones, vitamin derivatives, and metabolites is mediated by nuclear receptors (NRs), which play an important role in ligand-dependent gene expression and human health. NRs function as homodimers or heterodimers and are involved in a combinatorial, coordinated and sequentially orchestrated exchange between coregulators (corepressors, coactivators). The architecture of DNA-bound functional dimers positions the coregulators proteins. We previously demonstrated that retinoic acid (RAR-RXR) and vitamin D3 receptors (VDR-RXR) heterodimers recruit only one coactivator molecule asymmetrically without steric hindrance for the binding of a second cofactor. We now address the problem of homodimers for which the presence of two identical targets enhances the functional importance of the mode of binding. Using structural and biophysical methods and RAR as a model, we could dissect the molecular mechanism of coactivator recruitment to homodimers. Our study reveals an allosteric mechanism whereby binding of a coactivator promotes formation of nonsymmetrical RAR homodimers with a 21 stoichiometry. Ligand conformation and the cofactor binding site of the unbound receptor are affected through the dimer interface. A similar control mechanism is observed with estrogen receptor (ER) thus validating the negative cooperativity model for an established functional homodimer. Correlation with published data on other NRs confirms the general character of this regulatory pathway.

Towards high-throughput identification of endocrine disrupting compounds with mass spectrometry.

High-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking has the ability to monitor the ligand-dependent dimerization of the human estrogen receptor alpha ligand binding domain (hERalpha LBD) in solution. Because only ER ligands enhance the homodimer abundance, we evaluated the ability of this label-free approach for identifying endocrine disrupting compounds (EDCs) in a high-throughput manner. This was achieved by combining an automated liquid handler with an automated MS acquisition procedure, which allowed a five-fold gain in operator time compared to a fully manual approach. To detect ligand binding with enough confidence, the receptor has to be incubated with at least a 10 microM concentration of the test compound. Based on the increase of the measured homodimer intensity, eight compounds with a relative binding affinity (RBA, relative to the natural hormone estradiol) >7% were identified as ER ligands among the 28 chemicals tested. Two other compounds, quercetin and 4-tert-amylphenol, were also identified as ER ligands, although their RBAs have been reported to be only 0.01% and 0.000055%, respectively. This suggests that these two ligands have a higher affinity for hERalpha LBD than reported in the literature. The high-mass MALDI approach thus allows identifying high affinity EDCs in an efficient way.

Monitoring ligand modulation of protein-protein interactions by mass spectrometry: estrogen receptor alpha-SRC1.

Many drugs and chemicals exert their biological effect by modulating protein-protein interactions. In vitro approaches to characterize these mechanisms are often based on indirect measurements (e.g., fluorescence). Here, we used mass spectrometry (MS) to directly monitor the effect of small-molecule ligands on the binding of a coactivator peptide (SRC1) by the human estrogen receptor alpha ligand binding domain (hERalpha LBD). Nanoelectrospray mass spectrometry (nanoESI-MS) and high-mass matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with chemical cross-linking were employed to follow these processes. The chemical cross-linking protocol used prior to high-mass MALDI analysis allows detection of intact noncovalent complexes. The binding of intact hERalpha LBD homodimer with two coactivator peptides was detected with nanoESI-MS and high-mass MALDI-MS only in the presence of an agonist ligand. Furthermore, high-mass MALDI-MS revealed an increase of the homodimer abundance after incubating the receptor with a ligand, independent of the ligand character (i.e., agonist, antagonist). The binding characteristics of the compounds tested by MS correlate very well with their biological activity reported by cell-based assays. High-mass MALDI appears to be an efficient and simple tool for directly monitoring ligand regulation mechanisms involved in protein-protein interactions. Furthermore, the combination of both MS methods allows identifying and characterizing endocrine-disrupting compounds or new drug compounds in an efficient way.

Cleaved thioredoxin fusion protein enables the crystallization of poorly soluble ERalpha in complex with synthetic ligands.

The ligand-binding domain (LBD) of human oestrogen receptor alpha was produced in Escherichia coli as a cleavable thioredoxin (Trx) fusion in order to improve solubility. Crystallization trials with either cleaved and purified LBD or with the purified fusion protein both failed to produce crystals. In another attempt, Trx was not removed from the LBD after endoproteolytic cleavage and its presence promoted nucleation and subsequent crystal growth, which allowed the structure determination of two different LBD-ligand-coactivator peptide complexes at 2.3 A resolution. This technique is likely to be applicable to other low-solubility proteins.

Estrogen receptor-ligand complexes measured by chip-based nanoelectrospray mass spectrometry: an approach for the screening of endocrine disruptors.

In the present report, a method based on chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hERalpha LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine disruptors, can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. Using proper experimental conditions, the nanoESI-MS approach allowed for the detection of specific ligand interactions with hERalpha LBD. The relative gas-phase stability of selected hERalpha LBD-ligand complexes did not mirror the binding affinity in solution, a result that demonstrates the prominent role of hydrophobic contacts for stabilizing ER-ligand complexes in solution. The best approach to evaluate relative solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17beta-estradiol (E2) used as a reference ligand. Among the ligands tested, the relative binding affinity for hERalpha LBD measured by nanoESI-MS was 4-hydroxtamoxifen approximately diethylstilbestrol > E2 >> genistein >> bisphenol A, consistent with the order of the binding affinities in solution. The limited reproducibility of the bound to free protein ratio measured by nanoESI-MS for this system only allowed the binding constants (K(d)) to be estimated (low nanomolar range for E2). The specificity of nanoESI-MS combined with its speed (1 min/ligand), low sample consumption (90 pmol protein/ligand), and its sensitivity for ligand (30 ng/mL) demonstrates that this technique is a promising method for screening suspected endocrine disrupting compounds and to qualitatively evaluate their binding affinity.