Identification of Endocrine-Disrupting Compounds Using Nanoelectrospray Ionization Mass Spectrometry

A method using chip-based nanoelectrospray mass spectrometry (nanoESI-MS) is described to detect noncovalent ligand binding to the human estrogen receptor alpha ligand-binding domain (hER? LBD). This system represents an important environmental interest, because a wide variety of molecules, known as endocrine-disrupting compounds (EDCs), can bind to the estrogen receptor (ER) and induce adverse health effects in wildlife and humans. An efficient analytical method is therefore required to identify EDCs and characterize their solution-phase binding affinity and character (i.e. agonist or antagonist). Using proper experimental conditions, the nanoESI-MS approach allowed the detection of specific ligand interactions with hER? LBD. The best approach to evaluate solution-binding affinity by nanoESI-MS was to perform competitive binding experiments with 17?-estradiol (E2) as a reference ligand. Among the ligands tested, the relative binding affinity for hER? LBD measured by nanoESI-MS was 4-hydroxytamoxifen ? diethylstilbestrol > E2 ? genistein ? bisphenol A, consistent with the order of the binding affinities in solution. To discern agonist from antagonist, we used the specificity of a coactivator peptide for agonist-bound receptor. A specific coactivator-hER? LBD complex was detected only in the presence of an agonist ligand. Therefore, 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 method is promising for the identification and characterization of suspected ER ligands in a high-throughput manner

HIV-1 Pre-Integration Complexes. Structures, Functions and Drug Design

International audienc

Dual inhibition of HIV-1 replication by integrase-LEDGF allosteric inhibitors is predominant at the post-integration stage

BACKGROUND: LEDGF/p75 (LEDGF) is the main cellular cofactor of HIV-1 integrase (IN). It acts as a tethering factor for IN, and targets the integration of HIV in actively transcribed gene regions of chromatin. A recently developed class of IN allosteric inhibitors can inhibit the LEDGF-IN interaction. RESULTS: We describe a new series of IN-LEDGF allosteric inhibitors, the most active of which is Mut101. We determined the crystal structure of Mut101 in complex with IN and showed that the compound binds to the LEDGF-binding pocket, promoting conformational changes of IN which explain at the atomic level the allosteric effect of the IN/LEDGF interaction inhibitor on IN functions. In vitro, Mut101 inhibited both IN-LEDGF interaction and IN strand transfer activity while enhancing IN-IN interaction. Time of addition experiments indicated that Mut101 behaved as an integration inhibitor. Mut101 was fully active on HIV-1 mutants resistant to INSTIs and other classes of anti-HIV drugs, indicative that this compound has a new mode of action. However, we found that Mut101 also displayed a more potent antiretroviral activity at a post-integration step. Infectivity of viral particles produced in presence of Mut101 was severely decreased. This latter effect also required the binding of the compound to the LEDGF-binding pocket. CONCLUSION: Mut101 has dual anti-HIV-1 activity, at integration and post-integration steps of the viral replication cycle, by binding to a unique target on IN (the LEDGF-binding pocket). The post-integration block of HIV-1 replication in virus-producer cells is the mechanism by which Mut101 is most active as an antiretroviral. To explain this difference between Mut101 antiretroviral activity at integration and post-integration stages, we propose the following model: LEDGF is a nuclear, chromatin-bound protein that is absent in the cytoplasm. Therefore, LEDGF can outcompete compound binding to IN in the nucleus of target cells lowering its antiretroviral activity at integration, but not in the cytoplasm where post-integration production of infectious viral particles takes place

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

A new crystallization strategy: the presence of cleaved thioredoxin fusion is critical for crystallization of the estrogen nuclear receptor ligand binding domain in complex with synthetic ligands. This novel technique should be regarded as an interesting alternative for crystallization of difficult proteins

Production of unstable proteins through the formation of stable core complexes

International audiencePurification of proteins that participate in large transient complexes is impeded by low amounts, heterogeneity, instability and poor solubility. To circumvent these difficulties we set up a methodology that enables the production of stable complexes for structural and functional studies. This procedure is benchmarked and applied to two challenging protein families: the human steroid nuclear receptors (SNR) and the HIV-1 pre-integration complex. In the context of transcriptional regulation studies, we produce and characterize the ligand-binding domains of the glucocorticoid nuclear receptor and the oestrogen receptor beta in complex with a TIF2 (transcriptional intermediary factor 2) domain containing the three SNR-binding motifs. In the context of retroviral integration, we demonstrate the stabilization of the HIV-1 integrase by formation of complexes with partner proteins and DNA. This procedure provides a powerful research tool for structural and functional studies of proteins participating in non-covalent macromolecular complexes

The HIV-1 Pre-Integration Complexes: Structures, Functions and Dynamics

International audienc

Toward High Resolution Structures of the HIV-1 In/LEDGF/DNA Complex

International audienc