Agonist versus antagonist induce distinct thermodynamic modes of co-factor binding to the glucocorticoid receptor.

The glucocorticoid receptor (GR) is involved in the transcriptional regulation of genes associated with inflammation, glucose homeostasis, and bone turnover through the association with ligands, such as corticosteroids. GR-mediated gene transcription is regulated or fine-tuned via the recruitment of co-factors including coactivators and corepressors. Current therapeutic approaches to targeting GR aim to retain the beneficial anti-inflammatory activity of the corticosteroids while eliminating negative side effects. Towards achieving this goal the experiments discussed here reveal a mechanism of co-factor binding in the presence of either bound agonist or antagonist. The GR ligand binding domain (GR-LBD(F602S)), in the presence of agonist or antagonist, utilizes different modes of binding for coactivator versus corepressor. Coactivator binding to the co-effector binding pocket of GR-LBD(F602S) is driven both by favorable enthalpic and entropic interactions whereas corepressor binding to the same pocket is entropically driven. These data support the hypothesis that ligand-induced conformational changes dictate co-factor binding and subsequent trans-activation or trans-repression.

Development of a high-throughput assay to measure histidine decarboxylase activity.

Histamine is a well-known mediator of allergic, inflammatory, and neurological responses. More recent studies suggest a role for histamine and its receptors in a wide range of biological processes, including T-cell maturation and bone remodeling. Histamine serum levels are regulated mainly by the activity of the histamine-synthesizing enzyme histidine decarboxylase (HDC). Despite the importance of this enzyme in many physiological processes, very few potent HDC inhibitors have been identified. HDC assays suitable for high-throughput screening have not been reported. The authors describe the development of a fluorescence polarization assay to measure HDC enzymatic activity. They used a fluorescein-histamine probe that binds with high affinity to an antihistamine antibody for detection. Importantly, they show that probe binding is fully competed by histamine, but no competition by the HDC substrate histidine was observed. The automated assay was performed in a total volume of 60 muL, had an assay window of 80 to 100 mP, and had a Z' factor of 0.6 to 0.7. This assay provides new tools to study HDC activity and pharmacological modulation of histamine levels.

Characterization of the binding site for inhibitors of the HPV11 E1-E2 protein interaction on the E2 transactivation domain by photoaffinity labeling and mass spectrometry.

An indandione-containing class of inhibitors abrogates DNA replication of human papillomavirus (HPV) types 6 and 11 by binding reversibly to the transactivation domain (TAD) of the viral E2 protein and inhibiting its interaction with the viral E1 helicase. To locate the binding site of this class of protein-protein interaction inhibitors, a benzophenone derivative was used to generate an irreversibly labeled E2-TAD polypeptide. The single site of covalent modification of the E2-TAD was identified by proteolytic digestions using trypsin, LysC, and V8 proteases and characterization of the resulting peptides by LC-MS procedures. Through this methodology, the benzophenone attachment point was located at the terminal methyl of residue Met101. Evidence further pinpointed the site of photoaffinity attachment to the terminal carbon atom, which is significant in providing a definitive example of the ability to locate photoinduced cross-linking to a polypeptide with atomic resolution using solely mass spectrometric detection. The location of the inhibitor binding site vis-à-vis the Glu39 and Glu100 residues sensitive to mutation for HPV 11 E2-TAD is discussed in relation to the crystal structure of the E2-TAD from the related HPV type 16.

The design of potent hydrazones and disulfides as cathepsin S inhibitors.

The design and synthesis of dipeptidyl disulfides and dipeptidyl benzoylhydrazones as selective inhibitors of the cysteine protease Cathepsin S are described. These inhibitors were expected to form a slowly reversible covalent adduct of the active site cysteine of Cathepsin S. Formation of the initial adduct was confirmed by mass spectral analysis. The nature and mechanism of these adducts was explored. Kinetic analysis of the benzoyl hydrazones indicate that these inhibitors are acting as irreversible inhibitors of Cathepsin S. Additionally, the benzoylhydrazones were shown to be potent inhibitors of Cathepsin S processing of Class II associated invariant peptide both in vitro and in vivo.

Characterization of the allosteric inhibition of a protein-protein interaction by mass spectrometry.

The allosteric inhibition of the lymphocyte function associated antigen-1/intercellullar adhesion molecule (LFA-1/ICAM-1) interaction, by a class of small molecules, is characterized by a battery of mass spectrometric techniques. Binding of hydantoins to the I domain of LFA-1 is observed by size exclusion chromatography/mass spectrometry (SEC/MS) and by direct electrospray ionization mass spectrometry (ESI/MS). A photoactive hydantoin analog specifically labels an amino acid residue of LFA-1 I domain. Competition with this photoaffinity labeling by a panel of inhibitors is correlated with their Kd's for inhibition of the LFA-1/ICAM interaction. Alterations to the tertiary structure of LFA-1 I domain, upon compound binding, are inferred from perturbation in the ESI mass spectrum of the polypeptide's charge state distribution and by an altered level of nonspecific multimer formation. The results demonstrate specific, stoichiometric, reversible binding of the hydantoins to LFA-1. They further show correlation of this binding with activity and indicate alterations in the polypeptide's tertiary structure, on hydantoin binding, consistent with the proposed mechanism for inhibition of the protein-protein interaction.

Design and synthesis of dipeptide nitriles as reversible and potent Cathepsin S inhibitors.

The specificity of the immune response relies on processing of foreign proteins and presentation of antigenic peptides at the cell surface. Inhibition of antigen presentation, and the subsequent activation of T-cells, should, in theory, modulate the immune response. The cysteine protease Cathepsin S performs a fundamental step in antigen presentation and therefore represents an attractive target for inhibition. Herein, we report a series of potent and reversible Cathepsin S inhibitors based on dipeptide nitriles. These inhibitors show nanomolar inhibition of the target enzyme as well as cellular potency in a human B cell line. The first X-ray crystal structure of a reversible inhibitor cocrystallized with Cathepsin S is also reported.