Comparison of miniaturized time-resolved fluorescence resonance energy transfer and enzyme-coupled luciferase high-throughput screening assays to discover inhibitors of Rho-kinase II (ROCK-II).

Kinases are important drug discovery targets for a wide variety of therapeutic indications; consequently, the measurement of kinase activity remains a common high-throughput screening (HTS) application. Recently, enzyme-coupled luciferase-kinase (LK) format assays have been introduced. This format measures luminescence resulting from metabolism of adenosine triphosphate (ATP) via a luciferin/luciferase-coupled reaction. In the research presented here, 1536-well format time-resolved fluorescence resonance energy transfer (TR-FRET) and LK assays were created to identify novel Rho-associated kinase II (ROCK-II) inhibitors. HTS campaigns for both assays were conducted in this miniaturized format. It was found that both assays were able to consistently reproduce the expected pharmacology of inhibitors known to be specific to ROCK-II (fasudil IC50: 283 +/- 27 nM and 336 +/- 54 nM for TR-FRET and LK assays, respectively; Y-27632 IC50: 133 +/- 7.8 nM and 150 +/- 22 nM for TR-FRET and LK assays, respectively). In addition, both assays proved robust for HTS efforts, demonstrating excellent plate Z' values during the HTS campaign (0.84 +/- 0.03; 0.72 +/- 0.05 for LK and TR-FRET campaigns, respectively). Both formats identified scaffolds of known and novel ROCK-II inhibitors with similar sensitivity. A comparison of the performance of these 2 assay formats in an HTS campaign was enabled by the existence of a subset of 25,000 compounds found in both our institutional and the Molecular Library Screening Center Network screening files. Analysis of the HTS campaign results based on this subset of common compounds showed that both formats had comparable total hit rates, hit distributions, amount of hit clusters, and format-specific artifact. It can be concluded that both assay formats are suitable for the discovery of ROCK-II inhibitors, and the choice of assay format depends on reagents and/or screening technology available.

ISG15: a ubiquitin-like enigma.

ISG15 is a 17 kDa protein encoded by an interferon stimulated gene. Described in 1979, it was the first ubiquitin-like modifier to be identified, and its discovery followed the first reports of ubiquitin by only four years. While many important functions for ubiquitin have been reported, the functions for ISG15 and its conjugation are still largely unknown. Evidence suggests that ISG15 and its modification system play important roles in the innate immune response, regulation of interferon signaling, pregnancy, and several cancers. Modification of proteins by ISG15 occurs in a manner similar to that of ubiquitin and other ubiquitin-like modifiers. The enzymes which help perform the activation and conjugation of ISG15 have recently been identified. The conjugation enzyme identified for ISG15 was revealed to be an enzyme that was also involved in ubiquitin conjugation. Identification of an ISG15 specific protease has also been reported. Knockout of this protease in mice decreases the lifespan of these mice and makes them hypersensitive to treatment with interferon or lipopolysaccharide. The study of ISG15 and its modification system may yield a set of potentially useful therapeutic targets and thus, there is an increasing awareness and interest in this protein modifier. This review will highlight the history of its discovery, describe more recent observations about the enzymes involved in ISG15 modification, and summarize new findings which have important implications for the ISG15 system in signal transduction and immunology. This review will also point out important questions that remain to be answered and identify the major roadblocks which currently obstruct the understanding of ISG15 biologic functions.

Analysis of two acidic P6 pocket residues in the pH dependency of peptide binding by I-E(k).

Peptide binding to major histocompatibility complex (MHC) class II molecules is optimal at mildly acidic pH. X-ray crystal structures solved for the murine class II molecule I-E(k) revealed an interesting localization of negatively charged residues within the P6 pocket, which may have implications in the pH dependency of peptide binding. Protonation of these critical residues, under acidic conditions, has been proposed to be important for the formation of stable class II-peptide complexes. In this study, we address a possible role for these charged residues in the pH dependency of peptide binding. An I-E(k) mutant was generated in which two acidic residues of the P6 pocket were substituted with uncharged residues. This class II mutant was expressed, purified, and tested for its ability to bind peptides. The mutant I-E(k) was observed to load peptides optimally at mildly acidic pH. Peptide binding to the mutant was enhanced in the presence of DM, and optimal DM-enhanced binding occurred in the acidic pH range. These findings indicate that structural changes other than protonation of acidic residues in pocket 6 must play a dominant role in pH-regulated peptide binding to I-E(k).

A novel single chain I-A(b) molecule can stimulate and stain antigen-specific T cells.

Multimers of soluble major histocompatibility complex class I and II molecules have proven to be useful reagents in quantifying and following specific T cell populations. This study describes the design, generation, and characterization of a novel, single chain I-A(b) molecule which utilizes a unique linker derived from the murine invariant chain. A fragment of the invariant chain, residues 58-85, binds to a region proximal to the class II peptide binding groove and stabilizes occupancy of the class II invariant chain-associated peptide. We have utilized this fragment, replacing CLIP with the Ealpha peptide sequence, to lock the attached peptide into the class II binding groove. The single chain I-A(b) molecule was recognized by a panel of conformation-sensitive, I-A(b)-specific, monoclonal antibodies. Membrane-bound and soluble forms of the single chain I-A(b) stimulated an antigen-specific T cell hybridoma, and tetramers made from soluble monomers stained these cells. The unique features of this molecule may be useful in the design of recombinant T cell receptor ligands containing peptides with low affinity for MHC.

Phytochemical and immunomodulatory properties of an Echinacea laevigata (Asteraceae) tincture.

BACKGROUND: Echinacea preparations are consumed for the prevention or treatment of upper respiratory infections. OBJECTIVE: The objective of this study was to provide the first data regarding the in vitro immunomodulatory properties of the American federally endangered species Echinacea laevigata (Asteraceae). METHODS: Human peripheral blood mononuclear cells were cultured with root tinctures from E. laevigata, E. angustifolia, E. pallida, and E. purpurea. Cytokine production (tumor necrosis factor [TNF], interleukin [IL]-2, IL-10) and mononuclear cell proliferation were measured. High-performance liquid chromatography was used to assay levels of known bioactive compounds from all extracts tested to statistically determine whether there were relationships between extract phytochemical content and observed immune effects. RESULTS: E. laevigata extract was most similar to E. pallida extract and able to augment IL-10 and mononuclear cell proliferation, but not TNF or IL-2. Echinacoside, a caffeic acid derivative, correlated most strongly with results. CONCLUSIONS: This species may deserve continued investigation in both experimental and therapeutic contexts.

Retinoic acid-induced protein ISGylation is dependent on interferon signal transduction.

Interferon-stimulated gene 15 (ISG15) is a ubiquitin-like modifier that forms conjugates with target protein substrates. As its name suggests, its expression and conjugation to other proteins are highly regulated by interferon (IFN). It was recently demonstrated that ISG15 expression, ISG15 conjugation, and several enzymes involved in ISG15 modification are upregulated in an acute promyelocytic cell line following treatment with retinoic acid, suggesting a possible retinoic acid induced IFN-independent ISG15 modification pathway. In this study, we examined a possible link between IFN signaling and retinoic acid-induced ISG15 conjugation. We observed that ISGylation can be induced by retinoic acid in two myeloid leukemia cell lines. By sandwich ELISA, we detected increased IFN secretion into cell culture media following retinoic acid treatment. Blockade of the type I IFN receptor with a neutralizing antibody blocked retinoic acid induced ISG15 expression and ISG15 conjugation. Taken together, these data suggested that retinoic acid-induced secretion of IFN plays a fundamental role in retinoic acid promoted ISGylation.

ISG15 modification of ubiquitin E2 Ubc13 disrupts its ability to form thioester bond with ubiquitin.

ISG15 was the first ubiquitin-like modifier to be identified. However, the function of ISG15 modification has been an enigma for many years. At present, no data are available about the function of ISGylation for any target. In this paper, we report the identification of Ubc13, which forms a unique ubiquitin-conjugating enzyme (Ubc) complex with ubiquitin enzyme variant Mms2 and generates atypical Lys63-linked ubiquitin conjugates, as one of the targets of ISG15 modification. Furthermore, we identify Lys92 as the only ISG15 modification site in Ubc13, which is the first report about the ISG15 modification site. Using the "covalent affinity" purification assay, we found that unmodified Ubc13 can bind to the ubiquitin-agarose, whereas ISGylated Ubc13 cannot. This result indicates that ISGylation of Ubc13 disrupts its ability to form thioester bond with ubiquitin.

Modification of peptide interaction with MHC creates TCR partial agonists.

We report the creation of TCR partial agonists by the novel approach of manipulating the interaction between immunogenic peptide and MHC. Amino acids at MHC anchor positions of the I-E(k)-restricted hemoglobin (64-76) and moth cytochrome c (88-103) peptides were exchanged with MHC anchor residues from the low affinity class II invariant chain peptide (CLIP), resulting in antigenic peptides with altered affinity for MHC class II. Several low affinity peptides were identified as TCR partial agonists, as defined by the ability to stimulate cytolytic function but not proliferation. For example, a peptide containing methionine substitutions at positions one and nine of the I-E(k) binding motif acted as a partial agonist for two hemoglobin-reactive T cell clones (PL.17 and 3.L2). The identical MHC anchor substitutions in moth cytochrome c (88-103) also created a partial agonist for a mCC-reactive T cell (A.E7). Thus, peptides containing MHC anchor modifications mediated similar T cell responses regardless of TCR fine specificity or antigen reactivity. This data contrasts with the unique specificity among individual clones demonstrated using traditional altered peptide ligands containing substitutions at TCR contact residues. In conclusion, we demonstrate that altering the MHC anchor residues of the immunogenic peptide can be a powerful method to create TCR partial agonists.