Dieter R. Mueller

Inhibition of Dengue Virus Polymerase by Blocking of the RNA Tunnel

ABSTRACT Dengue virus (DENV) is the most prevalent mosquito-borne viral pathogen in humans. Neither vaccine nor antiviral therapy is currently available for DENV. We report here that N-sulfonylanthranilic acid derivatives are allosteric inhibitors of DENV RNA-dependent RNA polymerase (RdRp). The inhibitor was identified through high-throughput screening of one million compounds using a primer extension-based RdRp assay [substrate poly(C)/oligo(G)20]. Chemical modification of the initial “hit” improved the compound potency to an IC50 (that is, a concentration that inhibits 50% RdRp activity) of 0.7 μM. In addition to suppressing the primer extension-based RNA elongation, the compound also inhibited de novo RNA synthesis using a DENV subgenomic RNA, but at a lower potency (IC50 of 5 μM). Remarkably, the observed anti-polymerase activity is specific to DENV RdRp; the compound did not inhibit WNV RdRp and exhibited IC50s of >100 μM against hepatitis C virus RdRp and human DNA polymerase α and β. UV cross-linking and mass spectrometric analysis showed that a photoreactive inhibitor could be cross-linked to Met343 within the RdRp domain of DENV NS5. On the crystal structure of DENV RdRp, Met343 is located at the entrance of RNA template tunnel. Biochemical experiments showed that the order of addition of RNA template and inhibitor during the assembly of RdRp reaction affected compound potency. Collectively, the results indicate that the compound inhibits RdRp through blocking the RNA tunnel. This study has provided direct evidence to support the hypothesis that allosteric pockets from flavivirus RdRp could be targeted for antiviral development.

N-Sulfonylanthranilic Acid Derivatives as Allosteric Inhibitors of Dengue Viral RNA-Dependent RNA Polymerase

A novel class of compounds containing N-sulfonylanthranilic acid was found to specifically inhibit dengue viral polymerase. The structural requirements for inhibition and a preliminary structure-activity relationship are described. A UV cross-linking experiment was used to map the allosteric binding site of the compound on the viral polymerase.

Fluoroaluminate stimulates phosphorylation of p130 Cas and Fak and increases attachment and spreading of preosteoblastic MC3T3-E1 cells.

Fluoroaluminate is a G-protein activator, it stimulates osteoblastic cells in culture, and is a bone-forming agent in vivo. To elucidate the mechanisms of G-protein-mediated action of fluoroaluminate in osteoblasts, we studied protein tyrosine phosphorylation in the preosteoblastic cell line MC3T3-E1. Fluoroaluminate, lysophosphatidic acid (LPA; an agonist for G-protein-coupled receptor), or adhesion to type I collagen all stimulated phosphorylation of a similar set of proteins, including p130, p120, p110 (previously identified as proline-rich tyrosine kinase 2, Pyk2), and p70. The phosphorylation of these proteins was sensitive to an Src inhibitor, but not to a Gi-protein inactivator, pertussis toxin. By purification/mass spectrometry and by immunodepletion, p130 protein was identified as p130 Cas (Crk-associated protein), a Src substrate and a protein involved in signaling by cell-adhesion receptors, integrins. Phosphorylation of immunoprecipitated p130 Cas increased upon stimulation with fluoroaluminate and with agonists of G-protein-coupled receptors, but not with growth factors. By immunodepletion, the p120 protein was identified as focal adhesion kinase, Fak. The addition of fluoroaluminate during cell attachment to type I collagen further stimulated phosphorylation of p130 Cas and of Fak. Simultaneously, fluoroaluminate increased the number of attached MC3T3-E1 cells and their spreading. These novel aspects of fluoroaluminate action in cell culture may be important for the bone-forming action of fluoroaluminate in vivo.

LC-MALDI MS and MS/MS--an efficient tool in proteome analysis.

Liquid chromatography-matrix-assisted laser desorption/ionization mass spectrometry represents a sensitive, hyphenated MS- and MS/MS-technique with a broad range of applications in all areas ofproteome analysis. Whereas a number of interface types have been developed for coupling MALDI MS and liquid chromatography, in this chapter selected on-line and off-line types and techniques will be discussed with respect to their individual properties and performance. The technique is especially attractive in off-line mode where LC-separation and MS analyses are decoupled and each step can be performed at its individual optimum. Different speed of chromatographic separation and achievement of S/N criteria in MS or MS/MS mode can be optimized independently by individual adjustment of specific operating parameters. This flexibility makes LC-MALDI MS attractive for the analysis of peptide mixtures from low to medium complexity. Using sequential MS analysis of parallel LC runs (multiplexing), even highly complex samples can be handled. Quantitation at the MS and MS/MS level can be accomplished by a variety of labeling techniques, where the predominant formation of singly charged ions in MALDI alleviates the assignment of isotopomers. After discussing the level of complementarity between LC-MALDI and LC-ESI MS, selected applications of LC-MALDI MS are presented. Examples of membrane protein analysis applying 1D SDS PAGE are discussed in detail as well as applications in protein interaction analysis. These application examples clearly show that in all respects LC-MALDI MS and MS/MS are flexible and sensitive techniques which can be adapted to a wide range of different workflows.

Biophotonics Applied to Proteomics

Since the completion of the human genome sequencing, our understanding of gene and protein function and their involvement in physiopathological states has increased dramatically, partly due to technological developments in photonics. Photonics is a very active area where new developments occur on a weekly basis, while established tools are adapted to fulfill the needs of other disciplines like genomics and proteomics. Biophotonics emerged at the interface of photonics and biology as a very straightforward and efficient approach to observe and manipulate living systems. In this chapter, we review the current applications of photonics and imaging to proteomics from 2D gels analysis to molecular imaging.