Metabolic profiling of Vaccinium macrocarpon using 1H-NMR

In der Naturheilkunde wird Vaccinium macrocarpon (Großfrüchtige Moosbeere, „Cranberry“, Kultur-Preiselbeere) als Mittel zur Prophylaxe und Behandlung von Harnwegsentzündungen verwendet. Klinische Studien belegen eine positive Korrelation zwischen regelmäßigem (täglichen) Konsum von Cranberrysaft und geringerer Häufigkeit von Harnwegsentzündungen. Wird der Cranberrysaft aufgrund seiner Wirkung als gesundheitsförderndes Nahrungsergänzungsmittel konsumiert, sollte eine Qualitätskontrolle der bioaktiven Inhaltsstoffe, der phenolischen Sekundärmetaboliten, erfolgen. Im Rahmen dieser Arbeit wurde mithilfe von NMR-Spektroskopie ein Profil der phenolischen Sekundärmetaboliten in einem Vaccinium-macrocarpon-Extrakt erstellt. Anschließend wurden Cranberrysäfte von vier Herstellern mittels NMR-Spektroskopie untersucht und die Daten einer Multivariaten Datenanalyse unterworfen. Die untersuchten Cranberrysäfte zeigten unterschiedliche Konzentrationen von Benzoesäure, Benzoesäure-Glucosid, Quercetin und Catechin.In folklore and natural medicine, American cranberry is believed to be active in the prevention and treatment of urinary tract infections (UTIs). Clinical studies show a correlation of cranberry juice consumption and decreased occurrence of UTIs. Becoming important for the maintenance of human health, the quality of cranberry juice has to be monitored and assured. In the present work, a profile of the phenolic secondary metabolites of a Vaccinium macrocarpon extract, which seem to be the bioactive compounds, was established, using NMR spectrometry. Subsequently, cranberry juice samples of four different brands were analyzed by NMR in combination with multivariate data analysis. The analyzed juices showed differences depending on the manufacturer mostly by the concentrations of benzoic acid, benzoic acid glucoside, quercetin and catechin present in the juice

Development And Study Of Inhibitors Of Heat Shock Protein 70 Induction

Tumor and cancer cells that over express heat shock protein 70: HSP70) are found to be multidrug resistant and thermo tolerant, creating a hurdle to existing therapy. Although HSP70 is recognized as an increasingly important drug target, the protein structure of this highly conserved chaperone remains challenging for direct targeting. An alternative strategy is to inhibit the transcription of HSP70. Among known small molecule inhibitors of HSP70 induction, quercetin has very low toxicity and has the advantage of being easily modified for structure-activity studies. One part of the dissertation focuses on the identification of quercetin derivatives with improved specificity and activity, and to determine the protein targets of quercetin responsible for inhibition of heat shock induction of HSP70. A library of quercetin derivatives was synthesized and screened for their ability to inhibit HSP70 induction and at the same time not enhance HSP27 phosphorylation. The derivatives that inhibit HSP70 induction were also found to be inhibitors of both Ck2 kinase and CaMKII kinase that are known to activate heat shock transcription factor 1 that leads to HSP70 induction. A biotinylated quercetin affinity agent was also developed that was able to pull down the CK2 kinase target in vitro and several other proteins in vivo under UVA irradiation. In collaboration with mass spectrometry center, these unknown protein targets were identified by proteomic studies, and found to be previously identified chemotherapeutic targets. Another goal of this dissertation was to develop polyamide-based gene inhibitors of heat shock induction that interfere with the binding of the heat shock transcription factor. A series of polyamides that targeted the heat shock elements were designed and synthesized and demonstrated to bind the target DNA by DNase I footprinting. These polyamides were evaluated by gel shift assay for their ability to block binding of the heat shock factor and the most effective polyamide was shown to decrease HSP70 expression in Jurkat cells by western blot assay

Drosophila neprilysins control insulin signaling and food intake via cleavage of regulatory peptides

Insulin and IGF signaling are critical to numerous developmental and physiological processes, with perturbations being pathognomonic of various diseases, including diabetes. Although the functional roles of the respective signaling pathways have been extensively studied, the control of insulin production and release is only partially understood. Herein, we show that in Drosophila expression of insulin-like peptides is regulated by neprilysin activity. Concomitant phenotypes of altered neprilysin expression included impaired food intake, reduced body size, and characteristic changes in the metabolite composition. Ectopic expression of a catalytically inactive mutant did not elicit any of the phenotypes, which confirms abnormal peptide hydrolysis as a causative factor. A screen for corresponding substrates of the neprilysin identified distinct peptides that regulate insulin-like peptide expression, feeding behavior, or both. The high functional conservation of neprilysins and their substrates renders the characterized principles applicable to numerous species, including higher eukaryotes and humans. DOI: http://dx.doi.org/10.7554/eLife.19430.00

Characterization of protein-ligand interactions : the role of thermodynamic and structural data in the drug discovery process

Comparing biological activities and thermodynamic profiles obtained for one compound toward proteins, differing only in few residues is a promising strategy to increase the knowledge of protein-ligand interactions and active site topologies thereby enabling rational drug design. For this purpose, wild type proteins and their mutants carrying a set of point mutations can be used. Such an approach was applied in the present study to guide the development of small-molecule antagonists targeting PqsR, a novel target to limit Pseudomonas aeruginosa pathogenicity. It resulted in two chemically divers fragments with antagonistic activity and provided insights into their binding modes. The latter can be used to assist fragment optimization and therefore the identified PqsR antagonists are promising scaffolds for further drug design efforts against this important pathogen. Alternatively, proteins from various species, which are highly conserved in sequence and differ only in few residues, might be considered. This line of attack was also utilized employing human and marmoset monkey 17β-HSD1, a promising target for the treatment of estrogen-dependent diseases. By means of in silico methods a better understanding of 17β-HSD1 active site topologies and inhibitor binding modes was achieved that guided the elaboration of a lead compound. The described strategy was successfully applied for hit identification and lead optimization reflecting its benefit in drug design.Die möglichst genaue Kenntnis der räumlichen Gestalt der Ligand Bindungsstelle und der damit verbundenen Protein-Ligand-Wechselwirkungen ist eine Voraussetzung für rationales Wirkstoffdesign. Eine aussichtsreiche Strategie um diese Informationen zu erhalten, ist es biologische/biophysikalische Daten einer Verbindung gegenüber Proteinen zu vergleichen, die sich nur in wenigen Aminosäuren unterscheiden. Einerseits können dazu Wildtyp und Mutanten genutzt werden, was in dieser Studie zur Entwicklung von PqsR Antagonisten geführt hat. PqsR ist ein neues Target zur Limitierung der Pseudomonas aeruginosa Pathogenität. Zwei chemisch verschiedene, antagonistische Fragmente wurden entdeckt und Aufschlüsse über deren Bindungsmodi erhalten, die zur weiteren Optimierung genutzt werden können. Somit stellen die entdeckten Liganden vielversprechende Grundgerüste zur weiteren Wirkstoffentwicklung gegen dieses relevante Pathogen dar. Alternativ können hochkonservierte Proteine verschiedener Spezies in Betracht gezogen werden. Unter Verwendung von humaner und marmoset 17β-HSD1, einem erfolgversprechenden Target zur Behandlung estrogenabhängiger Erkrankungen, wurden durch in silico Methoden das aktive Zentrum der 17β-HSD1 sowie Bindungsmodi ausgewählter Hemmstoffe untersucht. Anhand der erstellten Modelle wurde eine Leitverbindung weiterentwickelt. Diese Strategie wurde erfolgreich für das Wirkstoffdesign bei der Hit Identifizierung sowie der Lead Optimierung angewendet

Molecular Science for Drug Development and Biomedicine

With the avalanche of biological sequences generated in the postgenomic age, molecular science is facing an unprecedented challenge, i.e., how to timely utilize the huge amount of data to benefit human beings. Stimulated by such a challenge, a rapid development has taken place in molecular science, particularly in the areas associated with drug development and biomedicine, both experimental and theoretical. The current thematic issue was launched with the focus on the topic of “Molecular Science for Drug Development and Biomedicine”, in hopes to further stimulate more useful techniques and findings from various approaches of molecular science for drug development and biomedicine

50th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 50th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-endorsed by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Breckenridge, Colorado, July 27-31, 2008