Detection and characterization of sirtuin 1-modulating natural products

Die zunehmende Alterung unserer Gesellschaft hat zur Folge, dass altersbedingte Erkrankungen, wie z. B. Typ-2 Diabetes, vermehrt auftreten. Dies bringt sowohl wirtschaftliche als auch große soziale Probleme mit sich. Ernährungsbezogene Präventionsansätze befassen sich daher u. a. mit dem Einfluss von Nahrungsbestandteilen auf die Expression krankheitsrelevanter Gene. Die Deacetylase SIRT1, die durch kleine Moleküle in ihrer Aktivität reguliert werden kann, steuert diverse Stoffwechselprozesse, die mit altersbedingten Erkrankungen in Verbindung stehen. In der Vergangenheit wurden verschiedene Screeningmethoden zur Detektion SIRT1-modulierender Substanzen entwickelt. Allerdings weisen viele der verfügbaren SIRT1-Screeningmethoden Nachteile auf, etwa die Generierung von Artefakten aufgrund von Fluoreszenzdetektion und/oder -markierung. Um dieses Problem zu lösen wurde in der vorliegenden Arbeit eine MALDI-TOF MS-basierte Screeningmethode entwickelt. Anhand dieser Methode kann die Deacetylierung eines Peptidsubstrates aufgrund seiner um 42 Da verringerten Masse detektiert werden, ohne dass eine Substratmarkierung erforderlich ist. Die Methode wurde so optimiert, um sie einerseits als Hochdurchsatz-Screeningmethode, und andererseits zur nachfolgenden Charakterisierung der ermittelten SIRT1-Modulatoren verwenden zu können. Die Methodenentwicklung sowie das Screening einer Naturstoffbibliothek wurden mit einem unmarkierten und mit einem fluoreszenzmarkierten Peptid durchgeführt. Das unmarkierte Peptid diente dabei der Erzielung artefaktfreier Ergebnisse. Das fluoreszenzmarkierte Peptid wurde hingegen zur Detektion artifizieller SIRT1-Aktivatoren eingesetzt, um strukturelle Ähnlichkeiten zu anderen SIRT1-Aktivatoren aufzeigen zu können. Aus dem Screening resultierten acht SIRT1-Inhibitoren und ein artifizieller SIRT1-Aktivator. Diese Substanzen wurden in vitro und in verschiedenen Zellsystemen überprüft. Dabei zeigte der stärkste SIRT1-Inhibitor NP1 im Zellsystem eine reduzierte p53-Deacetylierung. Der artifizielle SIRT1-Aktivator NP9, der mit einem IC50 -Wert von 1,7 µM zudem ein sehr starker p300-Inhibitor ist, wies eine stark antiinflammatorische Wirkung auf. Anhand der Histonacetyltransferase p300 wurde gezeigt, dass die MALDI-TOF MS Methode sich auch zur Detektion der Umkehrreaktion eignet. Zudem bietet die Methode die Möglichkeit, die Aktivität vieler weiterer posttranslationaler Enzyme zu messen.The progressive increase in life expectancies of our society results in an increasing prevalence of age-related diseases like type-2 diabetes. This causes economic as well as vast social problems. Therefore, nutrition related preventive approaches include for example the influence of dietary components on the expression of disease related genes. The deacetylase SIRT1 can be regulated by small molecules and controls several metabolic processes that influence age-related diseases. Several methods to screen for SIRT1 modulating compounds have been developed in the past. However, many of the available SIRT1 screening methods bear disadvantages such as artifact generation because of fluorescence detection and/or labeling. Hence, in the present work a more advantageous MALDI-TOF MS based screening method was developed. Applying this method, deacetylation of a peptide substrate can be detected by a 42 Da mass shift without the need of any substrate labeling. The method was optimized to serve as a high-throughput screening system as well as for subsequent characterization of detected SIRT1 modulators. Method development as well as screening of a natural product library was conducted using an unlabeled and a fluorescently labeled peptide. The unlabeled peptide was used to generate artifact-free results. In contrast, the fluorescently labeled peptide was applied for the detection of artificial SIRT1 activators, in order to demonstrate structural similarities to other SIRT1 activating compounds. The screening resulted in eight SIRT1 inhibitors and one artificial SIRT1 activator. These compounds were further tested in vitro and in different cell lines. Cell treatment with the most potent SIRT1 inhibitor NP1 revealed reduced p53 deacetylation. The artificial SIRT1 activator compound NP9, which is also a very strong inhibitor of p300 with an IC50 value of 1.7 µM, showed a strong antiinflammatory effect. By employing the histone acetyltransferase p300, the applicability of the MALDI-TOF MS based method for the reverse reaction was demonstrated. Moreover, the presented method provides the opportunity to measure the activity of many other post-translationally active enzymes

Rekombinante Allergene, Peptide und Virus-like Particles in der Immuntherapie von Allergien

Currently, extract-based therapeutic allergens from natural allergen sources (e.g., house dust mites, tree and grass pollen) are used for allergen-specific immunotherapy (AIT), the only causative therapy that can exhibit positive disease-modifying effects by tolerance induction and prevention of disease progression. Due to variations in the natural composition of the starting materials and different manufacturing processes, there are variations in protein content, allergen composition, and allergenic activity of similar products, which poses specific challenges for their standardization. The identification of the nucleotide sequences of allergenic proteins led to the development of molecular AIT approaches. This allows for the application of exclusively relevant structures as chemically synthesized peptides, recombinant single allergens, or molecules with hypoallergenic properties that potentially allow for an up-dosing with higher allergen-doses without allergic side effects leading more quickly to effective cumulative doses. Further modifications of AIT preparations to improve allergenic and immunogenic properties may be achieved, e.g., by including the use of virus-like particles (VLPs). To date, the herein described therapeutic approaches have been tested in clinical trials only. This article provides an overview of published molecular approaches for allergy treatment used in clinical AIT studies. Their added value and challenges compared to established therapeutic allergens are discussed. The aim of these approaches is to develop highly effective and well-tolerated AIT preparations with improved patient acceptance and adherence

Development and in-house validation of allergen-specific ELISA tests for the quantification of Dau c 1.01, Dau c 1.02 and Dau c 4 in carrot extracts (Daucus carota)

Even though carrot allergy is common in Europe, the amount of different allergens in carrots is still unknown due to a lack of methods for quantitative allergen measurements. The current study aimed at the development of quantitative ELISA tests for the known carrot allergens, namely Dau c 1.01, Dau c 1.02, and Dau c 4 in pure carrot extracts. Monoclonal antibodies targeting the major carrot allergen isoforms Dau c 1.01 and Dau c 1.02 were generated and combined in sandwich ELISA with rabbit antisera against Api g 1, the celery homologue of Dau c 1. A competitive ELISA for the carrot profilin Dau c 4 was based on a polyclonal rabbit antiserum. The three ELISA tests were allergen-specific and displayed detection limits between 0.4 and 6 ng allergen/ml of carrot extract. The mean coefficient of variation (CV) as a means of intraassay variability of the Dau c 1.01, Dau c 1.02 and Dau c 4 ELISA tests was 8.1%, 6.9%, and 11.9%, and the mean interassay CV 13.3%, 37.1% and 15.6%, respectively. Target recovery ranged between 93 and 113%. In conclusion, the specific, accurate and reproducible quantification of three important carrot allergens may help to identify less allergenic carrot varieties, as well as to standardize the amount of allergens in extracts used for carrot allergy diagnosi

Standardisation of allergen products: 4. Validation of a Candidate European Pharmacopoeia Standard Method for Quantification of Major Grass Pollen Allergen Phl p 5

BACKGROUND: The aim of the BSP090 project is the establishment of European Pharmacopoeia Chemical Reference Substances (CRSs) in combination with corresponding standard ELISA methods for quantification of major allergens in allergen products. Here, we present data of a Phl p 5-specific sandwich ELISA that proved suitable for the quantification of Phl p 5, one of the major Timothy grass (Phleum pratense) pollen allergens. METHODS: A Phl p 5-specific ELISA system was assessed with respect to accuracy, precision, inter-assay (within laboratory) and inter-laboratory variations, in a ring trial including 14 laboratories in Europe and the USA. Model samples containing recombinant Phl p 5a CRS as well as native grass pollen extracts were analysed. Each participant was instructed to perform at least one preliminary assay to familiarise with the protocol, followed by three independent assays. RESULTS: The candidate standard ELISA proved suitable to quantify recombinant and native Phl p 5 with satisfactory precision (93% of results within ±30% acceptance range). Inter-assay variation (max. GCV 24%) and especially inter-laboratory variation (max. GCV 13%) showed conclusive results. When assessing accuracy by means of recovery of recombinant spikes from a grass pollen extract matrix, similarly satisfactory spike recovery results were observed for the two spikes with higher concentrations (all within ±30% acceptance range), whereas recovery of the lowest concentration spike was slightly poorer with mean results of six laboratories exceeding acceptance range. CONCLUSIONS: Based on the collaborative study results, the assessed Phl p 5-specific immunoassay is appropriate to be proposed as European Pharmacopoeia standard method