Data di Pubblicazione:
2013
Citazione:
PROTEOMIC APPROACHES IN DRUGS AND BIOMARKERS DISCOVERY / A. Pancotti ; tutor: M. Carini ; co-tutor: S. Romeo ; coordinatore: E. Valoti. UNIVERSITA' DEGLI STUDI DI MILANO, 2013 Feb 22. 25. ciclo, Anno Accademico 2012. [10.13130/pancotti-andrea_phd2013-02-22].
Abstract:
DOTTORATO DI RICERCA IN CHIMICA DEL FARMACO (XXV CICLO)
Proteomic approaches in Drugs and Biomarkers Discovery
Dott. Andrea Pancotti
Tutor: Prof. Marina Carini
Co-tutor: Prof. Sergio Romeo
Abstract
My thesis project is focused on development and application of proteomic approaches in two fundamental research areas: the drug discovery process and the discovery of disease biomarkers. In particular, new and reliable strategies by mass spectrometry for the fast identification and validation of drug targets, lead compounds and disease biomarkers have been considered.
Proteomic Approaches in Drug Discovery
In Prof. Romeo’s group new compounds have been identified with high in vitro efficacy against the intraerythrocytic stages of Plasmodium falciparum (P.f.) (IC50 <10 nM),1 but the molecular target is unknown and preliminary results indicate that these compounds may exerts their activity against P.f. through multiple targets.
Proteomics offers a unique tool for target identification2 and several proteomic approaches are available, one of the most interesting is the so called chemical proteomics, which couple affinity purification methods with mass spectrometry and therefore permits to increase selectivity and sensitivity.3
In order to facilitate the mass spectrometric analysis, it would be necessary to know the target localization into P.f. of our compounds. Therefore last year, fluorescence compounds, as analytical tools for preliminary target identification, were designed.
These compounds have been tested in the Prof. Taramelli’s laboratory against D10 and W2 strains, but only few compounds resulted to be active, and activities were elicited only in a micromolar range. These activities were not sufficient to conduct fluorescent studies, therefore further optimizations were undertaken.
Considering the recent SAR obtained in our laboratory, compound 1 has been synthesized characterized by the presence of coumarin and a propylendiamine linker between leucine and the fluorescent group.
This compound was active against P.f (IC50 D10 and W2= 13 nM) and fluorescent studies are being performed. In the meanwhile we are focusing our attention on the target identification.
The adopted chemical proteomics approach requires the preparation of an alkyne derivate of the lead compound that will be incubated in a Pf lysate. Then trough click chemistry the protein bound alkyne derivative will be reacted with an azide containing a cleavable linker and a terminal biotin unit (compound 3). The lysate will be purified by affinity based chromatography using streptavidin beads. Then the cleavable linker will be cleaved and the protein-compound complex analyzed using HPLC-MS analysis.
Thus propargylic derivative 2 was synthesized. It was tested in the Professor Taramelli’s laboratory and resulted to be very active against Pf. (IC50 D10= 8nM/ IC50 W2= 12nM). Compound 3 has been synthesized and it is characterized by a biotin unit, an acylhydrazone cleavable linker, a poliethylenglicol spacer and a terminal azide.
In order to verify if compound 2 and 3 were useful tools to perform the procedure that we aimed to follow, the optimized click chemistry reaction was performed between the two compounds obtaining compound 4.
In order to test the whole proteomics procedure, compound 4 was immobilized on agarose streptavidin beads and after the selective cleavage of the acylhydrazone cleavable linker and reduction of intermediate, compound 5 was obtained and characterized through direct infusion ESI-MS spectrum.
After this successful result the final proteomics analysis is being conducted in collaboration with Professor Taramelli’s laboratory.
Proteomic Approaches in Biomarkers Discovery
One of the most exciting areas of prote
Tipologia IRIS:
Tesi di dottorato
Keywords:
MALARIA ; CHEMICAL PROTEOMICS ; TARGET IDENTIFICATION ; OXIDATIVE STRESS ; CYS 34 ; HIS 146 ; BIOMARKER DISCOVERY
Elenco autori:
A. Pancotti
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