PROTEOMIC APPROACHES IN DRUGS AND BIOMARKERS DISCOVERY
Creators: name: PANCOTTI, ANDREA
Publisher: name: Universit� degli Studi di Milano
Year: 2013

description: 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 &lt;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 proteomic research is the identification and validation of disease biomarkers which can be used as measurements within clinical studies and for the purpose of predictive diagnosis. The study of proteomics is considered to be a key for the characterization of human diseases and disease states, and mass spectrometry technology plays a crucial role in this disease research.
We will focus in particular on detection and quantization of post-translational modifications of proteins caused by oxidative and carbonyl stress. While the development of specific antibodies against modified proteins has made it possible to confirm the occurrence of oxidative stress in vivo and its involvement in several physio-pathological conditions, the resultant chemical modifications of proteins has not yet been explored. Hence we need proteomic tools, which can sensitively detect oxidative damage on peptides and proteins: these tools would be helpful to understand exactly when, how, and where the damage occurs, and to gain a deeper insights into the mechanism of onset, progression, and/or complication of the diseases.
Proteomics approaches have led to the identification of the protein/s showing high sensitivity to oxidation/carbonylation, and among them human serum albumin (HSA) was found to be the main target in the circulation.4-6 During the first year we demonstrated covalent modification of HSA-Cys34 by acrolein in patients undergoing hepatectomy surgery by using a mass spectrometric approach based on a triple quadruple mass spectrometer in precursor ion scan mode, able to identify unknown modifications of Cys34 by reactive carbonyl species (RCS) generated by lipid peroxidation. However, parallel studies performed in our laboratories demonstrated that in some pathologic conditions (i.e. uremic subjects), Cys34 undergoes disulfide formation by cysteinylation, thus limiting its availability for reaction with RCS. Hence, Cys 34-adducts couldn?t always be useful as carbonylation biomarkers. Therefore, I focused my attention on another HSA nucleophilic site (His-146), previously recognized as a potential oxidation/carbonylation target7. 
Digesting HSA with tripsin or tripsin+chimotripsin, two different peptides containing His-146 have been obtained: H*PYFYAPELLFFAK and H*PY, respectively. Anyway, analysis of tripsin or tripsin+chimotripsin digested HNE-HSA adduct did not lead to identification of any covalent modifications on His 146. Using an high-resolution, high mass accuracy mass spectrometer (LTQ-Orbitrap), performing a full scan analysis in data-dependent mode followed by data analysis with the SEQUEST algorithm it was possible to explain that the absence of any signals relative to RCS-H*PYFYAPELLFFAK adducts was due to different missed cleavages. The analyses relative to the H*PY peptide indicated the formation of one adduct only, H(HNE)PYF, but no significant fragmentation of the corresponding [M+H]+ was observed in MS/MS experiments, even working at high collision energy (40eV). Maybe the reason could be that the peptide is too short for an optimal fragmentation.
Taking into account these observations, we have considered as alternative approach the application of a new HSA digestion strategy, based on the use of chimotripsin only. In silico studies indicated the EIARRH*PY peptide as a new tag containing the target residue His 146 in the middle, exactly like Cys-34 in the LQQC*PF peptide, previously analyzed with satisfactory results. This new peptide has also the appropriate length to be analyzed by HPLC-MS/MS. Hence, the EIARRH*PY peptide seemed to possess the optimal characteristics to start a new MS strategy involving His-146 as a carbonyl adduction site on HSA.
Thus, HSA was isolated from human plasma by affinity chromatography, reduced with NaBH4 and digested with chimotripsin only in order to obtain the expected peptide. The peptide mixture was analyzed by LTQ-Orbitrap in data dependent scan mode and afterwards by the SEQUEST algorithm, comparing the obtained data with the primary sequence of the protein. The results of the analysis confirmed the formation of the expected peptide.
In this way we created the bases for the future studies on the EIARRH*PY peptide, in order to identify any covalent modifications of His146 induced by RCS.

References
1. Vaiana, N.; Marzahn, M.; Parapini, S.; Liu, P.; DelAgli, M.; Pancotti, A.; Sangiovanni, E.; Basilico, N.; Bosisio, E.; Dunn, B. M.; Taramelli, D.; Romeo, S. Bioorganic &amp; Medicinal Chemistry Letters 2012, 22, 5915.
2. Brown, D.; Superti-Furga, G. Drug Discov Today 2003, 8, 1067.
3. Godl, K.; Wissing, J.; Kurtenbach, A.; Habenberger, P.; Blencke, S.; Gutbrod, H.; Salassidis, K.; Stein-gerlach, M.; Missio, A.; Cotten, M.; Daub, H. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 15434.
4. Mera, K.; Anraku, M.; Kitamura, K.; Nakajou, K.; Maruyama, T.; Otagiri, M. Biochem. Biophys. Res. Commun. 2005, 334, 1322.
5. Anraku, M.; Kitamura, K.; Shinohara, A.; Adachi, M.; Suenaga, A.; Maruyama, T.; Miyanaka, K.; Miyoshi, T.; Shiraishi, N.; Nonoguchi, H.; Otagiri, M.; Tomita, K. Kidney Int. 2004, 66, 841.
6. Aldini, G.; Vistoli, G.; Regazzoni, L.; Gamberoni, L.; Facino, R. M.; Yamaguchi, S.; Uchida, K.; Carini, M. Chem. Res. Toxicol. 2008, 21, 824.
7. Aldini, G.; Gamberoni, L.; Orioli, M.; Beretta, G.; Regazzoni, L.; Facino, R. M.; Carini, M. J. Mass Spectrom. 2006, 41, 1149.