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Chemical synthesis and rational inhibitor design of the human chemokine CCL2

Durham University

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Course Description

Supervisor: Ehmke PohlSteven Cobb Commercial partner: Cambridge Research Biochemicals (CRB) This studentship is funded by the EPSRC Chemokines are a superfamily of more than 50 functionally related proteins secreted mostly by vertebrate cells. These small proteins of approximately 70-150 amino acids act primarily as chemoattractants to guide the migration of surrounding cells. Chemokines are involved in many vital biological functions ranging from the promotion of new blood vessels (angiogenesis) to inflammatory responses in the activation of lymphocytes. Consequently, Chemokines play key roles in the human immune response to diseases ranging from viral and bacterial infections, cancer to autoimmune diseases such as allergies and artherosclerosis. Our target is the monocyte chemoattractant protein 1 (MCP-1 or CCL2) which plays a major role in atheroscleoris and related conditions. The chemical synthesis of small proteins by a combination of solid-phase peptide synthesis (SPPS) and native chemical ligation offers the great advantage that various non-natural amino acids can be introduced. The target protein can therefore easily be modified to contain fluorescence andor 19F NMR active probes that can be utilized in various techniques such as high-throughput screening of chemical libraries. A limited number of chemokines (either natural, biotinylated or fluorescently tagged) are already commercially available. However, given the synthetic accessibility of the chemokines (~100 amino acids) and our ability to prepare novel amino acid probes (i.e. Fluorescent, 19F NMR active) it will be possible to prepare a range of new chemically tagged chemokines that will be of both academic and commercial value. Objective 1: Novel amino acid that can be used as chemical probes will be used to prepare several chemically labelled CCL2 proteins. Among the amino acids that will be used are unique multi-functional amino acid probes that are both 19F NMR and Raman active. Such probes will be of particular interest as CCL2 proteins that contain chemical tags like this are not currently available. During the total synthesis of the CCL2 proteins the student will gain considerable experience in solid-phase peptide synthesis (SPSS), native chemical ligation strategies, and peptide purification and characterisation. Objective 2: Once the full-length CCL2 proteins have been synthesised (Objective 1) we will confirm that they have the correct folding by determining their crystal structures. We have already prepared a novel library of peptide based CCL2 inhibitors (currently 12 compounds) and the student will investigate the binding properties of these compounds with CCL2 using a variety of biophysical techniques including 19F NMR and isothermal titration calorimetry (ITC). In addition we will perform co-crystallization experiments with our inhibitors and the CCL2 proteins prepared. Co-crystal structure determination will enable us to decipher the molecular basis of ligand binding and hence open the door for rational inihibitor improvement. Further information: ehmke.pohl@durham.ac.uk

Supervisor: Ehmke PohlSteven Cobb Commercial partner: Cambridge Research Biochemicals (CRB) This studentship is funded by the EPSRC Chemokines are a superfamily of more than 50 functionally related proteins secreted mostly by vertebrate cells. These small proteins of approximately 70-150 amino acids act primarily as chemoattractants to guide the migration of surrounding cells. Chemokines are...

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