ETD system

Electronic theses and dissertations repository


Tesi etd-10212013-134904

Thesis type
Tesi di laurea specialistica LC5
Elucidation of the binding site of antagonists for the human chemokine receptor CCR2b
Corso di studi
relatore Dott.ssa Betti, Laura
relatore Prof.ssa Heitman, Laura
correlatore Prof.ssa Giorgi, Irene
Parole chiave
  • PEI transfection
  • calcium phosphate transfection
  • antagonists of ccr2
  • ccr2
  • radioligand binding assays
Data inizio appello
Riassunto analitico
The present study focused his attention on the binding site of two small molecule antagonists of human CCR2b chemokine receptor.
Chemokine receptors belong to the family of G protein-coupled receptors. To date, the chemokine system is made up by 50 ligands and 21 receptors, classified on the basis of the cystein pattern of the chemokines. CCR2 and its endogenous ligand CCL2 have been found to be involved in several diseases such as multiple sclerosis, rheumatoid arthritis, metabolic diseases, cancer and neuropathic pain. In these years different treatments have been proposed from mAb to small molecules. Unfortunately, no cures are available on the market yet.

The aim of this project was to elucidate the binding site of two molecule antagonists of hCCR2 chemokine receptor: V-10-5191 and V-10-5299. Two different techniques of transfection in mammalian HEK-293 cells were performed: calcium phosphate and PEI transfection. Comparison between these two techniques leaded to the better expression of the receptors. Receptor expression was detected using 125I-CCL2, [3H]-V-10-5191 and [3H]-V-10-5299 in radioligand binding assays. Only the polycation PEI transfection method yielded CCR2 expression for which 125I-CCL2 binding could be detected. For this reason, the PEI method was chosen as a more efficient method to carry out following transfections using WT hCCR2b and in addition ten mutants of hCCR2b kindly provided by J. Peace. Receptor expression was detected using the radioligand binding assays mentioned previously.
The analysis of the effects of these mutants on specific binding added information on the chemical interactions that occurs between the residues involved in the binding site and the three molecules. In conclusion these data could support further studies in the design of new antagonists of chemokine receptor CCR2, improving the knowledge on their binding site and clarify the pharmacological profile of the molecules already known.