Identifying Lead Compounds for S100P in Pancreatic Cancer Therapy Using in Vitro Biophysical Techniques
Kekule Vithanage, Thamarasi Vithana
The protein S100P has been associated with the aggressiveness of several neoplastic disease, including pancreatic ductal adenocarcinoma (PDAC). S100 proteins are a family of calcium binding proteins that are small dimeric members of the EF-hand superfamily of Ca2+ binding proteins. In pancreatic ductal adenocarcinoma, protein S100P is overexpressed, and thereby meditate tumour growth, drug resistance and metastasis. Extracellular S100P binding to the cell surface receptor for advanced glycation end products (RAGE), triggers downstream signalling pathways and mediates cell growth and differentiation, cell cycle regulation and metabolic control. An effective treatment strategy would be to disrupt the formation of this S100P-RAGE complex by blocking the active binding site of S100P to the RAGE receptor. The aim of the present study was to investigate the interactions of drug compounds that are designed to block the S100P-RAGE complex formation. In order to perform this S100P protein was expressed in Escherichia. coli using a recombinant plasmid for wild type S100P and purified by affinity chromatography using a HisTrap column. The purity of the protein was assessed using sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The concentration of the purified protein was determined by either the Bradford assay or bicinchoninic acid assay (BCA). In this study, the interactions sites of the recombinant S100P with each of the compounds was assessed by nuclear magnetic resonance (NMR) spectroscopy. The interaction sites were modelled by a HADDOCK approach, using the interacting residues as constraints to model the structure. In conclusion the ligands C72, IBS409, IBS798 and IBS863 were identified to interact with S100P mainly in the hydrophobic pockets of the protein S100P. Binding sites of the ligands were identified via NMR, and the binding was found to be dominated by hydrophobic interactions. Further ITC experimentation may be required in order to gain an understanding of the stoichiometry and the binding affinity for these ligands.
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