Investigation of the action of new pentapeptides entities and the interaction between human group IIA phospholipase A2 and vimentin

Abstract

The cyclic pentapeptide c2 is a promising anti-cancer agent. The thesis investigates the mechanism of action of c2, focusing on its ability to prevent the interaction between hGIIA and vimentin. Computational studies using molecular dynamics and docking predicted that hGIIA would be stable as a monomer in physiological environments and provided possible binding conformations of c2 compound in hGIIA and vimentin. Using recombinant hGIIA and vimentin, assays were conducted to investigate the binding of hGIIA to vimentin and the role of c2 in inhibiting the interaction. Surface plasmon resonance and enzyme immunoassay studies showed evidence of hGIIA binding at the coil 2 domain of vimentin, and that c2 can inhibit this binding. Radiolabelled c2 displayed high affinity towards the coil 2 domain of vimentin relative to the coil 1 domain, suggesting that c2 prevents the interaction between hGIIA and vimentin possibly by binding to the hGIIA binding site on vimentin. From these results combined with the computational docking work, it is predicted that the most probable binding site of c2 on vimentin is located between Met347 and Asn357. In the advanced prostate cancer cell line PC-3, exogenously added hGIIA did not significantly increase the proliferation rate of the cells, and nor was this affected by vimentin silencing. The prostaglandin E2 level in PC-3 cells were reduced by the exogenously added hGIIA, and c2 had an opposing effect to hGIIA. Such results are a contrast to data that has come from early stage prostate cancer cells. The role of hGIIA potentially has changing roles depending on the stage and type of the cancer, and the mechanism of action of hGIIA in the prostate cancer cells has the potential to be highly complex. It was also demonstrated that c2 can bind to prostate cancer cells without the expression of hGIIA. It suggests the incorporation of c2 may be dependent on cell surface proteins such as vimentin to which it has affinity rather than hGIIA.