Over the last few decades, research on snake venom toxins has provided not only new tools to decipher molecular details of various physiological processes, but also inspiration to design and develop a number of therapeutic agents. Obtustatin represents the shortest known snake venom monomeric disintegrin isolated from the venom of Macrovipera lebetina obutusa (MLO) which is a specific inhibitor of 11 integrin. This low molecular weight peptide revealed a potent therapeutic effect on melanoma progression. Its oncostatic effect was related to the inhibition of angiogenesis. The aim of the proposed investigation is to obtain detailed information about the mechanism and topology of the synthesized and isolated obtustatin of the Macrovipera lebetina obtusa (MLO) venom in the membrane-binding process and to find out how the presence of such interaction changes the properties of membrane (GUVs) formed from the phospholipids and proteolipids isolated from the B16F10 cancer cells. GUVs (mean diameter 30 m) will be formed by the electroformation method. We will also use erythrocyte ghosts, which is the best comparable model with GUVs.
The investigation will also study the ability of synthesized and isolated obtustatin to inhibit proliferation of the metastatic melanoma cell line B16F10 in vitro and in vivo, as well as investigate the molecular mechanism. A B16F10 bearing mouse model and histochemical examination will be utilized to investigate the anti-tumor effects of these two obtustatins.
In addition, lactate dehydrogenase release assay, DNA retardation assay, PCR, Western blot, wound healing assay will be applied to elucidate the mechanism of synthesized and isolated obtustatin.