The main objective of this proposal is to gain a molecular understanding of the mechanism of inhibition of angiogenesis by the natural products fumagillin, ovalicin and the synthetic analog TNP-470, and to improve the efficacy and reduce the side effects of these drugs by targeting them to tumors. It was recently shown that both TNP-470 and ovalicin potently inhibit the enzymatic activity of the type 2 methionine aminopeptidase (MetAP2), suggesting that MetAP2 is a specific target for both TNP-470 and ovalicin. To delineate the molecular interactions between the drugs and MetAP2, the enzyme bound forms of the drugs and the residue in MetAP2 that is covalently modified by the drugs will be identified and characterized. Generating a random MetAP2 mutant library and performing genetic screens will identify additional residue from MetAP2 that are involved in drug binding. To confirm that MetAP2 is the target for the drugs in endothelial cells both wild type and drug-resistant MetAP2 mutants will be expressed in endothelial cells to examine whether they confer upon the endothelial cells resistance to the drugs. Two of the most obvious effects of inhibition of MetAP2 is blockade of N-terminal myristoylation and alteration of rates of protein turnover among MetAP2 substrates, either of which can account for the inhibition of endothelial cell growth by the drugs. To identify relevant MetAP2 substrates, proteins will be detected and identified whose myristoylation or turnover are affected by the drugs by labeling whole cell proteins with (3H)-myristic acid and (35S)-methionine. Once such MetAP2 substrates are identified, their roles in mediating the action of the drugs will be further investigated in endothelial cells. The ultimate goal will be to achieve a complete understanding of mechanism of inhibition of endothelial cell growth by the drugs through identification of both direct and indirect mediators of the action of the drugs. Finally, fumagillin and ovalicin will be conjugated with cyclic RGD peptides to target the drugs to endothelial cells at tumor sites which are known to express high level of the RGD binding alpha-v-beta-3 integrin. These drug-RGD peptide conjugates may prove to be much more selective and less toxic than TNP-470 and related angiogenesis inhibitors.
Showing the most recent 10 out of 13 publications
