) Anti-angiogenic therapy has immense clinical potential to not only impede tumor growth but also to inhibit tumor metastases. Despite a limited knowledge of its mode of action, the first small molecule antiangiogenic agent to enter clinical trials was the fumagillin derivative, TNP-470 (AGM1470). Our long-term goal is the exploration of TNP-470's molecular mechanism of action and the application of this information in the development of novel antiangiogenic agents. TNP-470 has proven to be an attractive lead compound due to its endothelial cell selectivity and cytostatic (versus cytotoxic) effect on proliferating endothelial cells. However, it is rapidly metabolized in vivo and is not orally available. Moreover, it has undesirable immunomodulatory, neurotoxicity, and weight loss inducing effects in vivo raising concerns over its ultimate utility as an antitumor compound. Since many of these issues may be unrelated to TNP-470's anti-angiogenic activity, we have attempted to dissect the molecular basis of its endothelial cell cytostatic effect. Towards that end, we have identified the methionine aminopeptidase 2 (MetAP-2) protein as a major TNP-470 target. TNP-470 selectively and covalently inhibits MetAP-2 enzymatic activity. Moreover, in collaboration, we have recently solved the crystal structure of MetAP-2 with and without bound fumagillin. This knowledge will greatly aid in the design of additional MetAP-2 inhibitors and several pharmaceutical companies have active programs focused on this goal. In order to confirm that MetAP-2 is responsible for the antiangiogenic effect of TNP-470, we propose here the generation of homozygous MetAP-2 null mice. Given our hypothesis that MetAP-2 activity is critical for endothelial cell proliferation and given the need for angiogenesis during fetal development, we predict that this mutation will be embryonic lethal. While such a result would validate MetAP-2 as a target for antiangiogenic development, we also want to investigate the role of MetAP-2 inhibition in mediating the known side effects of TNP-470. Therefore, using Tet(off) Cre recombinase technology, we propose to generate an inducible MetAP-2 knockout mouse that would grow normally to adulthood but then could be induced to become MetAP-2 nullizygous. These mice will be analyzed for the immunomodulatory and neurotoxicity phenotypes associated with TNP-470 addition and for their ability to support transplanted tumors and metastases.
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