Human angiogenin (Ang) is an unusual member of the pancreatic ribonuclease (RNase) superfamily that induces blood vessel formation in vivo. Recent studies have demonstrated that Ang plays a critical role in the establishment and growth of some human tumors. These results define as a medically important goal the identification or design of Ang inhibitors suitable for clinical applications. The approach to this problem presented here is based on recent breakthroughs in determining the 3D structure of Ang, achieved together with our collaborators, and on the continuing availability of new crystallographic data. Three classes of Ang antagonists will be developed: (1) Small nucleotide-based inhibitors of the ribonucleolytic activity of Ang that is known to be essential for its angiogenic action. The design of these compounds will take into account the unexpected blockage of the pyrimidine binding site observed in the Ang crystal structure (and confirmed by mutagenesis), and the consequent requirement for Ang to undergo an extensive conformational change in order to act on RNA. The nature of this conformational change will be explored by X-ray crystallography of Ang-nucleotide complexes, fluorescence spectroscopy, and mutagenesis. New nucleotide inhibitors will be synthesized and tested based on these findings and on kinetic results obtained with RNase A inhibitors and related molecules. These efforts will be aided by the mutagenic characterization of inhibitor/substrate binding subsites on Ang and by the development of an improved enzymatic assay for Ang. The antiangiogenic and antitumor activities of any inhibitors with low I/i values will be examined. The possibility will be investigated that molecules other than RNA are substrates for Ang; if so, additional inhibitors will be designed based on these substrates. (2) Derivatives of placental RNase inhibitor (PRI), an extremely tight- binding protein inhibitor of the enzymatic and angiogenic activities of Ang. Contact residues on PRI will be identified by X-ray crystallography and mutagenesis. The inhibitory properties of PRI domains containing critical residues will be tested. Mutagenesis will be used to improve the specificity and stability of PRI or any smaller active derivatives obtained. (3) Inhibitors of cellular interactions of Ang that are also required for angiogenic activity. Residues and regions of Ang that interact with Ang- binding proteins/receptors on endothelial cells will be identified by testing the capacities of Ang mutants to bind to these sites. The results will help to establish the mutual relationships of the enzymatic, angiogenic, and various cell-binding properties of Ang. The 3D structures of the complexes of Ang with any cell-surface Ang-binding proteins that can be obtained in quantity will be examined. The capacity of peptides derived from Ang/Ang-binding protein contact regions to inhibit the angiogenic activity of Ang will be tested.
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