Identification of small-molecule inhibitors of human angiogenin and characterization of their binding interactions guided by computational docking.

Angiogenin (ANG) is a potent inducer of angiogenesis and an RNase A homologue whose ribonucleolytic activity is essential for its biological action. Recently, we reported the identification of small non-nucleotide inhibitors of the enzymatic activity of ANG by high-throughput screening (HTS) [Kao, R. Y. T., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 10066-10071]. Two of the inhibitors that were obtained, National Cancer Institute compound NSC-65828 [8-amino-5-(4'-hydroxybiphenyl-4-ylazo)naphthalene-2-sulfonate] and ChemBridge compound C-181431 [4,4'-dicarboxy-3,3'-bis(naphthylamido)diphenylmethanone], were judged to be suitable for further development, and one of these (NSC-65828) was shown to possess antitumor activity in mice. Here we have used computational docking as a guide for the identification of available NSC-65828 and C-181431 analogues that bind more tightly to ANG, and for the characterization of inhibitor binding modes. Numerous analogues were found to have greater avidity than the HTS compounds or any small nucleotide inhibitors; four were considered to be of interest as potential leads (K(i) = 5-25 microM). Two of these analogues bind more tightly to ANG than to RNase A, and are the first small molecules shown to exhibit this selectivity. The predicted binding orientations of the HTS compounds and the new lead inhibitors were evaluated by determining the effects of ANG active site mutations on inhibitory potency. The results with ANG variants R5A, H8A, N68A, and des(121-123) are highly consistent with the docking models. Affinity changes observed with Q12A and Q117G reveal aspects of active site function that are not apparent from the free ANG crystal structure or from the modeled complexes. These findings should prove to be useful in the design of more effective and specific ANG antagonists.