James W. Fett

A small-molecule inhibitor of the ribonucleolytic activity of human angiogenin that possesses antitumor activity

The results of previous preclinical and clinical studies have identified angiogenin (ANG) as a potentially important target for anticancer therapy. Here we report the design and implementation of a high-throughput screening assay to identify small molecules that bind to the ribonucleolytic active site of ANG, which is critically involved in the induction of angiogenesis by this protein. Screening of 18,310 compounds from the National Cancer Institute (NCI) Diversity Set and ChemBridge DIVERSet yielded 15 hits that inhibit the enzymatic activity of ANG with Ki values <100 μM. One of these, NCI compound 65828 [8-amino-5-(4′-hydroxybiphenyl-4ylazo)naphthalene-2-sulfonate; Ki = 81 μM], was selected for more detailed studies. Minor changes in ANG or ligand structure markedly reduced potency, demonstrating that inhibition reflects active-site rather than nonspecific binding; these observations are consistent with a computationally generated model of the ANG⋅65828 complex. Local treatment with modest doses of 65828 significantly delayed the formation of s.c. tumors from two distinct human cancer cell types in athymic mice. ANG is the likely target involved because (i) a 65828 analogue with much lower potency against the enzymatic activity of ANG failed to exert any antitumor effect, (ii) tumors from 65828-treated mice had fewer interior blood vessels than those from control mice, and (iii) 65828 appears to have no direct effect on the tumor cells. Our findings provide considerable support for the targeting of the enzymatic active site of ANG as a strategy for developing new anticancer drugs.

Angiogenin mRNA in human tumor and normal cells

Angiogenin mRNA was characterized in HT-29 human colon adenocarcinoma cells and its distribution in other human cell types was studied. Several RNA species ranging from 800 to 6000 nucleotides hybridized to the angiogenin probes with the smallest being the major poly(A)-containing transcript. This transcript was detected in tumor cells of diverse cellular origin. Expression of angiogenin mRNA is not limited to neoplastic cells and is detected in normal epithelial cells, fibroblasts and peripheral blood cells. The latter, when induced by mitogens, expressed more mRNA than did unstimulated or HT-29 cells. Transformed fibroblasts did not contain higher levels of angiogenin mRNA than their normal counterparts, demonstrating that increased angiogenin mRNA expression does not necessarily occur upon neoplastic transformation. This study demonstrates that angiogenin mRNA is expressed in a wide spectrum of cells and is not correlated to a particular cell phenotype.

Inhibition of prostate carcinoma establishment and metastatic growth in mice by an antiangiogenin monoclonal antibody.

A neutralizing monoclonal antibody (MAb) 26‐2F to human angiogenin, a potent inducer of neovascularization, has been shown previously to prevent or delay the appearance of angiogenin‐secreting human colon, fibrosarcoma and lung tumor cell xenografts implanted subcutaneously (s.c.) into athymic mice. In an analogous model system, we report here that the antibody also prevents the establishment of PC‐3 androgen‐independent human prostate cancer tumors in, on average, 40% of treated mice (p < 0.0001, survivor analysis). Intriguingly, combining MAb 26‐2F together with cisplatin and suramin, 2 therapeutic agents that together showed little antitumor activity in the aforementioned model, resulted in an even greater degree of protection (71% protected, p = 0.009 compared to antibody treatment alone). This protective effect persisted several weeks after cessation of treatment. Additionally, prophylactic systemic administration of MAb 26‐2F dramatically reduced by 50% the formation of spontaneous regional metastasis originating from primary growth in the prostate gland of PC‐3M cells, highly metastatic variants of PC‐3. Protection from metastasis was still significant when treatment with MAb 26‐2F was delayed until after the primary tumor was well established. The antibody is not directly cytotoxic to either cell type, both of which secrete angiogenin in vitro and when growing as tumors in vivo, but changes the pattern of vascularity in primary tumors growing orthotopically. These findings, together with the observation that angiogenin protein and mRNA are apparently overexpressed in cancerous vs. normal human prostate tissues, demonstrate that angiogenin antagonism represents a promising new approach for preventing progression and metastasis of clinical prostate cancer.

Comparison of human and bovine brain derived heparin-binding growth factors

Two growth factors have been purified to homogeneity from human brain using heparin affinity chromatography. They have apparent molecular weights of 17 Kd and 18 Kd. Their amino acid compositions differ, but are similar to those of the two heparin-binding growth factors present in bovine neural tissue. These results suggest that the heparin-binding growth factors in neural tissue can be grouped into two distinct classes.

The Crystal Structure of Human Angiogenin in Complex with an Antitumor Neutralizing Antibody

The murine monoclonal antibody 26-2F neutralizes the angiogenic and ribonucleolytic activities of human angiogenin (ANG) and is highly effective in preventing the establishment and metastatic dissemination of human tumors in athymic mice. Here we report a 2.0 A resolution crystal structure for the complex of ANG with the Fab fragment of 26-2F that reveals the detailed interactions between ANG and the complementarity-determining regions (CDRs) of the antibody. Surprisingly, Fab binding induces a dramatic conformational change in the cell binding region of ANG at the opposite end of the molecule from the combining site; crosslinking experiments indicate that this rearrangement also occurs in solution. The ANG-Fab complex structure should be invaluable for designing maximally humanized versions of 26-2F for potential clinical use.

Lysates of two established human tumor lines contain heparin-binding growth factors related to bovine acidic brain fibroblast growth factor

Cell lysates of two established human tumor lines, a medulloblastoma (TE671), and a rhabdomyosarcoma (RD), contain mitogenic activity which elutes from heparin-Sepharose under conditions typical of class 1 heparin-binding growth factors, such as acidic brain fibroblast growth factor. The presence of this class of mitogen in both cell lines was confirmed by their chromatographic behavior on reversed-phase C3 columns, and by the ability of heparin to enhance their mitogenic activity. Using a specific synthetic DNA probe, RNAs were isolated from both cell lines by hybridization-selection, translated in vitro, and translated proteins affinity fractionated on heparin-Sepharose. The results demonstrate that TE671 and RD cell lysates contain mRNAs for mitogens related to acidic brain fibroblast growth factor, and also suggest that high molecular weight proteins exist that are closely related to, or are precursor forms of, the class 1 mitogens.

Induction of angiogenesis by mixtures of two angiogenic proteins, angiogenin and acidic fibroblast growth factor, in the chick chorioallantoic membrane

The chick chorioallantoic membrane assay was employed to assess the angiogenic response induced by mixtures of human angiogenin with bovine heparin-binding acidic fibroblast growth factor. Statistical evaluation of data accumulated at several molar ratios of the two proteins indicate that the angiogenic activity observed is neither an additive nor a synergistic resultant of the activities of the proteins separately. The possibility exists, however, that at an approximately 1:1 mole ratio an apparent inhibitory effect can be observed. Mechanisms which could underlie such observed effects are discussed.

C-terminal angiogenin peptides inhibit the biological and enzymatic activities of angiogenin

Synthetic peptides corresponding to the C-terminal region of angiogenin (Ang) inhibit the enzymatic and biological activities of the molecule while peptides from the N-terminal region do not affect either activity. The peptide Ang(108-121) transiently abolishes the inhibition of cell-free protein synthesis caused by angiogenin coincidentally with its cleavage of reticulocyte RNA. Several C-terminal peptides also inhibit nuclease activity of angiogenin when tRNA is the substrate. Furthermore, peptide Ang(108-123) significantly decreases neovascularization elicited by angiogenin in the chick chorioallantoic membrane assay.