In response to the RFA, we have focused on identifying a selective inhibitor of the target enzyme, Golgi alpha-mannosidase II. Many lines of evidence of demonstrate that inhibiting this s enzyme target can retard tumor progression in vivo. All of the inhibitors of this enzyme, however, have an unacceptable, serious side effect that precludes their further study as therapeutic drugs because they are active against another alpha-mannosidase located in the lysosome, as well as a-mannosidase II. Inhibition of the lysosomal enzyme causes a phenocopy of the deadly lysosomal storage disease, Golgi alpha-mannosidosis. Significant differences in the substrate binding specificities of these two mannosidases can now be exploited to develop an inhibitor that is selective for Golgi alpha-mannosidase II. The focus of this proposal is to develop a selective inhibitor of this enzyme that is active in vivo, thereby validating Golgi alpha-mannosidase II as a novel target for a potential anti-tumor therapeutic. Several key scientific advances make this proposal timely, and we have assembled a team of investigators at the Complex Carbohydrate Research Center, Univ. of Georgia, to take advantage of these advances. First, the human Golgi alpha-mannosidase II has been cloned and expressed by a member of our team, in order to be used in inhibitor screening experiments. The lysosomal enzyme has been expressed similarly, and recent data on the structure of the active sites of the enzymes will aid in inhibitor design. High through-put inhibitor assays have been developed to screen thousands of potential inhibitors. A novel class of inhibitor compounds, the sulfonium salts, have been developed by a third team member. These compounds, as well as the inhibitor mannostatin, will be modified by a directed combinatorial synthetic strategy by a fourth member to generate libraries of compounds that will be screened to find identify potent selective inhibitors of the Golgi a-mannosidase II in vitro. Lead compounds will be used again in modified combinatorial syntheses to refine inhibitory properties. All of these leads will be tested for in vivo inhibition using a cell-based rapid screening assay for selected inhibition of N-glycan processing. Compounds that show in vivo selective inhibition will then be tested for their abilities to inhibit the Golgi mannosidase in mouse tissues when delivered orally or by s.c. osmotic pumps. The experiments described in this proposal, therefore, will allow us to isolate lead compounds that selectively inhibit N-linked glycosylation events known to promote tumor progression and demonstrate the validity of the Golgi alpha-mannosidase II as a prime target for tumor therapeutic development.
