1. Development of a novel ELISA for the testing of glycobioconjugates as anti-HIV agents 2. Synthesis of potential inhibitors of the HIV entry mechanism 3. Probing the secondary structural characteristics of oligosaccharides utilizing circular dichroism

Abstract

AIDS, or acquired immunodeficiency syndrome, is caused by the human immunodeficiency virus (HIV). HIV is a retrovirus that is capable of rapid genetic mutation, which makes the virus and the disease difficult to treat. Several drug therapies are currently available, in the form of viral enzyme inhibitors. Other inhibitors of the viral entry and replication process are being investigated to enhance the drug therapy arsenal. Our research has focused on the development of HIV entry inhibitors. We are working towards the development of novel carbohydrate-based agents that are capable of binding the gp120 protein on the viral surface, such that viral entry into an uninfected host cell is prevented. In order for our research to progress, a qualitative method by which our synthetic compounds could be evaluated for gp120 binding was sought. We have developed a unique ELISA (enzyme-linked immunosorbent assay) that indicates whether or not a compound has binding affinity for the viral protein. A TIRF (total internal reflection fluorescence) microscopy method, has been developed as part of a collaborative effort with the laboratories of Professors Saavedra and O'Brien, to assess active compounds for quantitative equilibrium binding constants to gp120. We have synthesized several carbohydrate-based molecules targeted to one or more of the binding sites on the surface of gp120; the galactosylceramide site, the V3 loop, and the CD4 binding site. Utilizing both the ELISA and TIRF methods, we have succeeded in probing the binding profile of gp120. Circular dichroism studies have also been employed to evaluate the secondary structural characteristics of oligomeric carbohydrate materials. Molecules with helical properties have potential as CD4 binding site inhibitors. The long term goals of this project involve the synthesis and gp120 binding evaluation of novel carbohydrate-based materials to serve as entry inhibitors of the HIV replication process. A possible application of this project lies in the development of compounds capable of binding to more than one site on the protein. A variation of this goal involves the tethering of various compounds with specificities to different sites on gp120, for the purpose of inhibiting multiple binding sites on the protein.