The primary goal of this study is to determine the mechanism of action of an HIV entry inhibitor 1C9564 and its analogs. 1C9564 is a betulinic acid derivative that inhibits HIV at nanomolar concentrations. 1C9564 inhibits both HIV primary isolates and laboratory strains by preventing the viruses from entering the cells. However, the detailed mechanism of action is unknown. Our long-term goal is to understand how anti-HIV-l agents arrest the viral entry process and to develop an effective anti-HIV therapy. The objective of this study is to determine the anti-viral spectrum and the molecular target of 1C9564 and its analogs. The central hypothesis of this study is that 1C9564 interferes with the interaction of the HIV envelope and cellular receptors, and subsequently blocks HIV from entering the cells. This hypothesis has been formulated based on the observation that mutations in the HIV envelope were identified from an 1C9564 resistant strain. The hypothesis will be tested by pursuing four specific aims: 1) determine the antiviral potency against a panel of primary isolates, infection of macrophages, and drug resistant isolates. 2) identify the molecular target of 1C9564, 3) determine the role of cellular receptors in the inhibitory activity of 1C9564 and its analogs, and 4) identify and characterize the """"""""inhibited envelope intermediate"""""""" that is arrested by 1C9564. To achieve the proposed research, we will take advantage of the following: 1) 1C9564 can be labeled with radioisotope without affecting its anti-entry activity, 2) a series of HIV-l entry inhibitors that are derived from the lead compound 1C9564, and 3) the HIV-I strains that are resistant to 1C9564. The rationale underlying the proposed research is that 1C9564 targets one of the steps of the HIV-envelope-mediated membrane fusion and arrests the virus from entering the cells. The significance of identifying this mechanism of action is that it will not only provide a molecular target for small molecules such as 1C9564 but also could shed light on how the HIV-l envelope mediates viral entry. Highly active anti-retroviral therapy (HAART) has been very effective in suppressing HIV-l replication. However, the virus is suppressed rather than eradicated. Potent anti-HIV compounds, such as 1C9564, with a mode of action different from that of clinically available anti-HIV drugs could become a very useful addition to improve current anti-HIV therapy.
