Background:  Effective antiretroviral agents with novel mechanisms of action that lack cross-resistance to the currently available agents are essential to the future management of HIV-infected patients.  Viral entry, which occurs through a series of complex interactions with the cellular membrane and receptors, represents a new area for viral intervention.

Methods: A novel, small molecule, orally bioavailable HIV-1 inhibitor was identified that targets the viral envelope glycoproteins and blocks the entry of virus into cells.  The HIV-1 entry inhibitor was initially shown to block fusion between cells expressing either gp120 or CD4, suggesting that viral entry is the target.  Receptor binding assays were performed to determine whether the fusion inhibitory activity was the result of blocking the gp120/CD4 interaction. 

Results: Results showed that the entry inhibitor selectively inhibited the binding of recombinant envelope gp120_JRFL to sCD4 via a competitive mechanism. To ascertain whether the HIV-1 viral envelope is the binding site of the inhibitor, gp120 proteins were generated from resistant gp120_BRU recombinant viruses and used in gp120/CD4 binding assays.  Results indicated that binding was inhibited 43- to 61-fold less when gp120 _BRU variants containing resistance-associated mutations were used.  The fact that radiolabeled inhibitor binds selectively to purified gp120 protein with a K_d value of 52 nM provided additional evidence that the HIV-1 entry inhibitor directly targets gp120.  The direct binding of HIV entry inhibitor to gp120 was further validated using the Biacore 3000 in which a gp120 coated sensor chip directly measures inhibitor binding signal intensities.  Additional studies, using both biochemical and cellular approaches, demonstrated that the interaction of this compound with gp120 is reversible. 

Conclusions: Therefore, this novel class of HIV entry inhibitors block HIV-1 access into cells by binding to gp120 and inhibiting gp120/CD4 interactions.  This novel class of entry inhibitors has the potential to expand current treatment options and address many of the needs for improved antiretroviral therapy.