Dimerization of HIV-1 protease subunits is essential for its proteolytic activity, which plays a critical role in HIV-1 replication. Hence, the inhibition of protease dimerization represents a unique target for potential intervention of HIV-1. We developed an intermolecular fluorescence resonance energy transferbased HIV-1-expression assay employing cyan and yellow fluorescent protein-tagged protease monomers. Using this assay, we identified non-peptidyl small molecule inhibitors of protease dimerization. These inhibitors, including darunavir and two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 M and blocked HIV-1 replication with IC 50 values of 0.0002-0.48 M. These agents also inhibited the proteolytic activity of mature protease. Other approved anti-HIV-1 agents examined except tipranavir, a CCR5 inhibitor, and soluble CD4 failed to block the dimerization event. Once protease monomers dimerize to become mature protease, mature protease is not dissociated by this dimerization inhibition mechanism, suggesting that these agents block dimerization at the nascent stage of protease maturation. The proteolytic activity of mature protease that managed to undergo dimerization despite the presence of these agents is likely to be inhibited by the same agents acting as conventional protease inhibitors. Such a dual inhibition mechanism should lead to highly potent inhibition of HIV-1.
The Z- and E-isomers of fluoromethylenecyclopropane analogues 11a-d and 12a-d were synthesized, and their antiviral activities were evaluated. The purine (Z,E)-methylenecyclopropane carboxylates 13 and 24 were selectively fluorinated using lithium diisopropylamide, LiCl, and N-fluorobenzenesulfonimide to give (Z,E)-fluoroesters 22 and 25. Reduction with LiBH(4) or diisobutylaluminum hydride gave after chromatographic separation Z-isomers 11a and 11e and E-isomers 12a and 12e. The O-demethylation of 11e and 12e afforded guanine analogues 11b and 12b. Fluorination of (Z,E)-cytosine and thymine esters 15 and 16 afforded (Z,E)-fluoroesters 26 and 27, which were resolved before the reduction to analogues 11c and 11d and 12c and 12d. Adenine Z-isomer 11a was the most effective against Towne and AD169 strains of human cytomegalovirus (HCMV, EC(50) 3.6 and 6.0 microM, respectively), but it was less effective against murine virus (MCMV, EC(50) 69 microM). Thymine Z-isomer 11d was effective against HSV-1 in BSC-1 cells (ELISA, EC(50) 2.5 microM) but inactive against HSV-1 or HSV-2 in Vero or HFF cells. All of the analogues with the exception of 12d were effective at least in one of the assays against Epstein-Barr virus (EBV) in Daudi or H-1 cells in a micromolar or submicromolar range. Cytosine and thymine Z-isomers 11c and 11d were active against varicella zoster virus (VZV) with EC(50) 0.62 microM. Adenine Z- and E-isomers 11a and 12a were effective against HIV-1 in MT-2 or MT-4 cells with EC(50) 12-22 and 2.3-7.6 microM, respectively, whereas only 12a was effective against hepatitis B virus (HBV) with EC(50) 15 microM. Analogues 11a and 12a were weak substrates for adenosine deaminase.
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