In the period to be covered in the report, we demonstrate employing electrospray ionization mass spectrometry (ESI-MS) that DRV binds to protease monomer and inhibits dimerization but two conventional PIs (saquinavir and nelfinavir) that inhibit enzymatic activity but not dimerization failed to bind to monomers. Indeed, DRV binds to a monomer polyprotein consisting of 56 amino acid transframe region and PR (TFR-PRD25N), while saquinavir failed to bind to TFR-PRD25N. We also found that DRV failed to bind to mutant PR containing 4 amino acid substitutions (V32I, L33F, I54M, and I84V) that confer resistance to DRV on HIV. Three recombinant PR variants carrying T26A and R87K substitutions at the active site interface that were shown to disrupt PR dimerization in the FRET-based HIV-1 expression assay, remained as monomers, while a PR variant deficient of 4 carboxy-teminus amino acids that provide critical dimerization force (PR1-C95A) formed monomers and dimers. These data suggest that relatively weak intermolecular interactions of two monomers initially occurs at the active site interface and with subsequent interactions at the termini interface the weakly-dimerized two monomers are firmly dimerized, completing the PR dimerization process. We also demonstrated that tipranavir (TPV), a protease inhibitor (PI), which inhibits the enzymatic activity and dimerization of HIV-1 protease, exerts potent activity against multi-PI-resistant HIV-1s. When a mixture of eleven clinical multi-PI-resistant (but TPV-sensitive) HIV-1s isolates (HIV11MIX), including HIVB and HIVC, was selected against TPV, HIV11MIX rapidly (HIV11MIXP10) acquired high-level TPV resistance and replicated at high concentrations of TPV. HIV11MIXP10 contained various AA substitutions including I54V and V82T. The intermolecular FRET-based HIV-1-expression assay revealed that TPV's dimerization inhibition activity against a clone of HIVB (cHIVB) was substantially compromised. The addition of I54V and V82T to cHIVB (cHIVBI54V/V82T) did not further compromise TPV's dimerization inhibition but conferred TPV resistance on cHIVB. None of single AA substitutions including L33I, found responsible for TPV resistance of HIV11MIXP10, conferred TPV resistance on wild-type cHIVNL4-3 but they compromised TPV's dimerization inhibition in cHIVNL4-3. Reversion of Ile-33 to Leu rendered cHIVBI54V/V82T less resistant to TPV, suggesting L33I's contribution to HIVB's TPV resistance. cHIVC acquired TPV resistance when introduced with L24M, which compromised TPV's dimerization inhibition. When TPV-selected, cHIVNL4-3I54V/V82T most readily developed TPV resistance and acquired E34D, which compromised TPV's dimerization inhibition in cHIVNL4-3. The data demonstrate that certain AA substitutions do not compromise TPV's dimerization inhibition but confer TPV resistance, while others compromise TPV's dimerization inhibition, contributing to HIV's TPV resistance. The data that TPV's dimerization inhibition is compromised with a single AA substitution may explain at least in part why the genetic barrier of TPV against HIV's development of TPV resistance is relatively low. While we attempted to generate DRV-resistant variants, we designed, synthesized, and identified novel non-peptidyl PIs that exert potent activity even against DRV-resistant HIV-1 variants, in continuous collaboration with Professor Ghosh. We have identified several novel PIs that have potent activity against various drug-resistant HIV-1 variants with favorable virologic and pharmacologic features (Aoki, Ghosh, and Mitsuya: manuscript in preparation). GRL-04810 and GRL-05010, containing the structure-based designed privileged cyclic ether-derived non-peptide P2 ligand, bis-tetrahydrofuranylurethane (bis-THF) and a difluoride moiety, are among them, both of which are active against a laboratory strain HIV-1LAI (EC50: 0.0008 and 0.003 microM) with minimal cytotoxicity (CC50: 17.5 and 37.0 microM in CD4+ MT-2 cells), respectively (Gomez, Amano, Ghosh, and Mitsya, manuscript under revision). The two compounds were active against multi-PI-resistant clinical HIV-1 variants isolated from patients who had no response to various antiviral regimens. GRL-04810 and GRL-05010 also blocked the infectivity and replication of each of the HIV-1NL4-3 variants selected by up to 5 microM lopinavir (EC50: 0.03 and 0.03 microM, respectively) and atazanavir (EC50: 0.02 and 0.04 microM, respectively). Moreover, they were active against darunavir (DRV)-resistant variants (EC50: in 0.03-0.034 microM range for GRL-04810, and 0.026-0.043 microM for GRL-05010), while DRV had EC50 values between 0.02-0.174 microM. GRL-04810 had a favorable lipophilicity profile as determined with the partition (logP) and distribution coefficients (logD) of -0.14 and -0.29, respectively. The in vitro blood-brain-barrier (BBB) permeability assay revealed that GRL-04810 and GRL-05010 may have a greater advantage in terms of crossing BBB than the currently available PIs with apparent penetration indexes of 47.8 x 10-6 and 61.8 x 10-6 cm/s, respectively. The present data demonstrate that GRL-04810 and GRL-05010 exert potent activity against a wide spectrum of HIV-1 variants in vitro and suggest that two fluorine atoms added to their bis-THF moiety may well enhance their penetration across BBB.
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