Subproject 1. Development of Novel Protease Inhibitors and Reverse Transcriptase Inhibitors Our structure-based design of HIV-1 PIs that can specifically interact with the enzyme backbone atoms has led to the development of compounds with unprecedented potency and drug-resistance profiles. Particularly notable is the development of a stereochemically defined 3(R),3a(S),6a(R)-bis-tetrahydrofuranyl urethane (bis-THF) as the non-peptidic P2 ligand. Incorporation of this ligand has provided a variety of exceedingly potent HIV-1 PIs with superior activity against multi-PI-resistant variants relative to other FDA-approved PIs. Darunavir, a PI recently approved for the treatment of drug-resistant HIV, directly resulted from this strategy. Our detailed proteinﾐdarunavir X-ray crystal structures have clearly demonstrated extensive interactions with the protein backbone in the enzyme active site. This present design concept targeting the protein backbone may serve as a powerful strategy to combat drug resistance. One such compound is GRL-98065, which exerts highly potent activity against a wide spectrum of laboratory HIV-1 strains and primary clinical isolates including multi-PI-resistant variants with minimal cytotoxicity. GRL-98065 was also active against HIV-1 isolates of various subtypes as well as HIV-1 isolates examined. Structural analyses revealed that the close contact of GRL-98065 with the main chain of the protease active site amino acids (Asp29 and Asp30) is critical for its potency and wide-spectrum activity against multi-PI-resistant HIV-1 variants. We also examined the potential for clinical use of 4-ethynyl-2-fluoro-2-deoxyadenosine (EFdA), which is highly potent against multi-nucleoside reverse transcriptase inhibitor-resistant HIV-1 variants. In particular, we studied the intracytoplasmic anabolism and kinetics of antiviral activity against HIV-1 of a nucleoside reverse transcriptase inhibitor, 4-ethynyl-2-fluoro-2-deoxyadenosine (EFdA), which has a potent activity against wild-type and multi-drug resistant HIV-1 strains. When CEM cells were exposed to 0.1 μM 3H-labled EFdA (3H-EFdA) or 3H-labeled-3-azido-2,3-dideoxythymidine (3H-AZT) for 6 hours, the intracellular EFdA-triphosphate (TP) level was 91.6 pmol/109 cells, while that of AZT was 396.5 pmol/109 cells. When CEM cells were exposed to 10 μM 3H-EFdA, the amount of EFdA-TP increased by 22-fold (2,090 pmol/109 cells), while the amount of 3H-AZT-TP only moderately increased by 2.4-fold (970 pmol/109 cells). The intracellular T1/2 values of EFdA-TP and AZT-TP were 17 and 3 hours, respectively. When MT-4 cells were cultured with 0.01 μM EFdA for 24 hours, thoroughly washed to remove EFdA, further cultured without EFdA for various periods of time, exposed to HIV-1NL4-3, and further cultured for an additional 5 days, the % protection values were 75 and 47 after 24 and 48 hours no-drug incubation, while those with 1 μM AZT were 55 and 9.2, respectively. The IC50 values of EFdA-TP against human polymerases α, β and γ were >100, >100, and 10 μM, while those of ddA-TP were >100, 0.2, and 0.2 μM, respectively. These data suggest that EFdA might represent a potent agent with a possibility of once- or twice-a-day regimen and warrant further development as a potential therapeutic for those harboring wild-type HIV-1 and/or multi-drug resistant variants. Subproject 2. Study of HIV-1 protease dimerization and discovery efforts of protease dimerization inhibitors. 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 transfer-based 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 μM and blocked HIV-1 replication with IC50 values of 0.0002-0.48 μM. 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. Subproject 3. Structural and molecular study of human CCR5 and its interactions with CCR5 inhibitors. We have characterized the structural and molecular interactions of CC-chemokine receptor 5 (CCR5) with three CCR5 inhibitors active against R5-HIV-1 including a potent in vitro and in vivo CCR5 inhibitor aplaviroc (AVC). The data obtained with saturation binding assays and structural analyses delineated the key interactions responsible for the binding of CCR5 inhibitors with CCR5 and illustrated that their binding site is located in a predominantly lipophilic pocket in the interface of extracellular loops (ECLs) and within the upper transmembrane (TM) domain of CCR5. Mutations in the CCR5 binding sites of AVC decreased gp120 binding to CCR5 and the susceptibility to HIV-1 infection, while mutations in TM4 and TM5 that also decreased gp120 binding and HIV-1 infectivity had less effects on the binding of CC-chemokines, suggesting that CCR5 inhibition targeting appropriate regions might render the inhibition highly HIV-1-specific while preserving the CC chemokine-CCR5 interactions. The present data delineating residue-by-residue interactions of CCR5 with CCR5 inhibitors should not only help design more potent and more HIV-1-specific CCR5 inhibitors, but also give new insights into the dynamics of CC-chemokine-CCR5 interactions and the mechanisms of CCR5 involvement in the process of cellular entry of HIV-1. Subproject 4. Elucidation of the Mechanisms of the Emergence of Drug Resistant HIV-1 Variants We examined 28 children with HIV-1 infection who were not responding to existing antiviral regimens and enrolled into clinical trials conducted at the National Cancer Institute to provide salvage therapy. In 3 of the 28 patients (10.7%), the Q151M complex amino acid substitutions were identified. The 3 patients had received nucleoside reverse transcriptase inhibitor (NRTI) monotherapy and/or combination regimens with multiple NRTIs for 4.3 to 8.6 years prior to the study. Recombinant infectious clones generated by incorporating the RT-encoding region of HIV-1 isolated from each patients plasma were highly resistant to zidovudine, didanosine and stavudine, while they were intermediate resistant towards lamivudine and tenofovir disoproxil fumarate. TDF-containing regimens reduced HIV-1 viremia in 2 of the 3 children carrying the Q151M complex. These data suggest that the Q151M is prevalent in pediatric patients with long-term NRTI monotherapy and/or dual NRTI regimens and that well designed HAART regimens may be meritorious in such patients
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