Highly active antiretroviral therapy or HAART, which refers to the utilization of multiply combined antiretroviral drugs for treatment of HIV-1 infection, has made an immense impact on the quality of life and prognosis of HIV-1-inflicted individuals over the last ten years. Indeed, HAART significantly suppresses the replication of HIV-1 and substantially extends the life expectancy of HIV-1-infected individuals. Recent analyses have revealed that life expectancy in HIV-1-infected individuals treated with HAART increased between 1996 and 2005, that mortality rates for HIV-1-infected persons have become much closer to general mortality rates since the introduction of HAART, and that first line HAART with boosted PI-based regimens resulted in less resistance within and across drug classes. However, the ability to provide effective long-term antiretroviral therapy for HIV-1 infection has yet remained a complex issue since those who initially achieved favorable viral suppression to undetectable levels have experienced treatment failure. One major mechanism of such treatment failure is the emergence of drug-resistant HIV-1 variants. Of note, it has been reported that HIV-1 hardly acquires resistance to DRV in test tube. In the present study, we attempted to select HIV-1 variants resistant to darunavir (DRV), which potently inhibits the enzymatic activity and dimerization of protease and has high levels of genetic barrier to HIV-1 development of resistance to DRV. When a single HIV-1 strain was used as a starting virus for selection against DRV, highly DRV-resistant HIV-1 variants were not obtained. Thus, we employed a mixture of 8 HIV-1 clinical isolates resistant to multiple PIs, expecting that homologous recombination from one isolate to another among them takes place in the presence of escalating doses of DRV and can expedite the emergence of highly DRV-resistant HIV-1 variants. The 8 primary HIV-1 strains, including HIV-1C were isolated from patients with AIDS, who had failed various antiviral regimens after receiving 9 to 11 anti-HIV-1 drugs over 32 to 83 months and contained 9 to 14 amino acid substitutions in the protease-encoding region, which are associated with HIV-1 resistance to various PIs. HIV-1MIX became highly resistant to DRV with an EC50 value approximately 333-fold greater than that against HIV-1NL4-3. HIV-1MIX at passage 51 (HIV-1MIXP51) replicated well in the presence of 5 M DRV and contained 14 mutations. HIV-1MIXP51 was highly resistant to amprenavir, indinavir, nelfinavir, ritonavir, lopinavir, and atazanavir and moderately resistant to saquinavir and tipranavir. HIV-1MIXP51 had a resemblance with HIV-1C of the HIV-1MIX population, selection using HIV-1C was also performed;however, its DRV resistance acquisition substantially delayed. H219Q and I223V substitutions in Gag, lacking in HIV-1CP51, likely contributed to confer replication advantage on HIV-1MIXP51 by reducing the intra-virion cyclophilin A content. HIV-1MIXP51 apparently acquired the substitutions from other HIV-1 strain(s) of HIV-1MIX through possible homologous recombination. The present data suggest that the use of multiple drug-resistant HIV-1 isolates is of utility in selecting drug-resistant variants and that DRV would not easily permit HIV-1 to develop significant resistance;however, HIV-1 can develop high levels of DRV-resistance when a variety of PI-resistant HIV-1 strains are generated as seen in patients experiencing sequential PI failure and ensuing homologous recombination takes place. HIV-1MIXP51 should be useful in elucidating the mechanisms of HIV-1 resistance to DRV and related agents. We also examined the effects of various amino acid (AA) substitutions, which were seen in HIV-1MIXP51 and DRV-resistant clinical HIV-1 strains on protease dimerization using the above-mentioned FRET-based HIV-1 expression assay. A single AA substitution introduced into the N-, C- termini or active site of protease such as P1A, Q2A, T4A, D25N, D30N and N98A, allowed protease to undergo dimerization, which DRV effectively inhibited at 1 muM, suggesting that these AAs are not significantly involved in the binding of DRV to the protease monomer subunit. We also examined whether four AA substitutions (V32I, L33F, I54M and I84V), identified in clinical HIV-1 variants isolated from those who failed to respond to DRV-including antiviral regimens as well as in an in vitro-selected highly DRV-resistant HIV-1 variant we have recently generated, affected the dimerization of protease. None of single mutation of the four AA substitutions significantly impacted on protease dimerization and DRV effectively blocked the dimerization. Introduction of 2 or 3 combined substitutions (e.g., V32I/L33F, V32I/I84V, V32I/L33F/I84V, or V32I/L33F/I54M) allowed protease to undergo dimerization and DRV still effectively inhibited the dimerization. However, when all the four substitutions were introduced into the FRET-based HIV-1 expression assay, DRV was disabled from inhibiting the dimerization. The A28S substitution that HIV-1 acquires when selected against various bis-THF-containing PDIs also disabled DRV from inhibiting protease dimerization. The four AA substitutions and A28S are presumed to alter the conformation of the binding site for DRV in the monomer subunit. The present data that the four combined AA substitutions are required for disabling DRVs dimerization inhibition activity should explain at least in part why the acquisition of HIV-1s resistance to DRV is substantially delayed. The loss of protease dimerization inhibition activity should represent a novel mechanism of the acquisition of HIV-1s drug resistance. We are currently attempting to obtain crystals of HIV-1 protease monomer subunit complexed with DRV to better understand the interaction of DRV and HIV-1 protease monomer subunit.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
Application #
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
National Cancer Institute Division of Basic Sciences
Zip Code
Ghosh, Arun K; Osswald, Heather L; Glauninger, Kristof et al. (2016) Probing Lipophilic Adamantyl Group as the P1-Ligand for HIV-1 Protease Inhibitors: Design, Synthesis, Protein X-ray Structural Studies, and Biological Evaluation. J Med Chem 59:6826-37
Amano, Masayuki; Salcedo-Gómez, Pedro Miguel; Zhao, Rui et al. (2016) A Modified P1 Moiety Enhances In Vitro Antiviral Activity against Various Multidrug-Resistant HIV-1 Variants and In Vitro Central Nervous System Penetration Properties of a Novel Nonpeptidic Protease Inhibitor, GRL-10413. Antimicrob Agents Chemother 60:7046-7059
Ghosh, Arun K; Yu, Xufen; Osswald, Heather L et al. (2015) Structure-based design of potent HIV-1 protease inhibitors with modified P1-biphenyl ligands: synthesis, biological evaluation, and enzyme-inhibitor X-ray structural studies. J Med Chem 58:5334-43
Aoki, Manabu; Hayashi, Hironori; Yedidi, Ravikiran S et al. (2015) C-5-Modified Tetrahydropyrano-Tetrahydofuran-Derived Protease Inhibitors (PIs) Exert Potent Inhibition of the Replication of HIV-1 Variants Highly Resistant to Various PIs, including Darunavir. J Virol 90:2180-94
Ghosh, Arun K; Martyr, Cuthbert D; Osswald, Heather L et al. (2015) Design of HIV-1 Protease Inhibitors with Amino-bis-tetrahydrofuran Derivatives as P2-Ligands to Enhance Backbone-Binding Interactions: Synthesis, Biological Evaluation, and Protein-Ligand X-ray Studies. J Med Chem 58:6994-7006
Ghosh, Arun K; Yashchuk, Sofiya; Mizuno, Akira et al. (2015) Design of gem-difluoro-bis-tetrahydrofuran as P2 ligand for HIV-1 protease inhibitors to improve brain penetration: synthesis, X-ray studies, and biological evaluation. ChemMedChem 10:107-15
Ghosh, Arun K; Martyr, Cuthbert D; Kassekert, Luke A et al. (2015) Design, synthesis, biological evaluation and X-ray structural studies of HIV-1 protease inhibitors containing substituted fused-tetrahydropyranyl tetrahydrofuran as P2-ligands. Org Biomol Chem 13:11607-21
Ghosh, Arun K; Takayama, Jun; Kassekert, Luke A et al. (2015) Structure-based design, synthesis, X-ray studies, and biological evaluation of novel HIV-1 protease inhibitors containing isophthalamide-derived P2-ligands. Bioorg Med Chem Lett 25:4903-4909
Amano, Masayuki; Tojo, Yasushi; Salcedo-Gómez, Pedro Miguel et al. (2015) A novel tricyclic ligand-containing nonpeptidic HIV-1 protease inhibitor, GRL-0739, effectively inhibits the replication of multidrug-resistant HIV-1 variants and has a desirable central nervous system penetration property in vitro. Antimicrob Agents Chemother 59:2625-35
Hayashi, Hironori; Takamune, Nobutoki; Nirasawa, Takashi et al. (2014) Dimerization of HIV-1 protease occurs through two steps relating to the mechanism of protease dimerization inhibition by darunavir. Proc Natl Acad Sci U S A 111:12234-9

Showing the most recent 10 out of 31 publications