Highly active antiretroviral therapy (HAART) has had a major impact on the acquired immunodeficiency syndrome (AIDS) epidemic in industrially advanced nations. However, eradication of human immunodeficiency virus type 1 (HIV-1) does not appear to be currently possible, in part due to the viral reservoirs remaining in blood and infected tissues. Moreover, a number of challenges have been encountered, which include various adverse effects, only partial and limited immunologic restorations achieved, and the occurrence of various cancers as consequences of survival elongation with HAART. Moreover, such limitations of HAART are exacerbated by the development of drug-resistant HIV-1 variants. Thus, the identification of new classes of antiretroviral drugs that have a unique mechanism(s) of action and produce no or minimal adverse effects remains an important therapeutic objective. In the present study, we utilized the intermolecular fluorescence resonance energy transfer (FRET)-based HIV-1-expression assay that we for the first time established employing cyan and yellow fluorescent protein-tagged HIV-1 protease monomers. Using this assay, we identified a group of non-peptidyl small molecule inhibitors of HIV-1 protease dimerization. These inhibitors, including the recently approved protease inhibitor (PI) darunavir (DRV) as well as two experimental protease inhibitors, blocked protease dimerization at concentrations of as low as 0.01 μM and blocked HIV-1 replication in vitro with IC50 values of 0.0002-0.48 μM. These agents also inhibited the proteolytic activity of mature HIV-1 protease. Another PI, tipranavir (TPV), active against HIV-1 variants resistant to multiple PIs, also blocked protease dimerization, although all other existing FDA-approved anti-HIV-1 drugs examined in the present study failed to block the dimerization. In the period of this annual report, we characterized effects of amino acid residue substitutions on protease dimerization and determined interactions of mutant protease and various PDIs. When a single mutation was introduced into the N-, C- termini and active site of protease, P1A, Q2A, T4A, D25N, D30N and N98A allowed protease to undergo dimerization, which DRV effectively inhibited at 1 μM, suggesting that these amino acids are not significantly involved in the binding of DRV to the protease monomer subunit. A single mutation such as V32I, L33F, I54M and I84V that are known to be associated with HIV-1s DRV resistance in clinical settings and emerged in vitro selection experiments using mixture of HIV-1MDR also allowed protease to undergo dimerization and DRV effectively blocked dimerization. DRV also blocked dimerization of double or triple DRV-resistance associated mutants such as V32I/L33F, V32I/I84V, V32I/L33F/I84V, and V32I/L33F/I54M. Protease with an A28S mutation or 4 combinational mutations (V32I/L33F/I54M/I84V) underwent dimerization, which DRV and TPV failed to block, strongly suggesting that such mutations altered the conformation of the monomer subunit binding site of DRV and TPV. The present data show that the dual inhibitory activity of PDIs (inhibition of protease dimerization and the catalytic activity of mature protease) should render PDIs highly potent HIV-1 inhibitors and should also give new insights into the process and dynamics of HIV-1 protease dimerization. In the period of the report, we also examined the mechanism of the emergence of HIV-1 variants resistant to another PDI, tiprnavir (TPV), which is active against multi-PI-resistant HIV-1 (HIVMPIR) isolates. We have recently demonstrated that TPV blocks the dimerization of HIV-1 protease subunits. TPV-resistant HIV-1 (HIVTPVR) variants were generated by propagating a mixture of eleven clinical HIVMPIR (but TPV-susceptible) isolates (HIVMIX) in the presence of increasing concentrations of TPV. Protease dimerization inhibition was examined using an intermolecular fluorescence resonance energy transfer (FRET)-based HIV-1-expression assay. HIVMIX at passage 10 (HIVMIXP10) replicated in the presence of 15 μM TPV and the majority of its clones contained L33IPRO/I54VPRO/V82TPRO. Two clinical HIVMPIR variants, HIVB and HIVC, acquired the replicative ability at 15 μM by passages 10 and 15, respectively. HIVBP10 contained the three substitutions, while HIVCP15 lacked L33IPRO but contained L24MPRO/I54VPRO/V82TPRO. Other HIVMPIR variants, HIVG and HIVTM, ceased to replicate at 2 and 3 μM, respectively. HIVGP15 containied V82TPRO, but had failed to acquire L33IPRO/I54VPRO;HIVTM initially had I54VPRO and HIVTMP15 contained V82TPRO, lacking L24MPRO/L33IPRO. TPVs dimerization inhibition activity was lost in the presence of L24MPRO or L33IPRO. Neither of I54VPRO or V82TPRO affected TPVs dimerization inhibition;but, both were thought to be associated with TPVs loss to inhibit proteases catalytic activity. High levels of TPV resistance require I54VPRO/V82TPRO plus L24MPRO or L33IPRO. L24MPRO and L33IPRO are associated withTPVs loss of dimerization inhibition activity, while I54VPRO/V82TPRO TPVs loss of proteases catalysis inhibition activity. The data should have virologic and clinical relevance in the mechanism of TPVs anti-HIV-1 activity and the emergence of TPV resistance.

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