We attempted to select HIV-1 variants highly resistant to DRV, by propagating a wild-type laboratory HIV-1 strain, HIV-1NL4-3, in MT-4 cells in the presence of increasing concentrations of DRV. We simultaneously and independently selected HIV-1 variants in the presence of RTV, APV, LPV, or ATV. HIV-1 variants that were capable of replicating in the presence of 5 microM of RTV, APV, LPV, and ATV relatively quickly emerged, while HIV-1 exposed to DRV continued to replicate poorly and failed to further replicate in the presence of 0.1 microM DRV, indicating that the emergence of DRV-resistant HIV-1 variant was substantially delayed compared to other PIs examined and HIV-1 failed to acquire significant resistance to DRV. We subsequently employed a mixture of 8 HIV-1 clinical isolates resistant to multiple PIs, expecting that homologous recombination from one isolate to another among the mixed clinical isolates would take 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 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. The virus continuously replicated in the presence of increasing concentrations of DRV eventually even in the presence of 5 microM (passage 51, HIV8MIXP51). The protease-encoding region of the proviral DNA isolated from infected MT-4 cells was cloned and sequenced at various passages. HIV8MIXP51 contained substitutions: L10I, I15V, K20R, L24I, V32I, L33F, M36I, M46L, F53S, I54V, I62V, L63P, K70Q, V82I, I84V, and L89M.When we examined the susceptibility of HIV-18MIX described above to a variety of FDA-approved PIs including DRV in MT-4 cells, HIV-18MIX at passage 51 (HIV-18MIXP51) was found highly resistant to DRV [(EC50 value >333-fold greater than that against wild-type clinical strain (HIV-1ERS104pre)]. HIV-18MIXP51 was highly resistant to APV, IDV, NFV, RTV, LPV, and ATV (all IC50s >1 microM) and also had significant resistance against SQV (33-fold increases in IC50) and TPV (18-fold increases in IC50). We also determined replication kinetics of HIV-1NL4-3 along with HIV-18MIXP51, which turned out to be capable of replicating in the presence of 1.0 microM DRV. When HIV-18MIXP51 was propagated in MT-4 cells in the presence or absence of 0.1 or 1 microM DRV, there was no discernable difference observed in the replication kinetics of HIV-18MIXP51 compared to that of HIV-1NL4-3 in the absence of DRV.Since HIV8MIXP51 was highly resistant to DRV as described above, we examined whether DRV still blocked the dimerization of protease (PR) of HIV8MIXP51 containing 14 amino acid substitutions, using newly generated pHIV-18MIXP51/CFP and pHIV-18MIXP51/YFP in the FRET-based HIV-1 expression system. As shown in Figure 5, DRV failed to block the dimerization of the PR of HIV8MIXP51 at 0.1 microM. These data suggested that all or subsets of the 14 amino acid substitutions present in the PR of HIV8MIXP51 were associated with the compromised PR dimerization seen in HIV8MIXP51 and its acquisition of DRV resistance. TPV, another PI that inhibits the enzymatic activity as well as dimerization of HIV-1 protease, exerts reasonable activity against multi-PI-resistant HIV-1 variants. When a mixture of eleven multi-PI-resistant (but all TPV-sensitive) clinical HIV-1 isolates (HIV11MIX), including HIVB and HIVC, was selected against TPV, HIV11MIX rapidly (HIV11MIXP10) acquired high-level TPV resistance and replicated in the presence of high concentrations of TPV by passage 10 (HIV11MIXP10). HIV11MIXP10 contained various AA substitutions including I54V and V82T. The 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 amino acid 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 (Aoki &Mitsuya, manuscript under revision). Importantly, the data that TPV's dimerization inhibition is compromised mostly with a single AA substitution should 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).

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC011105-05
Application #
8552981
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
5
Fiscal Year
2012
Total Cost
$215,965
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
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
Aoki, Manabu; Hayashi, Hironori; Yedidi, Ravikiran S et al. (2016) 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; 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
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 25:4903-9
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; 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
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
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
Yedidi, Ravikiran S; Garimella, Harisha; Aoki, Manabu et al. (2014) A conserved hydrogen-bonding network of P2 bis-tetrahydrofuran-containing HIV-1 protease inhibitors (PIs) with a protease active-site amino acid backbone aids in their activity against PI-resistant HIV. Antimicrob Agents Chemother 58:3679-88
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

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