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. 2015 Nov 18;90(5):2180-94.
doi: 10.1128/JVI.01829-15.

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

Manabu Aoki  1 , Hironori Hayashi  2 , Ravikiran S Yedidi  3 , Cuthbert D Martyr  4 , Yuki Takamatsu  5 , Hiromi Aoki-Ogata  6 , Teruya Nakamura  7 , Hirotomo Nakata  8 , Debananda Das  5 , Yuriko Yamagata  7 , Arun K Ghosh  4 , Hiroaki Mitsuya  9
Affiliations

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

Manabu Aoki et al. J Virol. .

Abstract

We identified three nonpeptidic HIV-1 protease inhibitors (PIs), GRL-015, -085, and -097, containing tetrahydropyrano-tetrahydrofuran (Tp-THF) with a C-5 hydroxyl. The three compounds were potent against a wild-type laboratory HIV-1 strain (HIV-1(WT)), with 50% effective concentrations (EC50s) of 3.0 to 49 nM, and exhibited minimal cytotoxicity, with 50% cytotoxic concentrations (CC50) for GRL-015, -085, and -097 of 80, >100, and >100 μM, respectively. All the three compounds potently inhibited the replication of highly PI-resistant HIV-1 variants selected with each of the currently available PIs and recombinant clinical HIV-1 isolates obtained from patients harboring multidrug-resistant HIV-1 variants (HIVMDR). Importantly, darunavir (DRV) was >1,000 times less active against a highly DRV-resistant HIV-1 variant (HIV-1DRV(R) P51); the three compounds remained active against HIV-1DRV(R) P51 with only a 6.8- to 68-fold reduction. Moreover, the emergence of HIV-1 variants resistant to the three compounds was considerably delayed compared to the case of DRV. In particular, HIV-1 variants resistant to GRL-085 and -097 did not emerge even when two different highly DRV-resistant HIV-1 variants were used as a starting population. In the structural analyses, Tp-THF of GRL-015, -085, and -097 showed strong hydrogen bond interactions with the backbone atoms of active-site amino acid residues (Asp29 and Asp30) of HIV-1 protease. A strong hydrogen bonding formation between the hydroxyl moiety of Tp-THF and a carbonyl oxygen atom of Gly48 was newly identified. The present findings indicate that the three compounds warrant further study as possible therapeutic agents for treating individuals harboring wild-type HIV and/or HIVMDR.

Importance: Darunavir (DRV) inhibits the replication of most existing multidrug-resistant HIV-1 strains and has a high genetic barrier. However, the emergence of highly DRV-resistant HIV-1 strains (HIVDRV(R) ) has recently been observed in vivo and in vitro. Here, we identified three novel HIV-1 protease inhibitors (PIs) containing a tetrahydropyrano-tetrahydrofuran (Tp-THF) moiety with a C-5 hydroxyl (GRL-015, -085, and -097) which potently suppress the replication of HIVDRV(R) . Moreover, the emergence of HIV-1 strains resistant to the three compounds was considerably delayed compared to the case of DRV. The C-5 hydroxyl formed a strong hydrogen bonding interaction with the carbonyl oxygen atom of Gly48 of protease as examined in the structural analyses. Interestingly, a compound with Tp-THF lacking the hydroxyl moiety substantially decreased activity against HIVDRV(R) . The three novel compounds should be further developed as potential drugs for treating individuals harboring wild-type and multi-PI-resistant HIV variants as well as HIVDRV(R) .

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Figures

FIG 1
FIG 1
Structures of GRL-015, -085, and -097. The structures of compounds used in this study, containing Tp-THF (A) or bis-THF (B) at the P2 site, are shown. The structure of GRL-012 (C) is also shown.
FIG 2
FIG 2
X-ray crystal structures of HIV-1 protease complexed with GRL-0476, -015, -085, or -097. The profiles of polar contacts made by GRL-0476 (A), -015 (B), -085 (C), and -097 (D) with WTPRD25N are shown. In each structure, the inhibitor is bound in two alternate orientations with average occupancies of 0.49 and 0.51. In order to analyze the hydrogen bonds (H bonds), hydrogen atoms were added and their orientations were optimized sampling the crystallographic water molecules through the protein preparation wizard in Maestro (v9.0 Schrodinger LLC.). In each panel, the carbon atoms for the protease inhibitors are in white, while the carbon atoms of WTPRD25N are in green. Nitrogen, oxygen, and sulfur atoms are in blue, red, and yellow, respectively. Crystallographic water molecules are shown as red spheres.
FIG 3
FIG 3
The P1 methoxybenzene moiety of GRL-085 shows improved nonbonded interactions. The P1 phenyl moiety of GRL-015 (A) and P1 methoxybenzene moiety of GRL-085 (B) are shown (thick-stick representation). In both panels, the carbon atoms of the PIs are in light blue, hydrogens are in white, and oxygen is in red. The carbon atoms of the corresponding protease amino acid residues (thin-stick representation) are in green, nitrogen atoms are in blue, oxygen atoms are in red, and hydrogen atoms are in white.
FIG 4
FIG 4
Inhibition of HIV-1 protease dimerization. COS7 cells were exposed to each of the agents (GRL-015, -085, -097, and -012 and DRV) at various concentrations (0.01, 0.1, 1, and 10 μM) and subsequently cotransfected with plasmids encoding full-length molecular infectious HIV-1 (HIVNL4-3) clones, producing CFP- or YFP-tagged protease. After 72 h, cultured cells were examined in the FRET-based HIV-1 expression assay, and the CFPA/B ratios (y axis) were determined. The mean values of the ratios obtained are shown as horizontal bars. A CFPA/B ratio greater than 1 signifies that protease dimerization occurred, whereas a ratio less than 1 signifies the disruption of protease dimerization. All the experiments were conducted in a blinded fashion. Statistical differences were as follows: for the CFPA/B ratios in the absence of drug (CFPA/BNo Drug) versus the CFPA/B ratios in the presence of 0.01 μM DRV (CFPA/B0.01 DRV), P = 0.5846; for CFPA/BNo Drug versus CFPA/B0.1 DRV, P = 0.0002; for CFPA/BNo Drug versus CFPA/B0.1 GRL-015, P = 0.1784; for CFPA/BNo Drug versus CFPA/B1 GRL-015, P = 0.0021; for CFPA/BNo Drug versus CFPA/B0.1 GRL-085, P = 0.2006; for CFPA/BNo Drug versus CFPA/B1 GRL-085, P = 0.0033; for CFPA/BNo Drug versus CFPA/B0.1 GRL-097, P = 0.4482; for CFPA/BNo Drug versus CFPA/B1 GRL-097, P = 0.0006; for CFPA/BNo Drug versus CFPA/B1 GRL-012, P = 0.1189; and for CFPA/BNo Drug versus CFPA/B10 GRL-012, P = 0.0007.
FIG 5
FIG 5
In vitro selection of HIV-1 variants against GRL-015, -085, and -097. HIV-1NL4-3 (A), a mixture of 11 multi-PI-resistant HIV-1 isolates (HIV-111MIX) (B), a DRV-resistant HIV-1 variant obtained from in vitro passage 30 with DRV (HIV-1DRVRP30) (C), and an infectious molecular HIV-1 clone derived from heavily treated HIV-1-infected patient (rCLHIV-1T48) (D) were propagated in the presence of increasing concentrations of each agent in MT-4 cells. The selection was carried out in a cell-free manner by 50 weeks escalating from the EC50 of the compounds for each virus.
FIG 6
FIG 6
Amino acid sequences of the protease-encoding regions of HIV-1NL4-3, HIV-111MIX, HIV-1DRVRP30, and rCLHIV-1T48 in vitro, selected with various agents. Shown are the amino acid sequences deduced from the nucleotide sequences of the protease-encoding region of proviral DNA isolated from HIV-1NL4-3 at week 20 with APV and at week 50 with DRV or GRL-015, -085, or -097 (A); HIV-111MIX at week 0 and week 50 with DRV or GRL-015, -085, -097, or -012 (B); HIV-1DRVRP30 at week 0 and week 35 with GRL-012 and at week 50 with GRL-085 or -097 (C); and rCLHIV-1T48 at week 0 and week 50 with GRL-085, -097, or -012 (D). The consensus sequence of HIV-1NL4-3 is at the top as a reference. Identity with sequence at individual amino acid positions is indicated by dots. The fractions on the right are the number of viruses which each clone is presumed to have originated from over the number of clones examined.
FIG 6
FIG 6
Amino acid sequences of the protease-encoding regions of HIV-1NL4-3, HIV-111MIX, HIV-1DRVRP30, and rCLHIV-1T48 in vitro, selected with various agents. Shown are the amino acid sequences deduced from the nucleotide sequences of the protease-encoding region of proviral DNA isolated from HIV-1NL4-3 at week 20 with APV and at week 50 with DRV or GRL-015, -085, or -097 (A); HIV-111MIX at week 0 and week 50 with DRV or GRL-015, -085, -097, or -012 (B); HIV-1DRVRP30 at week 0 and week 35 with GRL-012 and at week 50 with GRL-085 or -097 (C); and rCLHIV-1T48 at week 0 and week 50 with GRL-085, -097, or -012 (D). The consensus sequence of HIV-1NL4-3 is at the top as a reference. Identity with sequence at individual amino acid positions is indicated by dots. The fractions on the right are the number of viruses which each clone is presumed to have originated from over the number of clones examined.
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