A novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI), GRL-98065, is potent against multiple-PI-resistant human immunodeficiency virus in vitro

Abstract

We designed, synthesized, and identified GRL-98065, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing the structure-based designed privileged cyclic ether-derived nonpeptide P2 ligand, 3(R),3a(S),6a(R)-bis-tetrahydrofuranylurethane (bis-THF), and a sulfonamide isostere, which is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC(50)], 0.0002 to 0.0005 microM) with minimal cytotoxicity (50% cytotoxicity, 35.7 microM in CD4(+) MT-2 cells). GRL-98065 blocked the infectivity and replication of each of the HIV-1(NL4-3) variants exposed to and selected by up to a 5 microM concentration of saquinavir, indinavir, nelfinavir, or ritonavir and a 1 microM concentration of lopinavir or atazanavir (EC(50), 0.0015 to 0.0075 microM), although it was less active against HIV-1(NL4-3) selected by amprenavir (EC(50), 0.032 microM). GRL-98065 was also potent against multiple-PI-resistant clinical HIV-1 variants isolated from patients who had no response to existing antiviral regimens after having received a variety of antiviral agents, HIV-1 isolates of various subtypes, and HIV-2 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 important for its potency and wide-spectrum activity against multiple-PI-resistant HIV-1 variants. The present data demonstrate that the privileged nonpeptide P2 ligand, bis-THF, is critical for the binding of GRL-98065 to the HIV protease substrate binding site and that this scaffold can confer highly potent antiviral activity against a wide spectrum of HIV isolates.

FIG. 1.

Structures of GRL-98065, darunavir, and amprenavir.

FIG. 2.

Synthesis of GRL-98065.

FIG. 3.

In vitro selection of PI-resistant HIV-1 variants. HIV-1_NL4-3 was propagated in MT-4 cells in the presence of increasing concentrations of amprenavir (○), lopinavir (•), atazanavir (▵), or GRL-98065 (▴). Each passage of virus was done in a cell-free fashion.

FIG. 4.

Amino acid sequences of protease-encoding regions of HIV-1_NL4-3 variants selected in the presence of GRL-98065. The amino acid sequence of protease, deduced from the nucleotide sequence of the protease-encoding region of each proviral DNA isolated at each indicated time, is shown. The amino acid sequence of wild-type HIV-1_NL4-3 protease is illustrated at the top as a reference.

FIG. 5.

Amino acid sequences of Gag-encoding regions of HIV-1 variants selected in the presence of GRL-98065. The amino acid sequence of Gag, deduced from the nucleotide sequence of the Gag-encoding region of each proviral DNA isolated at each indicated time, is shown. The amino acid sequence of wild-type HIV-1_NL4-3 Gag is illustrated at the top as a reference.

FIG. 6.

Replication kinetics of GRL-98065-resistant HIV-1 variant and HIV-1_NL4-3. MT-4 cells (2.4 × 10⁵) were exposed to an HIV-1_GRL98065p40 or wild-type HIV-1_NL4-3 preparation containing 30 ng p24 in six-well culture plates for 3 h, and these MT-4 cells were divided into three fractions, each cultured with or without GRL-98065 (final concentration of MT-4 cells, 10⁴/ml; drug concentrations, 0, 0.01, and 0.1 μΜ). Amounts of p24 were measured every 2 days for up to 9 days.

FIG. 7.

Selected hydrogen bond interactions of GRL-98065 with wild-type HIV-1 protease. (A) The bis-THF group forms hydrogen bond interactions with backbone atoms of Asp29 and Asp30. There is a hydrogen bond with the backbone atom of Gly27. The hydroxyl group forms hydrogen bonds with the side chains of the catalytic aspartates. One oxygen of the benzodioxole group forms a hydrogen bond interaction with Asp30′, and the other oxygen of the benzodioxole group forms a water-mediated hydrogen bond interaction with Gly48′. (B) Hydrogen bond interactions between DRV and protease (PDB identifier, 1S6G) are shown. Most interactions between GRL-98065 and DRV are similar, except for interactions with Asp30′ and Gly48′. GRL-98065 interacts with the Asp30′ amide, while DRV interacts with the Asp30′ carbonyl oxygen. The benzodioxole oxygen of GRL-98065 has a water-mediated interaction with Gly48′ in the flap. This interaction appears to stabilize the binding site more for GRL-98065 and may be partly responsible for its greater antiviral potency than that of DRV.

FIG. 8.

Interactions between GRL-98065 and wild-type protease. van der Waals surfaces of GRL-98065 (green), Val82 (red), and Ile85 (magenta) are shown. There are strong van der Waals interactions of GRL-98065 with Val82 and Val82′. Note that Val82 was replaced with isoleucine as a primary resistance mutation during in vitro passage of HIV-1 in the presence of GRL-98065. However, Ile85 does not have van der Waals contact with the inhibitor, suggesting that I85V emerged as a secondary mutation during in vitro selection with the inhibitor.