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. 2013 May;57(5):2036-46.
doi: 10.1128/AAC.02189-12. Epub 2013 Feb 12.

GRL-0519, a novel oxatricyclic ligand-containing nonpeptidic HIV-1 protease inhibitor (PI), potently suppresses replication of a wide spectrum of multi-PI-resistant HIV-1 variants in vitro

Masayuki Amano  1 , Yasushi Tojo, Pedro Miguel Salcedo-Gómez, Joseph Richard Campbell, Debananda Das, Manabu Aoki, Chun-Xiao Xu, Kalapala Venkateswara Rao, Arun K Ghosh, Hiroaki Mitsuya
Affiliations

GRL-0519, a novel oxatricyclic ligand-containing nonpeptidic HIV-1 protease inhibitor (PI), potently suppresses replication of a wide spectrum of multi-PI-resistant HIV-1 variants in vitro

Masayuki Amano et al. Antimicrob Agents Chemother. 2013 May.

Abstract

We report that GRL-0519, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI) containing tris-tetrahydrofuranylurethane (tris-THF) and a sulfonamide isostere, is highly potent against laboratory HIV-1 strains and primary clinical isolates (50% effective concentration [EC50], 0.0005 to 0.0007 μM) with minimal cytotoxicity (50% cytotoxic concentration [CC50], 44.6 μM). GRL-0519 blocked the infectivity and replication of HIV-1NL4-3 variants selected by up to a 5 μM concentration of ritonavir, lopinavir, or atazanavir (EC50, 0.0028 to 0.0033 μM). GRL-0519 was also potent against multi-PI-resistant clinical HIV-1 variants isolated from patients who no longer responded to existing antiviral regimens after long-term antiretroviral therapy, highly darunavir (DRV)-resistant variants, and HIV-2ROD. The development of resistance against GRL-0519 was substantially delayed compared to other PIs, including amprenavir (APV) and DRV. The effects of nonspecific binding of human serum proteins on GRL-0519's antiviral activity were insignificant. Our analysis of the crystal structures of GRL-0519 (3OK9) and DRV (2IEN) with protease suggested that the tris-THF moiety, compared to the bis-THF moiety present in DRV, has greater water-mediated polar interactions with key active-site residues of protease and that the tris-THF moiety and paramethoxy group effectively fill the S2 and S2' binding pockets, respectively, of the protease. The present data demonstrate that GRL-0519 has highly favorable features as a potential therapeutic agent for treating patients infected with wild-type and/or multi-PI-resistant variants and that the tris-THF moiety is critical for strong binding of GRL-0519 to the HIV protease substrate binding site and appears to be responsible for its favorable antiretroviral characteristics.

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Figures

Fig 1
Fig 1
Structures of GRL-0519, GRL-0529, amprenavir, and darunavir. M.W., molecular weight.
Fig 2
Fig 2
In vitro selection of PI-resistant HIV-1 variants. HIV-1NL4-3 was propagated in MT-4 cells in the presence of increasing concentrations of amprenavir (●), darunavir (△), GRL-0519 (▲), or GRL-0529 (○). Each passage of virus was conducted in a cell-free manner.
Fig 3
Fig 3
Amino acid sequences of the protease-encoding regions of HIV-1NL4-3 variants selected in the presence of GRL-0519. The amino acid sequences of protease, deduced from the nucleotide sequence of the protease-encoding region of each proviral DNA isolated at each indicated time, are shown. The amino acid sequence of the wild-type HIV-1NL4-3 protease is illustrated at the top as a reference.
Fig 4
Fig 4
Replication kinetics of HIV-1NL4-3 and HIV519RP37. MT-4 cells (3.2 × 105) were exposed to a HIV-1NL4-3 or HIV-1519RP37 preparation containing 10 ng/ml p24 in 6-well culture plates for 3 h, and the MT-4 cells were washed with fresh medium and divided into 4 fractions, each cultured with or without GRL-0519 (final concentration of MT-4 cells, 104/ml; drug concentrations, 0, 0.005, 0.01, and 0.015 μM). The amount of p24 in each culture flask was measured every 2 days for up to 7 days, once at each time point.
Fig 5
Fig 5
In vitro selection using a mixture of 8 multi-PI-resistant HIV-1MDR isolates. A mixture of 8 different multi-PI-resistant clinical HIV-1 isolates was propagated in MT-4 cells in the presence of increasing concentrations of amprenavir (●), darunavir (△), GRL-0519 (▲), or GRL-0529 (○). Each passage of virus was conducted in a cell-free manner. The amino acid substitutions identified in the protease-encoding regions of 2 different multi-PI-resistant clinical isolates compared to the consensus type B sequence cited from the Los Alamos database include L10I, I15V, E35D, N37E, K45R, I54V, L63P, A71V, V82T, L90M, I93L, and C95F in HIV-1MDR/A and L10R, N37D, M46I, I62V, L63P, A71V, G73S, V77IV82T, L90M, and I93L in HIV-1MDR/SS. The amino acid substitutions of the other 6 HIV-1MDR variants are given in Table 3, note a.
Fig 6
Fig 6
Hydrogen bond interactions of GRL-0519 and darunavir with protease. The interactions between protease–GRL-0519 (PDB ID, 3OK9) and protease-DRV (PDB ID, 2IEN) complexes as determined from their respective crystal structures were analyzed. (A and B) Similar interactions between protease–GRL-0519 (A) and protease-DRV (B) complexes. GRL-0519 has polar interactions with Asp-29, Asp-30, Gly-27, Asp-25, and Asp-25′. It has polar interactions with Ile-50 and Ile-50′ through a bridging water molecule. The protease-DRV complex also has these polar interactions. Both inhibitors have polar interactions with different backbone atoms of Asp-30′ in the S2′ site of the protease. GRL-0519 interacts with the backbone amide nitrogen, whereas DRV interacts with the carbonyl oxygen. (C and D) The interactions of tris-THF (C) and bis-THF (D) moieties with water molecules highlight differences in the polar interactions of the two inhibitors. The additional THF ring of GRL-0519 interacts with an extra water molecule and has four more polar interactions than DRV. The figures shown here were made with Maestro version 9.3 (Schrödinger, LLC, New York, NY, 2012).
Fig 7
Fig 7
van der Waals interactions of GRL-0519 and darunavir with protease. GRL-0519 is shown as sticks (green carbons), and the van der Waals surfaces of selected moieties of GRL-0519 and its complexed protease are shown in gray and blue, respectively. DRV is shown as sticks (gray carbons), and the van der Waals surfaces of selected moieties of DRV and its complexed protease are shown in yellow and plum, respectively. (A and B) van der Waals interactions between the benzomethoxy of GRL-0519 and Asp-29′ and Asp-30′ of protease (A) and interactions between the aniline of DRV and Asp-29′ and Asp-30′ of protease (B). The surface interactions suggest that the methoxy group of GRL-0519 makes stronger van der Waals interactions with Asp-29′ and Asp-30′ of protease than does DRV. (C and D) The molecules are rotated to show the van der Waals surface interactions of the tris-THF (C) and bis-THF (D) moieties with protease. The tris-THF group of GRL-0519 forms better van der Waals contacts at the S2 site of protease than the bis-THF of DRV. The figures were made with Maestro version 9.3 (Schrödinger, LLC, New York, NY, 2012).
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