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. 2014 Jul;58(7):3679-88.
doi: 10.1128/AAC.00107-14. Epub 2014 Apr 21.

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

Ravikiran S Yedidi  1 , Harisha Garimella  1 , Manabu Aoki  2 , Hiromi Aoki-Ogata  3 , Darshan V Desai  1 , Simon B Chang  1 , David A Davis  4 , W Sean Fyvie  5 , Joshua D Kaufman  6 , David W Smith  7 , Debananda Das  1 , Paul T Wingfield  6 , Kenji Maeda  1 , Arun K Ghosh  5 , Hiroaki Mitsuya  8
Affiliations

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

Ravikiran S Yedidi et al. Antimicrob Agents Chemother. 2014 Jul.

Abstract

In the present study, GRL008, a novel nonpeptidic human immunodeficiency virus type 1 (HIV-1) protease inhibitor (PI), and darunavir (DRV), both of which contain a P2-bis-tetrahydrofuranyl urethane (bis-THF) moiety, were found to exert potent antiviral activity (50% effective concentrations [EC50s], 0.029 and 0.002 μM, respectively) against a multidrug-resistant clinical isolate of HIV-1 (HIVA02) compared to ritonavir (RTV; EC50, >1.0 μM) and tipranavir (TPV; EC50, 0.364 μM). Additionally, GRL008 showed potent antiviral activity against an HIV-1 variant selected in the presence of DRV over 20 passages (HIVDRV(R)P20), with a 2.6-fold increase in its EC50 (0.097 μM) compared to its corresponding EC50 (0.038 μM) against wild-type HIV-1NL4-3 (HIVWT). Based on X-ray crystallographic analysis, both GRL008 and DRV showed strong hydrogen bonds (H-bonds) with the backbone-amide nitrogen/carbonyl oxygen atoms of conserved active-site amino acids G27, D29, D30, and D30' of HIVA02 protease (PRA02) and wild-type PR in their corresponding crystal structures, while TPV lacked H-bonds with G27 and D30' due to an absence of polar groups. The P2' thiazolyl moiety of RTV showed two conformations in the crystal structure of the PRA02-RTV complex, one of which showed loss of contacts in the S2' binding pocket of PRA02, supporting RTV's compromised antiviral activity (EC50, >1 μM). Thus, the conserved H-bonding network of P2-bis-THF-containing GRL008 with the backbone of G27, D29, D30, and D30' most likely contributes to its persistently greater antiviral activity against HIVWT, HIVA02, and HIVDRV(R)P20.

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Figures

FIG 1
FIG 1
Structures of protease inhibitors used in this study. GRL008 is a novel experimental HIV-1 PI, while DRV, tipranavir, lopinavir, saquinavir, indinavir, and ritonavir are FDA-approved PIs. Both GRL008 and DRV contain bis-THF as the P2 moiety. GRL008 and DRV contain benzene carboxamide and aniline, respectively, as the P2′ moieties.
FIG 2
FIG 2
Hydrogen bonding profiles of GRL008, DRV, and TPV. H-bonds formed by GRL008, DRV, and TPV with PRWT (a, b, and c, respectively) and with PRA02 (d, e, and f, respectively) in their corresponding crystal structures are shown as yellow dashed lines. In all panels, the carbon atoms of the inhibitors are shown as white thick sticks, while the corresponding carbon atoms of PRWT and PRA02 residues are shown as green and cyan thin sticks, respectively. Nitrogen, oxygen, sulfur, and fluorine atoms are shown as blue, red, yellow, and light cyan sticks, respectively. Crystallographic water molecules are shown as red spheres. All H-bonds were calculated as the distances between two heavy atoms, with a maximum distance cutoff of 3.0 Å and minimum angle cutoff for donor of 90° and for acceptor of 60°. The P2 bis-THF moiety of GRL008 and DRV shows three conserved H-bonds with the backbone amide nitrogen atoms of D29 and D30 in both PRWT (a and b) and PRA02 (d and e). While TPV shows similar three H-bonds with the backbone amide nitrogen atoms of D29 and D30 in PRWT (c), the sulfonyl oxygen of TPV shows two H-bonds, one each with the backbone amide nitrogen atoms of D29 and D30 and one H-bond with the side chain δ-oxygen atom of D30 from PRA02 (f). Additionally, the P2′ moieties of GRL008 and DRV show one conserved H-bond, each with the backbone of D30′ (a, b, d, and e). Both GRL008 and DRV have a direct H-bond with the backbone carbonyl oxygen atom of G27 (a, b, d, and e). No H-bonds with D30′ or G27 were seen with TPV; instead, three direct H-bonds, one each with the backbone carbonyl oxygen atom of G48 and the backbone amide nitrogen atoms of I50 and I50′, were seen with TPV (c and f). In the cases of GRL008 and DRV, bridging H-bonds were seen with the backbone amide nitrogen atoms of I50 and I50′ via a conserved water molecule (a, b, d, and e). While the P2′ benzene carboxamide moiety of GRL008 showed a conserved bridging H-bond with G48′ from both PRWT and PRA02 via a water molecule (a and d, respectively), such conserved bridging H-bonds were not seen for the P2′ aniline moiety of DRV with G48′ of PRA02 (e). Overall, no significant changes in the H-bonding profiles were seen for GRL008, DRV, and TPV against PRA02 compared to their profiles against PRWT in their respective crystal structures.
FIG 3
FIG 3
Hydrophobic interactions of GRL008 and DRV. (a and b) Profiles of hydrophobic interactions for GRL008 (a) and DRV (b) in the active site of PRA02 and PRWT. The protease binding pockets (S2, S1, S1′, and S2′) are shown as arcs and are labeled. The corresponding protease amino acids that form the hydrophobic contacts with either GRL008 or DRV are listed for each binding pocket accordingly. Residues shown in green are from PRWT structures (PDB IDs 4I8Z and 4HLA for GRL008 and DRV, respectively), while the residues in red are from PRA02. In both panels, the amino acid residues are labeled 1 to 99 for monomer 1 and 1′ to 99′ for monomer 2. Both GRL008 and DRV showed slightly altered but persistent contacts in all the binding pockets that were comparable to their corresponding wild-type profiles, thus supporting their higher antiviral activities.
FIG 4
FIG 4
Hydrogen bonds and hydrophobic contacts for RTV in the active site of PRA02. (a and b) H-bonds made by RTV-1 (a) and RTV-2 (b) (alternate conformations of the P2′ thiazolyl moiety of RTV, highlighted by red circles) in the active site of PRA02. In both panels, the carbon atoms of RTV are shown as white thick sticks, while the carbon atoms of corresponding amino acid residues from PRA02 are shown as green thin sticks. Nitrogen, oxygen, and sulfur atoms are shown as blue, red, and yellow sticks, respectively. Crystallographic water molecules are shown as red spheres, and the H-bonds are shown as yellow dashed lines. While RTV-1 (a) showed a profile comparable to its corresponding profile with PRWT (PDB ID 1HXW), RTV-2 (b) showed an altered binding orientation for the P2′ thiazolyl group, with an average root mean square deviation of 3 Å, resulting in loss of a critical H-bond with the backbone carbonyl oxygen atom of D30′. The P3 isopropyl group also showed a slightly altered binding orientation but was not biologically significant. (c) Profile of hydrophobic contacts made by RTV in the active sites of PRWT and PRA02. The binding pockets are represented as arcs and are labeled accordingly. The corresponding amino acids from PRWT and PRA02 that are involved in hydrophobic interactions with RTV are shown in green and red, respectively. For PRA02, only RTV-2 is shown here because the binding profile of RTV-1 in the active site of PRA02 is similar to that of its corresponding profile in the active site of PRWT. RTV-2 showed significant loss of contacts in the S2 and S2′ binding pockets of PRA02, thus supporting its weaker antiviral activity. Superposition of RTV-1 and RTV-2 is shown in Fig. S2 of the supplemental material.
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