Atomic resolution crystal structures of HIV‐1 protease and mutants V82A and I84V with saquinavir

Abstract

Saquinavir (SQV), the first antiviral HIV-1 protease (PR) inhibitor approved for AIDS therapy, has been studied in complexes with PR and the variants PR(I) (84V) and PR(V) (82A) containing the single mutations I84V and V82A that provide resistance to all the clinical inhibitors. Atomic resolution crystal structures (0.97-1.25 A) of the SQV complexes were analyzed in comparison to the protease complexes with darunavir, a new drug that targets resistant HIV, in order to understand the molecular basis of drug resistance. PR(I) (84V) and PR(V) (82A) complexes were obtained in both the space groups P2(1)2(1)2 and P2(1)2(1)2(1), which provided experimental limits for the conformational flexibility. The SQV interactions with PR were very similar in the mutant complexes, consistent with the similar inhibition constants. The mutation from bigger to smaller amino acids allows more space to accommodate the large group at P1' of SQV, unlike the reduced interactions observed in darunavir complexes. The residues 79-82 have adjusted to accommodate the large hydrophobic groups of SQV, suggesting that these residues are intrinsically flexible and their conformation depends more on the nature of the inhibitor than on the mutations in this region. This analysis will assist with development of more effective antiviral inhibitors.

Carbamylation of N-Terminal Proline

“…Other crystal structures of PR/SQV, including variants of PR, so far reported are orthorhombic and cubic forms. 7,9,[11][12][13][14] This new PR/SQV crystal structure is isomorphic with the structure reported for PR in complex with indinavir (IDV) (pdb code = 2AVV). 15 The electron density maps of PR/ SQV, obtained at 1.39 Å resolution, show the catalytic sites of both crystallographically independent dimers, each fully occupied by a SQV molecule statistically distributed in two different orientations, related by a pseudo-2-fold symmetry (Figure 1), with occupancies of 38-62% (AB dimer) and 25-75% (CD dimer), respectively.…”

“…On the contrary, several X-ray crystal structures of PR complexed with more asymmetric FDAapproved inhibitors, like darunavir, nelfinavir, and saquinavir (SQV), have the catalytic site occupied by the inhibitor oriented in two almost equivalent positions related by a pseudo-2-fold symmetry. [7][8][9][10][11][12] To investigate this order/disorder phenomenon and its possible relationship with crystal packing, we have undertaken a systematic search for new crystal forms of wild-type PR in complex with SQV. Single crystals of the PR/SQV complex were obtained by a vapor diffusion technique and analyzed by X-ray diffraction.…”

Critical differences in HIV‐1 and HIV‐2 protease specificity for clinical inhibitors

“…SQV-A was bound within subsites S3 to S2 0 of PR 1M and showed almost identical hydrogen bond interactions to those described for PR 1 -SQV. 26 The minor exception was that SQV had a shorter hydrogen bond to the main chain carbonyl oxygen of Gly27 in PR 1M with a distance of 3.2 Å compared to the weaker interaction with longer separation of 3.6 Å for PR 1 . A similar shorter interaction was described in the SQV complex with another mutant PR L76V, 27 however, the interaction in PR 1M may be influenced by the aromatic-aromatic interactions of the adjacent P1 and P3 groups of SQV-A with the quinoline rings of SQV-B [ Fig.…”

“…The following structures were chosen for the comparison: PR 1 -DRV (2IEN at 1.30 Å resolution), 24 PR 1 -APV (3NU3 at 1.02 Å resolution), 18 PR 1 -SQV (2NMY at 1.10 Å resolution), 26 and PR 2 -DRV (3EBZ at 1.20 Å resolution). 12 The PR 1M and PR 1 dimers are almost identical with small RMSDs of $0.2 Å for the pairs of structures in the same space group and unit cell, whereas larger differences of 0.7-0.8 Å were apparent between structures with nonisomorphous unit cells.…”

Insight into the structural similarity between HIV protease and secreted aspartic protease-2 and binding mode analysis of HIV-Candida albicans inhibitors

“…Also, a double hydrogen-bond system is displayed between inhibitors and a water molecule, which bridges the inhibitor carbonyl groups and Ile50/Ile50′ amide protons. Specifically, in saquinavir/HIV-1 protease complex, the hydroxyl function of hydroxyethylene isostere establishes hydrogen-bonds with side chain carboxylate oxygen atoms of both catalytic Asp25/Asp25′, and two water molecules establish three more hydrogenbonds with saquinavir and the enzyme 34 . The interaction between the inhibitor and flap residues is mediated by a water molecule interacting with the carbonyl oxygen of Gly27.…”