Effect of Flap Mutations on Structure of HIV-1 Protease and Inhibition by Saquinavir and Darunavir

doi:10.1016/j.jmb.2008.05.062

Journal of Molecular Biology

Volume 381, Issue 1, 1 August 2008, Pages 102-115

https://doi.org/10.1016/j.jmb.2008.05.062 Get rights and content

Abstract

HIV-1 (human immunodeficiency virus type 1) protease (PR) and its mutants are important antiviral drug targets. The PR flap region is critical for binding substrates or inhibitors and catalytic activity. Hence, mutations of flap residues frequently contribute to reduced susceptibility to PR inhibitors in drug-resistant HIV. Structural and kinetic analyses were used to investigate the role of flap residues Gly48, Ile50, and Ile54 in the development of drug resistance. The crystal structures of flap mutants PR_I50V (PR with I50V mutation), PR_I54V (PR with I54V mutation), and PR_I54M (PR with I54M mutation) complexed with saquinavir (SQV) as well as PR_G48V (PR with G48V mutation), PR_I54V, and PR_I54M complexed with darunavir (DRV) were determined at resolutions of 1.05–1.40 Å. The PR mutants showed changes in flap conformation, interactions with adjacent residues, inhibitor binding, and the conformation of the 80s loop relative to the wild-type PR. The PR contacts with DRV were closer in PR_G48V–DRV than in the wild-type PR–DRV, whereas they were longer in PR_I54M–DRV. The relative inhibition of PR_I54V and that of PR_I54M were similar for SQV and DRV. PR_G48V was about twofold less susceptible to SQV than to DRV, whereas the opposite was observed for PR_I50V. The observed inhibition was in agreement with the association of G48V and I50V with clinical resistance to SQV and DRV, respectively. This analysis of structural and kinetic effects of the mutants will assist in the development of more effective inhibitors for drug-resistant HIV.

Introduction

HIV-1 (human immunodeficiency virus type 1) protease (PR) is an effective drug target for antiviral inhibitors. Each subunit of the active PR homodimer has a glycine-rich region, including residues Lys45-Met-Ile-Gly-Gly-Ile-Gly-Gly-Phe-Ile-Lys55, termed the “flap,” which folds into two antiparallel β strands. The flexible flaps participate in the binding of a substrate or an inhibitor in the active site cavity of PR.1, 2, 3 The importance of residues in the flap for PR activity has been characterized through large-scale mutagenesis.⁴ The residues Met46, Phe53, and Lys55 are the most tolerant of substitutions; Ile47, Ile50, Ile54, and Val56 tolerate a few conservative substitutions only; and Gly48, Gly49, Gly51, and Gly52 are the most sensitive to mutation. Therefore, mutations in the flap residues may alter the enzyme activity, conformation, and flexibility of the flap. Mutations in flap residues 46, 47, 48, 50, 53, and 54 are frequently observed in drug-resistant mutants of HIV and show various levels of reduced drug susceptibility to different PR inhibitors (PIs).5, 6 Altered flap conformations have been reported for PR and its mutants in the unliganded form7, 8, 9 or with bound peptide.¹⁰ Previously, we analyzed the structures, activities, inhibition, and stability of PR variants with the single substitutions of flap residues M46L, G48V, I50V, and F53L.8, 11, 12, 13, 14, 15 These studies used PR from HIV-1 subtype B, which forms ∼ 10% of global infections with HIV-1 group M as compared with ∼ 50% for subtype C.¹⁶ Recently, the PRs from subtypes C and F were analyzed.17, 18 These subtypes contain 8–10 polymorphic substitutions in the sequence outside of the flaps and inhibitor binding site, which can influence catalytic activity and inhibition. The PR structures and interactions with inhibitors were very similar to those of the subtype B PR, with larger structural variations in surface residues 34–42 for the subtype F PR and surface residues 65–69 for the subtype C PR.

Saquinavir (SQV) was the first PI to be approved by the U.S. Food and Drug Administration in 1995, and it is still widely used in AIDS therapy, along with nine other inhibitors. The latest PI, darunavir (DRV, previously known as TMC114), a chemical derivative of the PI amprenavir, was approved in 2006 for salvage therapy and is extremely potent against the wild-type PR and most drug-resistant mutants in vitro and in vivo.11, 19, 20, 21 DRV boosted with ritonavir is recommended for treatment-experienced patients who respond poorly to other PIs. SQV was designed to target the wild-type PR, and its chemical structure contains a number of peptidic main chain groups mimicking a natural substrate of PR as shown in Fig. 1a.²² In contrast, DRV was designed to be less peptidic while introducing more hydrogen bond interactions with the main chain atoms of PR in order to maintain its effectiveness on PR variants.20, 23

In this study, PR variants with the individual flap mutations G48V, I50V, I54V, and I54M were analyzed to gain insight into their role in the development of drug resistance. G48V is one of the primary drug-resistant mutations selected during treatment with SQV.24, 25 I50V arises in treatment with amprenavir and confers resistance to DRV.⁵ Mutations of I54M and I54V are commonly observed during therapy with multiple PIs.5, 26, 27, 28 Several mutations of Ile54 are present in isolates with reduced susceptibility to SQV. Mutations I54M and I54L are frequent in clinical isolates resistant to DRV.²⁹ Moreover, Met was the most frequently identified substitution of residue 54 after treatment with amprenavir, which is chemically related to DRV.²⁸ Residue 50 lies at the tip of the PR flap, whereas residues 48 and 54 are located on opposite β strands of the flap (Fig. 1b). Previously, the crystal structure of the double-mutant G48V/L90M with SQV was analyzed,³⁰ and we reported the structure of PR_I50V (PR with I50V mutation) with DRV.¹¹ Here, the crystal structures of flap mutants PR_G48V (PR with G48V mutation), PR_I50V, PR_I54V (PR with I54V mutation), and PR_I54M (PR with I54M mutation) were solved in complexes with SQV and DRV. Comparison of the mutant and wild-type structures revealed changes in the flap conformation, interactions between flap residues from the two PR subunits, inhibitor binding, and conformation of residues 78–82 (the 80s loop). The kinetic data are discussed in relation to the structural changes. This analysis confirmed the important roles of residues in the flaps and enhanced our understanding of the drug-resistant mechanisms used by the flap mutants.

Section snippets

Kinetics

The wild-type HIV-1 PR in these studies contains mutations Q7K, L33I, and L63I to diminish autoproteolysis as well as C67A and C95A to prevent cysteine–thiol oxidation, and it showed kinetic parameters, stability, and dimer dissociation almost identical with those in the unmutated wild-type PR.³¹ Kinetic parameters were measured for the resistant mutants and the wild-type PR using the fluorescence substrate based on the p2-NC cleavage site of HIV-1 (Table 1). The mutants PR_G48V, PR_I50V, and PR

Preparation of HIV-1 PR mutants

The optimized HIV-1 PR clone with mutations Q7K, L33I, and L63I to diminish the autoproteolysis of the PR as well as mutations C67A and C95A to prevent cysteine–thiol oxidation was used as the initial template for adding drug-resistant mutations.³¹ This optimized PR had kinetic parameters and stability almost identical with those in the mature PR (GenBank accession code HIVHXB2CG).³¹ Plasmid DNA (pET11a, Novagen) encoding PR was utilized to construct mutant PR with the use of a QuickChange

Acknowledgements

The research was supported in part by the Georgia State University Molecular Basis of Disease Program, the Georgia Research Alliance, the Georgia Cancer Coalition, and the National Institutes of Health through grants GM062920 and GM053386. We thank the staff at the SER-CAT beamline at the Advanced Photon Source, Argonne National Laboratory, for providing assistance during X-ray data collection. Use of the Advanced Photon Source was supported by the Office of Basic Energy Sciences of the U.S.

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Drug resistance is a serious problem for controlling the HIV/AIDS pandemic. Current antiviral drugs show several orders of magnitude worse inhibition of highly resistant clinical variant PRS17 of HIV-1 protease compared with wild-type protease. We have analyzed the effects of a common resistance mutation G48V in the flexible flaps of the protease by assessing the revertant PRS17_V48G for changes in enzyme kinetics, inhibition, structure, and dynamics. Both PRS17 and the revertant showed about 10-fold poorer catalytic efficiency than wild-type enzyme (0.55 and 0.39 μM⁻¹min⁻¹ compared to 6.3 μM⁻¹min⁻¹). Clinical inhibitors, amprenavir and darunavir, showed 2-fold and 8-fold better inhibition, respectively, of the revertant than of PRS17, although the inhibition constants for PRS17_V48G were still 25 to 1,200-fold worse than for wild-type protease. Crystal structures of inhibitor-free revertant and amprenavir complexes with revertant and PRS17 were solved at 1.3–1.5 Å resolution. The amprenavir complexes of PRS17_V48G and PRS17 showed no significant differences in the interactions with inhibitor, although changes were observed in the conformation of Phe53 and the interactions of the flaps. The inhibitor-free structure of the revertant showed flaps in an open conformation, however, the flap tips do not have the unusual curled conformation seen in inhibitor-free PRS17. Molecular dynamics simulations were run for 1 μs on the two inhibitor-free mutants and wild-type protease. PRS17 exhibited higher conformational fluctuations than the revertant, while the wild-type protease adopted the closed conformation and showed the least variation. The second half of the simulations captured the transition of the flaps of PRS17 from a closed to a semi-open state, whereas the flaps of PRS17_V48G tucked into the active site and the wild-type protease retained the closed conformation. These results suggest that mutation G48V contributes to drug resistance by altering the conformational dynamics of the flaps.
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It has been shown according to PR-darunavir cocrystallization data that one molecule of darunavir can be captured not only in the active site of PR, but also in FLAP region [7] or on the hydrophobic interface surface. In that study [7] darunavir showed dual inhibition potency via a competitive–noncompetitive mechanism. These properties may determine extremely low KI for the majority of PR variants and the outstanding ability of darunavir to block PR activity almost completely.
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The major structural proteins and enzymes essential for the replication of retroviruses, including the human immunodeficiency virus (HIV), are high–molecular weight polyproteins. The polyproteins are cleaved by a viral protease (PR) to give the mature structural proteins and enzymes, including the viral PR itself. The HIV-1 encodes a PR that is essential for the production of the infectious virus. Without effective HIV PR, HIV virions remain uninfectious. HIV-1 aspartyl PR consists of two identical 99–amino acid subunits. Each monomer unit has a flap, which is an extended β-sheet that plays an important role in substrate binding by acting as a part of substrate-binding site. The active site is situated within the core and consists of three residues Asp-Thr-Gly from each of the monomers, and lies beneath the flaps or the two identical hairpin loops (residue 45–55 in each monomer). In the free form of the PR, these flaps are very flexible and adopt open and closed conformations. The substrate binds to the enzyme in extended conformation and its interaction with different amino acids determines the specificity of the enzyme. The HIV-1 PR is essential for the replication of the infective virus, and is therefore an attractive target for the design of specific inhibitors or potential antiviral therapeutics. PR inhibitors have successfully been used as a therapy for HIV-infected individuals to reduce their viral loads and slow down the progress of Acquired Immune Deficiency Syndrome. This chapter presents a detailed account of the structure and function of HIV-1 PR and the development of its inhibitors.
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¹: Present addresses: F. Liu, Department of Structural Biology, Stanford University School of Medicine, Stanford, CA 94305, USA. A.Y. Kovalevsky, Bioscience Division, MS M888, Los Alamos National Laboratory, Los Alamos, NM 87545, USA.

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Journal of Molecular Biology

Effect of Flap Mutations on Structure of HIV-1 Protease and Inhibition by Saquinavir and Darunavir

Abstract

Introduction

Section snippets

Kinetics

Preparation of HIV-1 PR mutants

Acknowledgements

FEBS Lett.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

J. Mol. Biol.

Bioorg. Med. Chem.

J. Clin. Virol.

J. Mol. Biol.

Methods Enzymol.

Methods Enzymol.

J. Mol. Graphics Modell.

Structure of complex of synthetic HIV-1 protease with a substrate-based inhibitor at 2.3 Å resolution

Science

Flap opening and dimer-interface flexibility in the free and inhibitor-bound HIV protease

Structure

Sequence requirements of the HIV-1 protease flap region determined by saturation mutagenesis and kinetic analysis of flap mutants

Proc. Natl Acad. Sci. USA

Genotypic testing for human immunodeficiency virus type 1 drug resistance

Clin. Microbiol. Rev.

HIV-1 protease and reverse transcriptase mutations for drug resistance surveillance

AIDS

Conformational flexibility in the flap domains of ligand-free HIV protease

Acta Crystallogr., Sect. D: Biol. Crystallogr.

Crystal structures of a multidrug-resistant human immunodeficiency virus type 1 protease reveal an expanded active-site cavity

J. Virol.

Mechanism of substrate recognition by drug-resistant human immunodeficiency virus type 1 protease variants revealed by a novel structural intermediate

J. Virol.

Effectiveness of nonpeptide clinical inhibitor TMC-114 on HIV-1 protease with highly drug resistant mutations D30N, I50V, and L90M

J. Med. Chem.

Structural and kinetic analysis of drug resistant mutants of HIV-1 protease

Eur. J. Biochem.

Potent new antiviral compound shows similar inhibition and structural interactions with drug resistant mutants and wild type HIV-1 protease

J. Med. Chem.