Molecular Characterization of Clinical Isolates of ... - Journal of Virology

JOURNAL OF VIROLOGY, Sept. 2009, p. 8810–8818 0022-538X/09/$08.00⫹0 doi:10.1128/JVI.00451-09 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 83, No. 17

Molecular Characterization of Clinical Isolates of Human Immunodeficiency Virus Resistant to the Protease Inhibitor Darunavir䌤† ˇasˇkova´,1,2 Milan Kozˇísˇek,1,2 Pavlína R ˇ eza´ˇcova´,1,3 Jirˇ´ı Brynda,1,3 Tatyana Yashina,4 Kla´ra Grantz S 5 Ron M. Kagan, and Jan Konvalinka1,2,3* Gilead Sciences and IOCB Research Center, Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic, v.v.i., Flemingovo n. 2, 166 10 Prague 6, Czech Republic1; Department of Biochemistry, Faculty of Science, Charles University, Hlavova 8, Prague 2, Czech Republic2; Institute of Molecular Genetics of the Academy of Sciences of the Czech Republic, v.v.i., Videnska 1083, 140 00 Prague 4, Czech Republic3; Department of Infectious Diseases, Specialty Laboratories, Valencia, California 913554; and Department of Infectious Diseases, Quest Diagnostics Inc., San Juan Capistrano, California 926755 Received 4 March 2009/Accepted 11 June 2009

Darunavir is the most recently approved human immunodeficiency virus (HIV) protease (PR) inhibitor (PI) and is active against many HIV type 1 PR variants resistant to earlier-generation PIs. Darunavir shows a high genetic barrier to resistance development, and virus strains with lower sensitivity to darunavir have a higher number of PI resistance-associated mutations than viruses resistant to other PIs. In this work, we have enzymologically and structurally characterized a number of highly mutated clinically derived PRs with high levels of phenotypic resistance to darunavir. With 18 to 21 amino acid residue changes, the PR variants studied in this work are the most highly mutated HIV PR species ever studied by means of enzyme kinetics and X-ray crystallography. The recombinant proteins showed major defects in substrate binding, while the substrate turnover was less affected. Remarkably, the overall catalytic efficiency of the recombinant PRs (5% that of the wild-type enzyme) is still sufficient to support polyprotein processing and particle maturation in the corresponding viruses. The X-ray structures of drug-resistant PRs complexed with darunavir suggest that the impaired inhibitor binding could be explained by change in the PR-inhibitor hydrogen bond pattern in the P2ⴕ binding pocket due to a substantial shift of the aminophenyl moiety of the inhibitor. Recombinant virus phenotypic characterization, enzyme kinetics, and X-ray structural analysis thus help to explain darunavir resistance development in HIV-positive patients. ritonavir (46), D30N and N88D for nelfinavir (39), and I47A for lopinavir (14). Other mutations, such as I84V, lead to some level of resistance against almost all known PIs. These “primary” mutations often compromise the ability of the mutated PR to process its cognate substrates, the Gag and Gag-Pol polyproteins. So-called compensatory mutations are selected outside the PR active site (55) or even outside the PR coding region (11, 33). These compensatory mutations improve the ability of the mutated PR to bind to and cleave its substrates. Recently, resistance-conferring gag mutations have been identified even without any mutations in the PR coding region (38). Finally, resistance development might involve not only mutation but also insertion of one or more amino acids into the PR coding region (25, 54). A detailed understanding of the mechanism of resistance development for individual clinically available PIs is essential for early detection of treatment failure. Moreover, it could lead to the design of a new generation of PIs capable of inhibiting even the highly resistant PR species from AIDS patients (for a review, see reference 41; 8, 57). Darunavir (previously known as TMC114) (21, 28, 49, 50), recently approved by the FDA, is extremely potent against wild-type HIV as well as a large panel of PI-resistant clinical isolates and shows a high genetic barrier to the development of antiretroviral resistance (9, 48). It was suggested that the major structural features of the compound responsible for these favorable properties are a picomolar binding affinity to the wild-

Human immunodeficiency virus (HIV) requires active protease (PR) for processing Gag and Gag-Pol polyprotein precursors into mature structural proteins and replicative enzymes (22). HIV PR has therefore become one of the major targets for anti-HIV treatment, and PR inhibitors (PIs) have proven to be highly effective antiretroviral drugs. Indeed, nine PIs have been approved by the U.S. Food and Drug Administration (FDA) as antiviral drugs over the last 13 years, and their widespread use, in combination with potent reverse transcriptase inhibitors (RTIs), brought about significant decreases in AIDS mortality in the developed world (for recent reviews, see references 5, 37, and 52). However, due to rapid viral replication and the evolution of drug-resistant PR variants, drug resistance development remains a major complication of antiretroviral treatment. Most PIs select viral species with one or more specific mutations in the PR coding region that confer resistance, such as mutations G48V or L90M for saquinavir (16), V82A for

* Corresponding author. Mailing address: Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic, v.v.i., Flemingovo n. 2, 166 10 Prague 6, Czech Republic. Phone: 420-220183218. Fax: 420-220183578. E-mail: konval@uochb .cas.cz. † Supplemental material for this article may be found at http://jvi .asm.org/. 䌤 Published ahead of print on 17 June 2009. 8810

VOL. 83, 2009

CHARACTERIZATION OF HIV RESISTANT TO DARUNAVIR

type PR binding site, the ability to form numerous backboneto-backbone hydrogen bonds with the PR substrate binding cleft, and the ability to adopt a conformation that fits within the “substrate envelope” of the active site (5, 28). In vitro selection studies suggest that development of resistance against darunavir requires more individual mutations and develops more slowly than development of resistance to other PIs (9). A close structural homolog of darunavir, TMC126, selected a resistant viral strain with the unusual combination of A28S and I50V in vitro (58). Darunavir itself selects mutations R41T and K70E in vitro (9). However, sitespecific mutants of HIV type 1 (HIV-1) carrying one or both of these mutations in the PR coding sequence showed no resistance. Koh et al. reported the following mix of PR mutations leading to increased resistance after serial passages in the presence of darunavir: L10I, I15V, K20R, L24I, V32I, L33F, M36I, M46L, I54M, L63P, K70Q, V82A, I84V, and L89M. The resulting virus showed cross-resistance to ritonavir, nelfinavir, lopinavir, and amprenavir (20). In clinical studies with treatment-experienced (POWER) and naive (ARTEMIS) individuals, patients developing darunavir resistance tend to have a high number of PI resistance-associated mutations. It was suggested that the substitutions critical for development of darunavir resistance were V11I, V32I, L33F, I47V, I50V, I54L/M, G73S, T74P, L76V, I84V, and L89V (6, 10, 32). Finally, based on genotypic characterization of 48 patients experiencing treatment failure with a darunavir-containing regimen, Lambert-Niclot et al. identified mutations in the Gag-Pol reading frame (specifically resulting in I437T and I437V in p1) that influence the selection of darunavir-specific mutations (27). Due to their structural similarity, darunavir and amprenavir might share similar genotypic determinants of resistance, and there is a high degree of correlation between darunavir and amprenavir phenotypic susceptibility. However, on average darunavir treatment produces smaller changes of replication fitness for the same resistant mutant viruses, i.e., darunavir seems to have a higher genetic barrier to resistance development (40). No structural or enzymological analysis of a PR variant from HIV-positive patients failing darunavir therapy has yet been provided. In the present work we report structural and enzymological analysis of a panel of clinically derived mutant PRs with high levels of phenotypic resistance to darunavir. We obtained six DNA clones from deidentified clinical samples submitted to a U.S. reference laboratory for HIV-1 resistance testing that carried as many as 21 amino acid substitutions compared to the wild-type enzyme and exhibited high levels of predicted phenotypic resistance to darunavir according to the VirtualPhenotype (Virco) assay (10). One of the six clones contained no darunavirassociated mutations and also carried the I50L mutation, which may confer increased darunavir susceptibility (47). To analyze the contribution of these mutations to viral resistance on a molecular level, we characterized their effects on the virus by assaying PR phenotype and replicative capacity. Additionally, we conducted an enzymologic and structural analysis of the recombinant PRs in the presence and absence of darunavir, amprenavir, and lopinavir, a structurally unrelated PI (Fig. 1).

8811

FIG. 1. Chemical formulas of PIs used in this study.

MATERIALS AND METHODS Database mining. The prevalence of darunavir resistance mutations was surveyed in the Quest Diagnostics clinical laboratory database for samples submitted for PR and reverse transcriptase sequencing between June 2006, when darunavir was approved for clinical use by the U.S. FDA, and June 2008. The presence or absence of predicted antiretroviral resistance, as determined by the Quest Diagnostics resistance algorithm, was used as a surrogate marker for treatment. Predicted resistance to PIs and RTIs was determined as previously described (18). Darunavir resistance-associated mutations were considered to be those in the recently updated darunavir mutation index: V11I, V32I, L33F, I47V, I50V, I54L/M, T74P, L76V, I84V, and L89V (10). Phenotypic analysis. Phenotyping was performed as previously described using the Phenoscript single-cycle recombinant virus assay (7, 42). This assay is based on reverse transcription and PCR amplification of HIV-1 viral RNA, recombination into an HIV-1 molecular clone, and evaluation of PI susceptibility in a single cycle of infection in producer cells treated with different concentrations of PI. Results for each PI are expressed as the change in the 50% inhibitory concentration (IC50) for a patient-derived virus compared with that for a reference isolate (NL4-3) for the same drug. Susceptibilities to amprenavir, atazanavir, darunavir, indinavir, lopinavir, nelfinavir, saquinavir, and tipranavir were assayed. DNA amplification for recombinant protein expression. The HIV-1 PR coding regions from patients were amplified from the recombinant virus clones. Amino acid substitutions with respect to the HIV sequence database consensus B sequence (www.hiv.lanl.gov) were summarized (see Table 4). The amplifications were performed by PCR using the forward primer 5⬘-ATCCTTTCATATGCCT CAGATCACTCTTTGG-3⬘, which is specific to the 5⬘ end of the PR coding region and includes an NdeI site, and the reverse primer 5⬘-TTGAATTCGAT ATCATTAAAAATTTAAAGTGCAGCC-3⬘, which contains an EcoRI site. The PCR products were subsequently ligated into the expression vector pET24a (Novagen, Darmstadt, Germany). Protein expression and purification. The PRs were overexpressed in Escherichia coli BL21(DE3)RIL (Novagen, Darmstadt, Germany) and purified from inclusion bodies as previously described (24). After refolding and purification, they were all active site titrated to quantify the exact amount of the active enzyme in the preparation (typically, 80 to 90% of the total protein). Activity and inhibition assay. PR kinetic parameters (Km and kcat) and inhibition constants for inhibitors (Ki) were determined spectrophotometrically based on decrease in absorbance at 305 nm upon cleavage of the chromogenic peptide substrate KARVNle*NphEANle-NH2, as previously described (14). Typically, 8 pmol of PR was added to 1 ml 0.1 M sodium acetate buffer, pH 4.7, 0.3 M NaCl, and 4 mM EDTA containing substrate at a concentration near the Km of the enzyme and various concentrations of inhibitor dissolved in dimethyl sulfoxide. The data were analyzed using the equation for competitive inhibition according to Williams and Morrison (53). Crystallization and data collection. The PR-inhibitor complexes were prepared by mixing the enzyme with a fivefold molar excess of darunavir dissolved in dimethyl sulfoxide, followed by concentration of the enzyme up to 5 mg/ml by ultrafiltration using Microcon-10 filters (Millipore). Crystals were grown by the hanging drop vapor diffusion technique at 19°C. The crystallization drops had a 2:1 ratio by volume of protein to reservoir solution. Optimized crystallization conditions for the PRDRV1-darunavir complex were 0.6 M NaCl and 0.1 M MES

8812

ˇAS ˇKOVA ´ ET AL. GRANTZ S

J. VIROL.

TABLE 1. Crystal data and diffraction data collection and refinement statistics

TABLE 2. The darunavir mutation score is associated with a high number of PR mutations

Valuea for: Parameter

Data collection statistics Space group ˚) Cell parameters (A ˚) Wavelength (A ˚) Resolution (A No. of unique reflections Redundancy Completeness (%) Rmerge (%)b Avg I/␴(I) ˚ 2)c Wilson B (A Refinement statistics ˚) Resolution range (A No. of reflections in working set No. of reflections in test set R (%)d Rfree (%)e ˚) RMSD bond length (A RMSD angle (°) No. of water molecules ˚ 2) Mean B (A Ramachandran plot statistics Residues in favored regions (%) Residues in allowed regions (%) a b

冘冘

PRDRV1 (PDB code 3GGT)

PRDRV5 (PDB code 3GGU)

P61 62.54, 62.54, 82.64 1.54 45.31–2.05 (2.16–2.05) 11,575 (1,701) 5.8 (5.6) 99.9 (98.7) 6.7 (34.0) 9.8 (2.2) 23.65

P61 62.59, 62.59, 81.92 0.98 50–1.8 (1.85–1.80) 16,954 5.1 (5.2) 99.2 (99.9) 3.1 (20.2) 42.6 (7.18) 26.4

50–2.05 (2.1–2.05) 10,994 (813)

22.6–1.8 (1.85–1.8) 15,989 (1,189)

553 (37) 18.83 (22) 26.7 (31.9) 0.012 1.76 173 25.8

857 (68) 20.44 (23.4) 24.6 (28.2) 0.013 1.55 142 26.1

93.0

93.8

7.0

6.2

冘冘

The data in parentheses refer to the highest-resolution shell. Rmerge ⫽ 兩I 共hkl兲 ⫺ ⬍ I共hkl兲 ⬎ 兩 / hkl i Ii共hkl兲, where Ii(hkl) is hkl i i

an individual intensity of the ith reflection hkl and ⬍I(hkl)⬎ is the average intensity of the reflection hkl with summation over all data. c B, temperature factor (i.e., atomic displacement parameter). d R ⫽ 㛳Fo兩 ⫺ 兩Fc㛳/兩 Fo兩, where Fo and Fc are the observed and calculated structure factors, respectively. e Rfree is equivalent to R but is calculated for 5% of the reflections chosen at random and omitted from the refinement process.

(morpholineethanesulfonic acid), pH 6.0; conditions for the PRDRV5-darunavir complex crystallization were 0.8 M ammonium sulfate and 0.1 M sodium acetate, pH 5.0. For data collection, crystals were soaked with 25 to 30% (vol/vol) glycerol in the reservoir buffer solution and cryogenically cooled in liquid nitrogen. X-ray diffraction data for the PRDRV1 complex were collected at 120 K on a Mar345 image plate system using a Nonius FR591 rotating anode generator. Diffraction data were integrated and reduced using MOSFLM (29) and scaled using SCALA (13) from the CCP4 suite (1). Diffraction data for the PRDRV5 complex were collected at 100 K at beam line 19-BM of the Structural Biology Center at the Advanced Photon Source, Argonne National Laboratory, Argonne, IL. Diffraction data were processed using the HKL-3000 suite of programs (34). Crystal parameters and data collection statistics are summarized in Table 1. Structure refinement and analysis. Since the hexagonal crystals we obtained appeared to be isomorphous with all other P61 crystals of HIV-1 protease complexes, structure determination was performed by the difference-Fourier method using Protein Data Bank (PDB) structure 1U8G (4) as the initial model. Initial rigid-body refinement and subsequent restrained refinement were performed with the program REFMAC 5.1.24 (36) from the CCP4 package (1). The atomic coordinates for darunavir were obtained from PDB structure 1T3R (19). The program Coot (12) was used for manual model rebuilding and inhibitor building. Final translation, libration, and screw (TLS) refinement (3) was done with TLS groups corresponding to HIV PR subdomains (45). The quality of the final models was validated with Molprobity (31). The final refinement statistics are summarized in Table 1. All figures showing structural representations were prepared with the program PyMOL (version 0.99; DeLano Scientific LLC, San Carlos, CA [http://www .pymol.org]). The following services were used to analyze the structures: the PISA server (26) and the Protein-protein interaction server (17).

Darunavir mutation score ⱖ4 3 2 1 0 a

Samples with predicted resistance to ⱖ1 RTI or PI

Samples with predicted resistance to ⱖ1 PI

No. (%) of samples

Median no. of PR mutations (IQR)

No. (%) of samples

Median no. of PR mutationsa (IQR)

602 (2.5) 574 (2.3) 1,031 (4.2) 1,821 (7.5) 20,401 (83.5)

18 (15–20) 16 (13–18) 14 (11–17) 12 (9–15) 5 (3–7)

602 (7.4) 574 (7.0) 1,015 (12.4) 1,678 (20.5) 4,302 (52.6)

18 (15–20) 16 (13–18) 14 (12–17) 12 (9–15) 9 (7–11)

Differences from HIV-1 subtype B wild-type consensus sequence.

Protein structure accession numbers. Atomic coordinates and structure factors have been deposited in the PDB with accession codes 3GGT and 3GGU for PRDRV1 and PRDRV5, respectively.

RESULTS Prevalence of darunavir resistance mutations in a large clinical database. We obtained six DNA clones from deidentified clinical samples submitted to a U.S. reference laboratory for HIV-1 resistance testing. For the 2-year period between June 2006 (darunavir FDA approval) and June 2008, 40.3% (n ⫽ 60,643) of the samples had predicted resistance to a RTI or PI, while PI resistance was predicted for 13.5% of all samples tested. Only 1.9% of all samples tested (4.8% of samples with any predicted resistance) had a darunavir mutation score of ⱖ3, which is associated with reduced response to darunavir (10). The median number of PR mutations in PI-resistant samples with no darunavir index mutations was 9 (interquartile range [IQR]: 7 to 11) but was 18 (IQR: 15 to 20) in sequences with a darunavir mutation index of ⱖ4 (Table 2). In samples with resistance to any RTI or PI, the prevalence of mutations associated with a threefold or higher decrease in phenotypic susceptibility to darunavir (51) ranged from 0.6% (for V82F) to 4.5% (for V32I) (Table 3). Phenotypic susceptibility to PIs. The impact of darunavir resistance mutations on the phenotypic susceptibility to seven PIs was analyzed by a recombinant virus assay and is summarized in Table 4. Samples PRDRV1, PRDRV2, PRDRV4, PRDRV5, and PRDRV6, which carry between three and six darunavir resistance-associated mutations and 18 to 21 non-wildtype PR amino acid substitutions, exhibited high phenotypic changes in susceptibility to all PIs (Table 4). The changes in darunavir susceptibility ranged from 32-fold (PRDRV5) to ⬎193-fold (PRDRV1), and the changes for the structurally related PI amprenavir ranged from 24-fold (PRDRV5) to 75-fold (PRDRV2) for the same samples. The key mutations associated with darunavir resistance (V32I, I54L, and I54M [51]) were found in the three samples with the largest changes in darunavir susceptibility (PRDRV1, PRDRV2, and PRDRV4) but not in PRDRV5 or PRDRV6, which had smaller phenotypic changes in darunavir susceptibility. The relative ratios of experimentally determined Ki values for both structurally related PIs (darunavir, 32 to 2,000; amprenavir, 60 to 750) were significantly increased relative to that for the wild-type virus (Table 4). Sample PRDRV3, which carries no mutations associated with darunavir resistance, had nearly wild-type phenotypic suscep-

VOL. 83, 2009


TABLE 3. Prevalence of darunavir resistance mutations in a clinical database Mutationa

V11I V32I* L33F I47V I50V* I54L* I54M* T74P L76V* V82F* I84V L89V

No. of samples

% of samples with resistance to ⱖ1 RTI or PI

328 1,096 2,010 760 267 661 460 279 253 146 1,788

1.3 4.5 8.2 3.1 1.1 2.7 1.9 1.1 1.0 0.6 7.3

473

1.9

TABLE 5. Enzyme characteristics of PR variants analyzed in this study Sample

Km (␮M)

kcat (s⫺1)

kcat/Km (mM⫺1 s⫺1)

PRWTa PRDRV1 PRDRV2 PRDRV3 PRDRV4 PRDRV5 PRDRV6

15 ⫾ 1 68 ⫾ 3 30 ⫾ 2 13 ⫾ 2 132 ⫾ 4 113 ⫾ 9 53 ⫾ 2

30 ⫾ 1.8 14.5 ⫾ 0.2 14.0 ⫾ 0.3 14.2 ⫾ 0.6 13.4 ⫾ 0.7 12.2 ⫾ 0.5 9.6 ⫾ 0.2

1,990 ⫾ 210 214 ⫾ 9 462 ⫾ 34 1,082 ⫾ 132 102 ⫾ 6 108 ⫾ 10 180 ⫾ 7

Associated PI resistanceb

DRV, FPV DRV, DRV, DRV, DRV,

TPV FPV, LPV FPV, LPV, NFV FPV, LPV, NFV

a

DRV, FPV, LPV IDV, LPV, NFV ATV, FPV, IDV, LPV, NFV, SQV, TPV

ⴱ, mutation shown to cause a threefold or greater change in phenotypic susceptibility in a background of multiple PI resistance-associated mutations. V82F is not part of the darunavir mutation score, but mutants with this variant exhibit a nearly fivefold decrease in darunavir susceptibility (51). b Drugs for which the mutation reduces in vitro susceptibility or in vivo virological response (Stanford Drug Resistance Database 关http://hivdb.stanford .edu兴). DRV, darunavir; FPV, fosamprenavir; TPV, tipranavir; LPV, lopinavir; NFV, nelfinavir; IDV, indinavir; ATV, atazanavir; SQV, saquinavir. a

tibilities to darunavir, amprenavir, indinavir, lopinavir, and tipranavir. The reduced phenotypic susceptibilities to atazanavir and nelfinavir seen for this sample can be accounted for by the presence of I50L and D30N, mutations associated with resistance to atazanavir and nelfinavir, respectively. Enzymatic analysis of recombinant PRs. In order to characterize the enzymatic activities of PRs from samples PRDRV1 to PRDRV6, we cloned and expressed these PR sequences in E. coli. The recombinant PRs were purified and characterized in

8813

WT, wild type.

vitro by monitoring cleavage of a chromogenic peptide substrate in the presence and absence of a panel of specific PIs. The data are summarized in Tables 5 and 6. Somewhat surprisingly, the very high number of mutations did not dramatically affect the kcat values for the mutants. Most of the mutated PRs retained kcat values at least 30 to 50% of the wild-type value. However, a significantly larger effect was observed for the Km values, which were four- to eightfold higher for mutants PRDRV1, PRDRV4, and PRDRV6 than for the wild-type PR. The specific activities of the recombinant PRs are as low as 5% of the wild-type value (PRDRV5). Inhibition constants were determined by kinetic analysis using a chromogenic peptide substrate and the appropriate inhibitor. Inspection of the inhibition data summarized in Table 6 reveals that mutated PRs PRDRV4, PRDRV1, PRDRV2, and PRDRV6 show the highest increase in Ki values for darunavir relative to the wild-type value. PRDRV5 shows a much smaller difference in Ki value for darunavir (relative to the wild type), even though this PR species accumulated 20 mutations, including those responsible for cross-resistance to many other PIs

TABLE 4. Genotypes and phenotypic changes analyzed with a recombinant virus assay Sample

Phenotypic changeb (fold) in resistance toc:

PR mutationsa DRV

PRDRV1 PRDRV2

PRDRV3 PRDRV4 PRDRV5

PRDRV6

a

T12V, I13V, I15V, K20M, V32I, L33F, K43T, I54L, K55N, I62V, L63P, A71V, I72V, G73S, V77I, V82L, I84V, L89V, L90M I13V, K20R, V32I, L33F, E35D, M36I, R41K, K43T, I47V, I54M, I62V, L63V, A71V, I72T, G73S, T74P, V82L, L89V, I93L L10F, I13V, D30N, K45I, I50L, L63P, A71I, V77I, N88D L10I, I13V, K14R, V32I, L33F, K43T, M46I, I47V, I54L, I62V, L63P, A71T, I72T, G73T, V77I, P79S, I84V, L90M L10I, I13V, G16E, L33F, M36L, N37T, P39S, K45R, M46L, I54V, K55R, I62V, L63P, A71V, G73D, V82T, I84V, L89V, L90M, I93L L10I, I13V, I15V, K20R, L33F, E34N, E35D, M36I, K43T, I47V, I50L, F53L, Q58E, I62V, L63P, I66F, A71V, V77I, V82A, I84V, L89V

APV

LPV

ATV

IDV

NFV

SQV

TPV

⬎193 (700)

42 (370)

46 (11)

⬎121

48

62

88 (180)

75 (190)

155 (32)

⬎40

48

23

3.3

0.5 (1)

1.5 (2)

0.9 (1)

35

196

6.6

140 (2,000)

42 (750)

⬎133 (470)

86

36

26

36

32 (32)

24 (60)

⬎133 (18)

⬎121

⬎189

38

⬎223

⬎96

43 (320)

34 (300)

⬎40

18

100

45

81 (140)

1.1

4.4

30

13 23

0.4 7.7

Darunavir (DRV) resistance-associated mutations are underlined. Phenotypic change is expressed as a ratio of the sample IC50 to the IC50 of a reference isolate (NL4-3). The relative changes in Ki values for darunavir, the structurally related PI amprenavir (APV), and lopinavir (LPV) are shown in parentheses. Ki values were determined with a spectrophotometric assay. c TPV, tipranavir; NFV, nelfinavir; IDV, indinavir; ATV, atazanavir; SQV, saquinavir. b


8814

J. VIROL.

TABLE 6. Ki values for the inhibition of PR mutants a

Ki (pM) for inhibition by :

HIV-1 PR b

PRWT PRDRV1 PRDRV2 PRDRV3 PRDRV4 PRDRV5 PRDRV6

LPV

APV

DRV

18 ⫾ 9 190 ⫾ 40 570 ⫾ 40 19 ⫾ 3 8,500 ⫾ 200 320 ⫾ 30 2,600 ⫾ 100

180 ⫾ 20 68,000 ⫾ 6,000 36,000 ⫾ 3,000 440 ⫾ 80 140,000 ⫾ 3,000 11,000 ⫾ 500 56,000 ⫾ 3,000

5.3 ⫾ 3.6 3,700 ⫾ 300 960 ⫾ 80 4.9 ⫾ 7.2 11,000 ⫾ 1,000 170 ⫾ 20 1,700 ⫾ 100

a LPV, lopinavir; APV, amprenavir; DRV, darunavir. The inhibition constants were determined by spectrophotometric assay at the pH optimum of the PR (pH 4.7). b WT, wild type.

(L10I, L33F, M46L, I54V, A71V, V82T, I84V, L89V, and L90M). However, all three of the amprenavir- and darunavirassociated mutations, V32I, I47V, and I54L/M, are absent in PRDRV5, whereas all three are present in PRDRV1 and PRDRV4, the two PRs with the highest resistance values (Table 6). The resistance profile of the structurally similar PI amprenavir mirrors that of darunavir. Vitality describes the relative ability of a given PR species to hydrolyze its substrate in the presence of an inhibitor. The higher the vitality value, the greater the advantage given to that PR species (15). The relative vitality data for recombinant PRs analyzed in vitro are summarized in Fig. 2. The relative vitalities correlate with the corresponding phenotypic changes in DRV susceptibility to an even greater extent than do the enzymatically determined Ki values. The high number of darunavir resistance-associated mutations in PRDRV6, PRDRV4, PRDRV1, and PRDRV2 translates into the highest vitality values of all the PRs analyzed, and the corresponding recombinant viruses show the highest phenotypic changes in darunavir susceptibility. The relative vitalities and the phenotypic analysis reveal the same order of decreasing DRV susceptibility: PRDRV4 ⬎ PRDRV1 ⬎ PRDRV2 ⬎ PRDRV6. The vitality values for amprenavir follow a similar pattern in terms of phenotypes, although the vitalities for species PRDRV1, PRDRV2, PRDRV4, and PRDRV6 do not show significant variation (Fig. 2). The different pattern of vitality values observed for lopinavir reflects its different structure and resistance profile. X-ray structure analysis of PRDRV1 and PRDRV5. The crystal structures of drug-resistant mutants PRDRV1 and PRDRV5 in complex with darunavir were determined to 2.05-Å and 1.8-Å resolutions, respectively. Both complexes crystallized in the same hexagonal form with one PR dimer in the asymmetric unit, and structures were refined with two inhibitor molecules bound in alternative orientations related by 180° with 50% relative occupancy. All amino acid residue changes identified by DNA sequencing could be modeled into well-defined electron density maps, with the exception of surface residues R45 and R55 in PRDRV5, which had disordered side chains. The positions of mutations in the HIV PR structure are depicted in Fig. 3. In PRDRV1, there are three mutations in residues directly involved in interaction with the substrate and darunavir, namely, the V32I, V82L, and I84V mutations, while the PRDRV5 variant contains two such mutations, V82T and I84V. Other mutations are located outside the substrate bind-

ing cleft. Nevertheless, some of the associated residues are in direct contact with substrate/inhibitor binding residues (e.g., those associated with L10I, K20M, L33F, I54L/V, and L90M). In order to decipher changes in protein structure contributing to the impaired inhibitor binding, we compared our structures with the structure of wild-type PR in complex with darunavir (PDB code 1T3R) (19). The overall superposition with the wild-type enzyme main-chain atoms yielded root mean square deviations (RMSDs) of 0.75 Å and 0.78 Å for PRDRV1 and PRDRV5, respectively. Detailed comparison of pairwise RMSDs of the main-chain C␣ atoms pointed to regions that contribute the most to the structural differences in PR mutants relative to wild-type PR (Fig. 4A; see Fig. S1 in the supplemental material). Only structural differences in the position of main-chain atoms with an RMSD greater than 0.7 Å were considered to be significant (2). The structural differences are predominantly located in regions with amino acid residue changes; however, some mutations provoke long-range structural changes and affect residues of the enzyme substrate binding cleft (Fig. 4A). In PRDRV1, the structural change in the inhibitor-contacting residues comprising the S2/S2⬘ subsite residues 79 to 83 is likely due to the V82L and I84V mutations. Additional structural changes in the flap region (residues 40 to 49 and 51 to 56) (Fig. 4B), with residues 47 and 49 directly contacting the inhibitor, can be attributed to the flap mutations K43T, I54L, and K55N and mutations in structurally adjacent residues, V32I, L33F, and V77I. Similarly, in PRDRV5 the shape and character of the S2/S2⬘ subsites are directly affected by mutations V82T and I84V (Fig. 5B), and structural changes in the flap region (residues 51 to 55) including the flap hinge (residues 34 to 47) (Fig. 4B) can probably be attributed to mutations L33F, M36L, N37T, P39S, K45R, M46I, I54V, and K55R. In response to the change in the shape and character of the substrate binding pockets, the inhibitor substituents adjust their positions to various extents. The P2⬘ aminophenyl moiety undergoes the most profound structural change (Fig. 5). Darunavir was designed to form polar interactions predominantly with the PR backbone in order to withstand drug resistance

FIG. 2. Relative vitality values, defined as v ⫽ (Kikcat/Km)MUT/(Ki kcat/Km)WT, for recombinant PRs and PRIs, where MUT represents the mutant and WT represents the wild type. LPV, lopinavir; APV, amprenavir; DRV, darunavir.

VOL. 83, 2009


8815

FIG. 3. Positions of the mutations in PR variants used for structural studies. (A) Each monomer of PR is used to depict the mutations in the PRDRV1 and PRDRV5 variants in red and blue, respectively. Mutated residues are represented with their C␣ atoms (spheres) and their transparent solvent-accessible surfaces. Active-site aspartates are shown as sticks; darunavir bound to the active site is represented by sticks, and its solvent-accessible surface area is green. (B) Sequence alignment of PRDRV1 and PRDRV5 with the wild-type (WT) sequence. Primary mutations in residues interacting directly with darunavir are in boldface italics.

mutations (21). In our structures, no major differences in enzyme-inhibitor polar interactions were observed in mutant variants relative to the wild-type complex (19). The hydrogen bonds between the bis-tetrahydrofuranyl moiety and the backbone atoms of residues D29 and D30, which are believed to be responsible for the very favorable binding enthalpy of darunavir (28), are maintained in both variants studied (Fig. 5). The only difference in the darunavir hydrogen bonding network can be seen at the P2⬘ group of the PRDRV1 complex. Due to the aminophenyl NH2 group shift of almost 2.3 Å, the hydrogen bond interactions with the D30⬘ main chain found in the wild-type HIV PR structure are lost. Instead, the aminophenyl NH2 group in PRDRV1-darunavir interacts with the D30⬘ side chain (Fig. 5A). This direct hydrogen bond replaces a water-mediated contact in the wild-type PR structure used for comparison (PDB code 1T3R) (19). Interestingly, an alternative conformation of the D30 side chain forming a direct interaction with the p-NH2 group of the aniline has also been observed in another structure of darunavir bound to the wildtype enzyme (PDB code 2IEN) (50). A detailed structural and biophysical analysis of the PRinhibitor interactions in the PRDRV1 and PRDRV5 complexes will be published elsewhere (M. Kozˇísˇek et al., unpublished data).

DISCUSSION Darunavir is a PI with extremely high in vitro activity against HIV-1 species resistant to other PIs. It has been shown to have a high genetic barrier to resistance development and is efficient in the treatment of AIDS patients failing previous antiretroviral therapy involving multiple PIs (10). Since darunavir was introduced into clinical use in 2006, information on resistance development in patients is scarce, derived mostly from POWER 1, 2, and 3 studies of patients with a failing PIcontaining regimen (6, 35) and several subsequent studies (10). The aim of this study was to analyze the prevalence of mutations implicated in darunavir resistance (“darunavir mutation score”) in a clinical laboratory database; to quantify the in vitro resistance of those patient-derived virus species to darunavir, amprenavir, and lopinavir by a recombinant virus assay and enzyme kinetics; and to identify the structural features responsible for the decreased susceptibility to darunavir by X-ray structure analysis. Analysis of the Quest Diagnostics clinical laboratory database for samples submitted for HIV sequencing between June 2006 and June 2008 (total of 60,643 samples) showed that the prevalence of darunavir resistance remains low. Only 5.8% of the samples bearing a mutation(s) conferring resistance to any retroviral drug showed a darunavir mutation score of 3 or

8816


FIG. 4. Structural changes in PRDRV mutants relative to wild-type PR. (A and B) HIV PR in ribbon representations showing regions with structural differences in the protein main chain. On the background of the yellow PRwt-darunavir complex (PDB code 1T3R), the residues differing by RMSDs of ⬎0.7 Å for the superposition of the main-chain atoms are red and blue for the PRDRV1-darunavir (A) and PRDRV5darunavir (B) complexes, respectively. (C) Top view of the superposition of the flap region of PRDRV5 (blue), PRDRV1 (red), and wild-type (yellow) PRs. Significant changes are located within residues 34 to 37 in a part of the so-called flap elbow and between residues 52 and 54. Darunavir is represented by its solvent-accessible surface (green).

more, which is suggestive of darunavir resistance. This finding is comparable to data from Rinehart et al., who showed that, of samples received for routine antiretroviral resistance testing with reduced PI susceptibility, 6.7% revealed three or more darunavir resistance mutations (44). A darunavir mutation score above 3 was generally accompanied by a high number of background or compensatory mutations (the median number of PR mutations in PI-resistant samples was 18 [IQR: 15 to 20] in sequences with a darunavir mutation index higher than 4). It has been shown that mutation of at least 34 of the 99 residues of HIV-1 PR has clinical significance (56, 43). However, the typical number of mutations in resistant patients ranges from 5 to 15 mutations in the PR gene (37). With 18 to 21 amino acid residue changes, the PRs studied in this work exhibit the highest number of mutations in an HIV PR studied by means of enzyme kinetics and X-ray crystallography to date. Not surprisingly, this very high number of mutations compro-

J. VIROL.

mises the ability of the mutated enzymes to bind substrate, as documented by an eight- to ninefold increase in the Km values compared to wild-type HIV PR. The substrate turnover, on the other hand, is much less affected (the kcat values are reduced by a factor of only 2 to 3). The overall catalytic efficiency of the corresponding recombinant PRs is as low as 5% of that of the wild-type enzyme. It is remarkable that a PR species with such a low activity can still support efficient polyprotein processing and particle maturation of the corresponding virus. The enzymatic activities and inhibition constants of individual viral enzymes determined in vitro are often difficult to correlate with the replication capacity and viral fitness of the corresponding viruses. In this paper we document a very high level of agreement between the relative vitality values and the phenotypic resistance changes of these mutants. PR mutations have been associated with a lower viral replication capacity (15). Although actual replication capacity data were not available for this study, the mutants studied in this work show significantly higher enzymatic Km values and a much lower catalytic efficiency, which would be consistent with reduced replication capacity. Compensatory mutations in Gag cleavage sites have been observed to partially restore replication capacity of mutated virus species (27, 33). Moreover, certain Gag mutations might lead to the resistance to PIs per se, without accompanying mutations in the PR region (38). Therefore, fitness studies with intact virus and/or recombinant virus containing the mutant PR sequences and the Gag cleavage sequences would be needed to further delineate the effects of these on the PR activity and the replication capacity of these mutants. The data summarized in Tables 4 and 6 and in Fig. 2 confirm the usefulness of the darunavir mutation score: a high number of specific mutations leads to high relative Ki values (measured with purified recombinant PR) and, consequently, to reduced darunavir susceptibility, as quantified by the recombinant virus assay. Our data confirm the importance of the mutations V32I, I47V, and I54L/M, which previous studies have found to be implicated in amprenavir and darunavir resistance. Indeed, these mutations were present in PRDRV1, PRDRV2, and PRDRV4, the three PRs with the highest vitality and phenotypic resistance values. Correspondingly, PRDRV5, which has none of these signature mutations, shows medium resistance to darunavir and amprenavir both in terms of the in vitro vitality and relative replication capacity. In an attempt to rationalize and explain the structural role of the critical amino acid exchanges in the darunavir resistance score, we performed X-ray structure analysis of two darunavirresistant PR complexes. The significantly lower affinity and higher Ki value of the PRDRV1 variant with respect to darunavir could be explained by the loss of two PR-inhibitor hydrogen bonds between the inhibitor and PR main chain and one water-mediated contact in the P2⬘ binding pocket due to a substantial shift of the aminophenyl moiety (Fig. 5A). The shift of the aminophenyl ring weakens the interaction with the D30 main chain and has been described for PR variants containing the single mutation I50V or I54M (23, 30); however, in these cases the structural change was not dramatic enough to completely disrupt the interactions. Based on comparison with the PRDRV5-darunavir structure (Fig. 5B), we propose that the possible cause of the darunavir aminophenyl moiety movement

VOL. 83, 2009


8817

FIG. 5. Detailed view of the darunavir-enzyme interactions. The darunavir structure and substrate binding pockets of the wild-type complex are compared with the PRDRV1 (A) and PRDRV5 (B) complex structures. The wild-type structure is yellow, PRDRV1 is red, and PRDRV5 is blue. Residues 32, 82, and 84 are represented by their solvent-accessible surfaces (solid yellow for the wild type, mesh for the mutant variant). Dashed lines indicate hydrogen bond interactions, and the water molecule involved in water-mediated hydrogen contacts in the wild-type complex is shown as a yellow sphere. Interactions are shown for one darunavir orientation; however, they are conserved for both alternative conformations.

is the simultaneous presence of mutations V32I and I84V, resulting in a distinctive change in the shape of the S2/S2⬘ subsites. The complex interplay between the V32I and I84V mutations was indeed recently revealed using a bioinformatic resistance determination approach (51). Changes in the hydrogen bonding pattern similar to those observed in the PRDRV1-darunavir structure can be also expected for amprenavir, since it shares the P2⬘ aminophenyl substituent with darunavir. This assumption is supported by the relatively high resistance values of PRDRV1 for amprenavir (Table 6). On the other hand, the different profiles of the resistance of PR variants tested in this work to darunavir and amprenavir suggest that interaction of other inhibitor substituents which differ in amprenavir and darunavir may also play a role in darunavir resistance.

6.

7.

8. 9.

10.

ACKNOWLEDGMENTS This work was supported by a grant from the Ministry of Education (MSMT) of the Czech Republic within Program 1M0508, Research Centre for new Antivirals and Antineoplastics, by Research Plans AV0Z40550506 and AV0Z50520514 from the Academy of Sciences of the Czech Republic, and by the Sixth Framework of the European Commission (LSHP-CT-2007-037693). We thank members of the Structural Biology Center at Argonne National Laboratory for their help with conducting data collection at the 19-ID and 19-BM beam lines and Hillary Hoffman for critical editing of the manuscript and language corrections.

11.

12. 13.

14.

REFERENCES 1. Bailey, S. 1994. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. Sect. D Biol. Crystallogr. 50:760–763. 2. Betts, M. J., and M. J. Sternberg. 1999. An analysis of conformational changes on protein-protein association: implications for predictive docking. Protein Eng. 12:271–283. 3. Borman, A. M., S. Paulos, and F. Clavel. 1996. Resistance of human immunodeficiency virus type 1 to protease inhibitors: selection of resistance mutations in the presence and absence of the drug. J. Gen. Virol. 77:419–426. 4. Brynda, J., P. Rezacova, M. Fabry, M. Horejsi, R. Stouracova, M. Soucek, M. Hradilek, J. Konvalinka, and J. Sedlacek. 2004. Inhibitor binding at the protein interface in crystals of a HIV-1 protease complex. Acta Crystallogr. Sect. D Biol. Crystallogr. 60:1943–1948. 5. Chellappan, S., G. S. Kiran Kumar Reddy, A. Ali, M. N. Nalam, S. G. Anjum,

15.

16.

17. 18.

H. Cao, V. Kairys, M. X. Fernandes, M. D. Altman, B. Tidor, T. M. Rana, C. A. Schiffer, and M. K. Gilson. 2007. Design of mutation-resistant HIV protease inhibitors with the substrate envelope hypothesis. Chem. Biol. Drug Des. 69:298–313. Clotet, B., N. Bellos, J. M. Molina, D. Cooper, J. C. Goffard, A. Lazzarin, A. Wohrmann, C. Katlama, T. Wilkin, R. Haubrich, C. Cohen, C. Farthing, D. Jayaweera, M. Markowitz, P. Ruane, S. Spinosa-Guzman, and E. Lefebvre. 2007. Efficacy and safety of darunavir-ritonavir at week 48 in treatmentexperienced patients with HIV-1 infection in POWER 1 and 2: a pooled subgroup analysis of data from two randomised trials. Lancet 369:1169–1178. Dam, E., V. Obry, P. Jouvenne, J.-L. Meynard, F. Clavel, and E. Race. 2001 Definition of clinically relevant cut-offs for the interpretation of phenotypic data obtained using Phenoscript. Antivir. Ther. 6(Suppl. 1):123. De Clercq, E. 2007. Anti-HIV drugs. Verh. K. Acad. Geneeskd. Belg. 69: 81–104. De Meyer, S., H. Azijn, D. Surleraux, D. Jochmans, A. Tahri, R. Pauwels, P. Wigerinck, and M. P. de Bethune. 2005. TMC114, a novel human immunodeficiency virus type 1 protease inhibitor active against protease inhibitorresistant viruses, including a broad range of clinical isolates. Antimicrob. Agents Chemother. 49:2314–2321. De Meyer, S., I. Dierynck, E. Lathouwers, B. Van Baelen, T. Vangeneugden, S. Spinosa-Guzman, M. Peeters, G. Picchio, and M. P. de Bethune. 2008. Phenotypic and genotypic determinants of resistance to darunavir: analysis of data from treatment-experienced patients in POWER 1, 2, 3 and DUET-1 and 2. Antivir. Ther. 13:A33. Doyon, L., G. Croteau, D. Thibeault, F. Poulin, L. Pilote, and D. Lamarre. 1996. Second locus involved in human immunodeficiency virus type 1 resistance to protease inhibitors. J. Virol. 70:3763–3769. Emsley, P., and K. Cowtan. 2004. Coot: model-building tools for molecular graphics. Acta Crystallogr. Sect. D Biol. Crystallogr. 60:2126–2132. Evans, P. R. 1993. Data reduction. In Proceedings of CCP4 Study Weekend on Data Collection and Processing, p. 114–122, Daresbury Laboratory, Warrington, United Kingdom. Grantz Saskova, K., M. Kozisek, M. Lepsik, J. Brynda, P. Rezacova, J. Vaclavikova, R. M. Kagan, L. Machala, and J. Konvalinka. 2008. Enzymatic and structural analysis of the I47A mutation contributing the reduced susceptibility to HIV protease inhibitor lopinavir. Protein Sci. 17:1555–1564. Gulnik, S. V., L. I. Suyorov, B. Lju, B. Yu, B. Anderson, H. Mitsuya, and J. W. Erickson. 1995. Kinetic characterization and cross-resistance patterns of HIV-1 protease mutants selected under drug pressure. Biochemistry 34: 9282–9287. Jacobsen, H., M. Hanggi, M. Ott, I. B. Duncan, S. Owen, M. Andreoni, S. Vella, and J. Mous. 1996. In vivo resistance to a human immunodeficiency virus type 1 proteinase inhibitor: mutations, kinetics, and frequencies. J. Infect. Dis. 173:1379–1387. Jones, S., and J. M. Thornton. 1996. Principles of protein-protein interactions. Proc. Natl. Acad. Sci. USA 93:13–20. Kagan, R. M., P. K. Cheung, T. K. Huard, and M. A. Lewinski. 2006. Increasing prevalence of HIV-1 protease inhibitor-associated mutations cor-

8818

19.

20.

21.

22.

23.

24.

25.

26. 27.

28. 29. 30.

31.

32.

33.

34.

35.

36.

37.

38.


relates with long-term non-suppressive protease inhibitor treatment. Antivir. Res. 71:42–52. King, N. M., M. Prabu-Jeyabalan, E. A. Nalivaika, P. Wigerinck, M. P. de Bethune, and C. A. Schiffer. 2004. Structural and thermodynamic basis for the binding of TMC114, a next-generation human immunodeficiency virus type 1 protease inhibitor. J. Virol. 78:12012–12021. Koh, T., T. Towata, A. K. Ghosh, and H. Mitsuya. 2007. Selection in vitro of HIV-1 variants highly resistant to darunavir using a mixture of HIV-1 isolates resistant to multiple PI, abstr 606. Abstr. 14th Conf. Retrovir. Opportunistic Infect., Los Angeles, CA. Koh, Y., H. Nakata, K. Maeda, H. Ogata, G. Bilcer, T. Devasamudram, J. F. Kincaid, P. Boross, Y. F. Wang, Y. Tie, P. Volarath, L. Gaddis, R. W. Harrison, I. T. Weber, A. K. Ghosh, and H. Mitsuya. 2003. Novel bis-tetrahydrofuranylurethane-containing nonpeptidic protease inhibitor (PI) UIC-94017 (TMC114) with potent activity against multi-PI-resistant human immunodeficiency virus in vitro. Antimicrob. Agents Chemother. 47:3123–3129. Kohl, N. E., E. A. Emini, W. A. Schleif, L. J. Davis, J. C. Heimbach, R. A. F. Dixon, E. M. Scolnick, and I. S. Sigal. 1988. Active human immunodeficiency virus protease is required for viral infectivity. Proc. Natl. Acad. Sci. USA 85:4686–4690. Kovalevsky, A. Y., Y. Tie, F. Liu, P. I. Boross, Y. F. Wang, S. Leshchenko, A. K. Ghosh, R. W. Harrison, and I. T. Weber. 2006. Effectiveness of nonpeptide clinical inhibitor TMC-114 on HIV-1 protease with highly drug resistant mutations D30N, I50V, and L90M. J. Med. Chem. 49:1379–1387. Kozisek, M., J. Bray, P. Rezacova, K. Saskova, J. Brynda, J. Pokorna, F. Mammano, L. Rulisek, and J. Konvalinka. 2007. Molecular analysis of the HIV-1 resistance development: enzymatic activities, crystal structures, and thermodynamics of nelfinavir-resistant HIV protease mutants. J. Mol. Biol. 374:1005–1016. Kozisek, M., K. G. Saskova, P. Rezacova, J. Brynda, N. M. van Maarseveen, D. De Jong, C. A. Boucher, R. M. Kagan, M. Nijhuis, and J. Konvalinka. 2008. Ninety-nine is not enough: molecular characterization of inhibitorresistant human immunodeficiency virus type 1 protease mutants with insertions in the flap region. J. Virol. 82:5869–5878. Krissinel, E., and K. Henrick. 2005. Detection of protein assemblies in crystal. Computational Life Sci. 3695:163–174. Lambert-Niclot, S., P. Flandre, I. Malet, A. Canestri, C. Soulie, R. Tubiana, C. Brunet, M. Wirden, C. Katlama, V. Calvez, and A. G. Marcelin. 2008. Impact of gag mutations on selection of darunavir resistance mutations in HIV-1 protease. J. Antimicrob. Chemother. 62:905–908. Lefebvre, E., and C. A. Schiffer. 2008. Resilience to resistance of HIV-1 protease inhibitors: profile of darunavir. AIDS Rev. 10:131–142. Leslie, A. G. 1999. Integration of macromolecular diffraction data. Acta Crystallogr. Sect. D Biol. Crystallogr. 55:1696–1702. Liu, F., A. Y. Kovalevsky, Y. Tie, A. K. Ghosh, R. W. Harrison, and I. T. Weber. 2008. Effect of flap mutations on structure of HIV-1 protease and inhibition by saquinavir and darunavir. J. Mol. Biol. 381:102–115. Lovell, S. C., I. W. Davis, W. B. Arendall III, P. I. de Bakker, J. M. Word, M. G. Prisant, J. S. Richardson, and D. C. Richardson. 2003. Structure validation by Calpha geometry: phi, psi and Cbeta deviation. Proteins 50: 437–450. Madruga, J. V., D. Berger, M. McMurchie, F. Suter, D. Banhegyi, K. Ruxrungtham, D. Norris, E. Lefebvre, M. P. de Bethune, F. Tomaka, M. De Pauw, T. Vangeneugden, and S. Spinosa-Guzman. 2007. Efficacy and safety of darunavir-ritonavir compared with that of lopinavir-ritonavir at 48 weeks in treatment-experienced, HIV-infected patients in TITAN: a randomised controlled phase III trial. Lancet 370:49–58. Mammano, F., C. Petit, and F. Clavel. 1998. Resistance-associated loss of viral fitness in human immunodeficiency virus type 1: phenotypic analysis of protease and gag coevolution in protease inhibitor-treated patients. J. Virol. 72:7632–7637. Minor, W., M. Cymborowski, Z. Otwinowski, and M. Chruszcz. 2006. HKL3000: the integration of data reduction and structure solution—from diffraction images to an initial model in minutes. Acta Crystallogr. Sect. D Biol. Crystallogr. 62:859–866. Molina, J. M., C. Cohen, C. Katlama, B. Grinsztejn, A. Timerman, R. J. Pedro, T. Vangeneugden, D. Miralles, S. D. Meyer, W. Parys, and E. Lefebvre. 2007. Safety and efficacy of darunavir (TMC114) with low-dose ritonavir in treatment-experienced patients: 24-week results of POWER 3. J. Acquir. Immune Defic. Syndr. 46:24–31. Murshudov, G. N., A. A. Vagin, and E. J. Dodson. 1997. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. Sect. D Biol. Crystallogr. 53:240–255. Nalam, M. N. L., and C. A. Schiffer. 2008. New approaches to HIV protease inhibitor drug design II: testing the substrate envelope hypothesis to avoid drug resistance and discover robust inhibitors. Curr. Opin. HIV AIDS 3:642–646. Nijhuis, M., N. M. van Maarseveen, S. Lastere, P. Schipper, E. Coakley, B. Glass, M. Rovenska, D. de Jong, C. Chappey, I. W. Goedegebuure, G. Heilek-Snyder, D. Dulude, N. Cammack, L. Brakier-Gingra, J. Konvalinka, N. Parkin, H.-G. Kra ¨usslich, F. Brun-Vezinet, and C. A. Boucher. 2007. A novel substrate based HIV-1 protease inhibitor drug resistance mechanism. PLoS Med. 4:152–163.

J. VIROL. 39. Patick, A. K., M. Duran, Y. Cao, D. Shugarts, M. R. Keller, E. Mazabel, M. Knowles, S. Chapman, D. R. Kuritzkes, and M. Markowitz. 1998. Genotypic and phenotypic characterization of human immunodeficiency virus type 1 variants isolate from patients treated with the protease inhibitor nelfinavir. Antimicrob. Agents Chemother. 42:2637–2644. 40. Picchio, G., T. Vangeneugden, B. Van Baelen, E. Lefebvre, D. Miralles, and M.-P. de Be´thune. 2007. Prior utilization/resistance to amprenavir at screening has minimal impact on the 48-week response to darunavir/r in the POWER 1, 2 and 3 studies, abstr. 609. Abstr. 14th Conf. Retrovir. Opportunistic Infect., Los Angeles, CA. 41. Prejdova, J., M. Soucek, and J. Konvalinka. 2004. Determining and overcoming resistance to HIV protease inhibitors. Curr. Drug Targets Infect. Disord. 4:137–152. 42. Race, E., E. Dam, V. Obry, S. Paulous, and F. Clavel. 1999. Analysis of HIV cross-resistance to protease inhibitors using a rapid single-cycle recombinant virus assay for patients failing on combination therapies. AIDS 13:2061–2068. 43. Rhee, S. Y., W. J. Fessel, A. R. Zolopa, L. Hurley, T. Liu, J. Taylor, D. P. Nguyen, S. Slome, D. Klein, M. Horberg, J. Flamm, S. Follansbee, J. M. Schapiro, and R. W. Shafer. 2005. HIV-1 protease and reverse-transcriptase mutations: correlations with antiretroviral therapy in subtype B isolates and implications for drug-resistance surveillance. J. Infect. Dis. 192:456–465. 44. Rinehart, A. R., G. Picchio, M.-P. de Be´thune, L. B. Bacheler, T. Pattery, B. Wasikowski, and R. Falcon. 2007. Prevalence of darunavir resistance-associated mutations in samples received for routine clinical resistance testing, abstr. 44. Abstr. 5th Eur. HIV Drug Resist. Workshop, Cascais, Portugal, 28 to 30 March 2007. 45. Rose, R. B., C. S. Craik, and R. M. Stroud. 1998. Domain flexibility in retroviral proteases: structural implications for drug resistant mutations. Biochemistry 37:2607–2621. 46. Schmit, J. C., L. Ruiz, B. Clotet, A. Raventos, J. Tor, J. Leonard, J. Desmyter, E. DeClercq, and A. M. Vandamme. 1996. Resistance-related mutations in the HIV-1 protease gene of patients treated for 1 year with the protease inhibitor ritonavir (ABT-538). AIDS 10:995–999. 47. Sista, P., B. Wasikowski, P. Lecocq, T. Pattery, and L. Bacheler. 2008. The HIV-1 protease resistance mutation I50L is associated with resistance to atazanavir and susceptibility to other protease inhibitors in multiple mutational contexts. J. Clin. Virol. 42:405–408. 48. Surleraux, D. L., H. A. de Kock, W. G. Verschueren, G. M. Pille, L. J. Maes, A. Peeters, S. Vendeville, S. De Meyer, H. Azijn, R. Pauwels, M. P. de Bethune, N. M. King, M. Prabu-Jeyabalan, C. A. Schiffer, and P. B. Wigerinck. 2005. Design of HIV-1 protease inhibitors active on multidrug-resistant virus. J. Med. Chem. 48:1965–1973. 49. Surleraux, D. L., A. Tahri, W. G. Verschueren, G. M. Pille, H. A. de Kock, T. H. Jonckers, A. Peeters, S. De Meyer, H. Azijn, R. Pauwels, M. P. de Bethune, N. M. King, M. Prabu-Jeyabalan, C. A. Schiffer, and P. B. Wigerinck. 2005. Discovery and selection of TMC114, a next generation HIV-1 protease inhibitor. J. Med. Chem. 48:1813–1822. 50. Tie, Y. F., P. I. Boross, Y. F. Wang, L. Gaddis, A. K. Hussain, S. Leshchenko, A. K. Ghoshl, J. M. Louis, R. W. Harrison, and I. T. Weber. 2004. High resolution crystal structures of HIV-1 protease with a potent non-peptide inhibitor (UIC-94017) active against multi-drug-resistant clinical strains. J. Mol. Biol. 338:341–352. 51. Van Marck, H., I. Dierynck, G. Kraus, S. Hallenberger, T. Pattery, G. Muyldermans, H. Van Vlijmen, K. Hertogs, and M. P. de Bethune. 2007. Unravelling the complex resistance pathways of darunavir using bioinformatics resistance determination (BIRD). Antivir. Ther. 12:S141. 52. Volarath, P., R. W. Harrison, and I. T. Weber. 2007. Structure based drug design for HIV protease: from molecular modeling to cheminformatics. Curr. Top. Med. Chem. 7:1030–1038. 53. Williams, J. W., and J. F. Morrison. 1979. The kinetics of reversible tightbinding inhibition. Methods Enzymol. 63:437–467. 54. Winters, M. A., and T. C. Merigan. 2005. Insertions in the human immunodeficiency virus type 1 protease and reverse transcriptase genes: clinical impact and molecular mechanisms. Antimicrob. Agents Chemother. 49:2575–2582. 55. Wlodawer, A., and J. Vondrasek. 1998. Inhibitors of HIV-1 protease: a major success of structure-assisted drug design. Annu. Rev. Biophys. Biomol. Struct. 27:249–284. 56. Wu, T. D., C. A. Schiffer, M. J. Gonzales, J. Taylor, R. Kantor, S. Chou, D. Israelski, A. R. Zolopa, W. J. Fessel, and R. W. Shafer. 2003. Mutation patterns and structural correlates in human immunodeficiency virus type 1 protease following different protease inhibitor treatments. J. Virol. 77:4836–4847. 57. Yin, P. D., D. Das, and H. Mitsuya. 2006. Overcoming HIV drug resistance through rational drug design based on molecular, biochemical, and structural profiles of HIV resistance. Cell. Mol. Life Sci. 63:1706–1724. 58. Yoshimura, K., R. Kato, M. F. Kavlick, A. Nguyen, V. Maroun, K. Maeda, K. A. Hussain, A. K. Ghosh, S. V. Gulnik, J. W. Erickson, and H. Mitsuya. 2002. A potent human immunodeficiency virus type 1 protease inhibitor, UIC-94003 (TMC-126), and selection of a novel (A28S) mutation in the protease active site. J. Virol. 76:1349–1358.