Helena Danielson1 1 Department of Biochemistry and Organic Chemistry, Uppsala University, Sweden 2 Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University
Trang 1residues in hepatitis C virus nonstructural protein 3
Go¨ran Dahl1, Anja Sandstro¨m2, Eva A˚ kerblom2
and U Helena Danielson1
1 Department of Biochemistry and Organic Chemistry, Uppsala University, Sweden
2 Department of Medicinal Chemistry, Organic Pharmaceutical Chemistry, Uppsala University, Sweden
The bifunctional nonstructural protein 3 (NS3,
EC 3.4.21.98) from hepatitis C virus (HCV) is an
inter-esting enzyme biochemically, and is of importance for
anti-HCV drug discovery It has an N-terminal domain
that constitutes a serine protease with a typical
chymo-trypsin fold, and a C-terminal superfamily 2 DExH⁄
D-box RNA helicase domain Furthermore, the
prote-ase domain contains a structural zinc atom and needs
to interact with the viral nonstructural protein 4A
(NS4A) in order to be fully functional The protease
activity of NS3 is responsible for cleaving the viral
polyprotein of HCV, whereas the helicase is
responsi-ble for unwinding douresponsi-ble-stranded RNA Thus, the
two enzyme activities of the NS3 protein are involved
in critical steps of viral replication, making them both
attractive anti-HCV drug targets (for reviews, see [1] and [2])
From a biochemical point of view, it is not clear why the two enzymes are fused into a single protein The active sites of each of the enzymes are located in the respective domains, and truncated variants of NS3, containing either the protease or the helicase alone, are functional on their own However, the truncated protease has slightly different kinetic prop-erties and the helicase has a lower activity compared with the full-length enzyme [3–5], suggesting that there may be a functional justification for the con-struction
Nevertheless, for practical reasons, most researchers working on the discovery of drugs against either of the
Keywords
full length; hepatitis C virus; inhibition;
nonstructural protein 3; protease
Correspondence
U H Danielson, Department of
Biochemistry and Organic Chemistry,
Uppsala University, BMC, Box576,
SE-751 23 Uppsala, Sweden
Fax: +46 18 558431
Tel: +46 18 4714545
E-mail: Helena.Danielson@bioorg.uu.se
(Received 4 May 2007, revised 17 August
2007, accepted 25 September 2007)
doi:10.1111/j.1742-4658.2007.06120.x
This study of the full-length bifunctional nonstructural protein 3 from hep-atitis C virus (HCV) has revealed that residues in the helicase domain affect the inhibition of the protease Two residues (Q526 and H528), appar-ently located in the interface between the S2 and S4 binding pockets of the substrate binding site of the protease, were selected for modification, and three enzyme variants (Q526A, H528A and H528S) were expressed, puri-fied and characterized The substitutions resulted in indistinguishable Km values and slightly lower kcatvalues compared to the wild-type The Ki val-ues for a series of structurally diverse protease inhibitors were affected by the substitutions, with increases or decreases up to 10-fold The inhibition profiles for H528A and H528S were different, confirming that not only did the removal of the imidazole side chain have an effect, but also that minor differences in the nature of the introduced side chain influenced the charac-teristics of the enzyme These results indicate that residues in the helicase domain of nonstructural protein 3 can influence the protease, supporting our hypothesis that full-length hepatitis C virus nonstructural protein 3 should be used for protease inhibitor optimization and characterization Furthermore, the data suggest that inhibitors can be designed to interact with residues in the helicase domain, potentially leading to more potent and selective compounds
Abbreviations
HCV, hepatitis C virus; NS3, nonstructural protein 3; NS4A, nonstructural protein 4A.
Trang 2itors is not feasible, and many have apparently
discon-tinued their anti-HCV NS3 protease drug discovery
programmes However, when the first and, so far, only
crystal structure of the full-length NS3 was published,
it revealed that the protease active site was situated in
the interface between the helicase and the protease
domains, creating a well-defined binding cleft (Fig 1A)
[7] Indeed, it appears that certain residues in the
heli-case domain may interact directly with substrates or
product-based inhibitors binding in this cleft [7] As a
consequence, we have concluded that the full-length
protein is the most relevant model system for NS3
Moreover, despite the lower yields and stabilities
com-pared with those obtained with the truncated protease,
it is the protein that has been used in all of our studies
of the NS3 protease [8–13]
Nevertheless, we are interested in determining the
functional importance of the helicase domain for the
activity and inhibition of the protease, and have
focused on the identification of specific helicase
resi-dues which may be involved In the crystal structure of
the full-length enzyme, the C-terminus is located in the
substrate binding site and the helicase clearly interacts
with the C-terminus of NS3 (Fig 1B) [7] By structural
modelling of an inhibitor binding to full-length NS3,
In order to experimentally determine the importance
of these residues, we have substituted the side chains
of these residues for side chains with other functional properties, and have analysed how this affects protease activity and inhibition For the analysis of modified inhibition characteristics, a set of 11 inhibitors with varying structure, mechanism and potency was used (Fig 2) BILN 2061 was the first HCV NS3 protease inhibitor to reach clinical trials [14], but was later with-drawn because of cardiac toxicity in rhesus monkeys [15] Nevertheless, it is a useful model compound as it
is one of the most potent inhibitors of HCV NS3 today, and is a macrocyclic compound, in contrast to other inhibitors used here VX-950 was the second inhibitor to reach clinical trials and is therefore also a useful reference compound [16] It is a linear mecha-nism-based inhibitor with an electrophilic C-terminus, and is thus different mechanistically from BILN 2061 and the other compounds studied here The other inhibitors were selected from the compounds previ-ously tested against the wild-type enzyme They include tri- and tetrapeptides with either a carboxylic acid or an acyl sulfonamide as their C-terminus [11– 13], and hexapeptides with P2 and P4 residues of dif-ferent polarity and size [8,10]
H528
Q526 D81 H57 S139
Fig 1 Structure of full-length HCV NS3 (A) Complete HCV NS3 with the protease domain (red), helicase domain (blue) and NS4A cofactor fused to the N-terminus (green) (PDB: 1CU1) [7] The structural zinc atom in the protease domain and a phos-phate group in the helicase domain are also visible The interface region between the helicase and the protease domains is framed and shown in detail in (B) The cata-lytic triad, H57, D81 and S139, in the pro-tease domain, and the conserved helicase residues, Q526 and H528, are shown in a ball-and-stick representation The blue strand represents the C-terminus of NS3, and shows the substrate binding site of the protease.
Trang 3Fig 2 Structures of the studied inhibitors The arrows indicate the a-carbon of the P1 residue of the inhibitor The P2, P3, P4, P5 and P6 side chains can be deduced from the nomenclature by Schechter and Berger [20].
Trang 4Q526 and H528 were substituted for residues for side
chains with different interaction characteristics Three
single mutants, coding for the enzyme variants Q526A,
H528A and H528S, were created The alanine
substitu-tion replaces the original side chain with a small
hydrophobic moiety, whereas the serine substitution
also introduces alternative hydrogen bonding
capabili-ties
Full-length Q526A, H528A and H528S HCV NS3
were successfully constructed, expressed and purified
Expression resulted in around 150–200 lg of enzyme
per litre of cell culture for all variants and the
wild-type Purities were over 98% in all cases, as judged by
the single band on a silver-stained SDS⁄ PAGE gel
(Fig 3) An automatic chromatographic procedure
gave a highly reproducible purification
Kinetic characterization of NS3 protease variants
The three substitutions in the helicase domain of NS3
did not influence the Km values significantly, but kcat
values were reduced (Table 1) Assuming a normal
dis-tribution, the wild-type has a significantly higher kcat
value compared with the helicase variants at a 95% confidence level
Inhibition of NS3 protease variants The effect of changes in the helicase residues on the inhibition of the protease was investigated for the three variants and a series of inhibitors Inhibition constants (Ki) were determined for all variants and inhibitors (Fig 4)
The data showed that the substitutions of Q526 and H528 did not generally have a large or consistent effect
on the inhibition, but there were some notable effects and trends The Q526A and H528A substitutions had little impact on the inhibition of the enzyme by BILN 2061, but the H528S substitution reduced the potency of the inhibitor As a result, the Ki value for BILN 2061 differed 10-fold depending on whether the residue in position 528 was alanine or serine A similar profile was also seen with compound 9, the second most potent inhibitor By contrast, VX-950 was not affected by any of the investigated substitutions The effect of substituting either Q526 or H528 was found to be more diverse for the less potent com-pounds The inhibition by compounds 4 and 6 was not affected to any large extent by any of the substitutions, whereas compounds 2 and 5 became more effective for all variants The H528A substitution decreased the Ki values for compounds 1 and 3, but the Q526A and H528S substitutions had no effect All substitutions reduced the inhibition by compound 7, and both Q526A and H528A reduced it for compound 8 It should be noted that the differences between the values were larger than those deduced from the differences in bar lengths in the logarithmic graph
Discussion
This study has shown that residues in the helicase domain of full-length NS3 from HCV can directly influence the protease Modifications of the side chains
of two residues (Q526 and H528) were found to influ-ence the effect of some protease inhibitors The Km
97 kDa 66 45
30 20.1 14.4
Fig 3 Purification of HCV NS3 protease A silver-stained
SDS ⁄ PAGE gel from a typical purification procedure (in this case
the H528A variant): Lane 1, immobilized ion affinity chromatography
(IMAC) flow-through; lane 2, IMAC eluate; lane 3, IMAC eluate
after buffer change; lane 4, PolyU flow-through; lane 5, PolyU
elu-ate; lane 6, molecular weight marker (LMW-SDS Marker kit, GE
Healthcare).
kcat(s ) 0.53 ± 0.03 0.30 ± 0.04 0.32 ± 0.02 0.37 ± 0.04
k cat ⁄ K m (l M )1Æs)1)
1.96 ± 0.45 0.79 ± 0.37 1.10 ± 0.28 0.84 ± 0.58
Trang 5and kcat⁄ Kmvalues for the enzyme variants were
indis-tinguishable from those of the wild-type, even though
the kcat values were significantly lower However, this
is not considered to be of great importance and could
be a result of differences in the active enzyme
concen-tration
The effect of changing residues in the helicase
domain on inhibition was dependent on both the
sub-stitution and the inhibitor Subsub-stitution of Q526 with
alanine had only minor effects, despite earlier
specula-tions that this residue is located in the interface
between the S2 and S4 binding pockets of the
full-length enzyme, and therefore would be critical for
interaction with product-based inhibitors [7,10]
Substi-tuting H528 with alanine resulted in increased
inhibi-tion for all compounds except three, whose inhibiinhibi-tion
was more or less unchanged The increase was largest
for compounds with a P4 side chain Under the present
conditions (pH 7.4), H528 is expected to be essentially
uncharged and to act either as a hydrogen bond donor,
interacting with the carbonyl oxygen of the P4 residue
on the inhibitor, or as a hydrogen bond acceptor for
the P4 NH of the inhibitor Although some of the
inhibitors were only tripeptides, they all had at least
the equivalent of a P4 carbonyl group, and the
substi-tution of H528 with alanine or serine could thus be
expected to weaken the inhibition, but not to increase
it Apparently, other interactions play a significant
role, where, for example, the hydrophobic alanine
resi-due contributes more than a potential hydrogen bond
from the histidine When H528 was replaced with
ser-ine, the effect was more varied; both enhanced and
reduced inhibition were detected However, compounds
lacking a P4 residue and containing large P2 side
chains were affected the most by this substitution,
lead-ing to reduced potencies This effect became less and
less pronounced as the overall potency of the inhibitors
decreased It is not clear why the nature of the residue
at position 528 results in such different effects, espe-cially for BILN 2061, but these observations imply that the initial structural model [10] does not adequately describe the forces involved It should be noted that, even though a 10-fold change in Ki is significant, the change in binding energy is small and equal to approx-imately the loss of a hydrogen bond [17]
Drug discovery is a technique in which modified variants of enzymes are often used in order to increase stability, yield or other parameters to produce an effi-cient process However, it is critical that the modifica-tion does not compromise the informamodifica-tion obtained when using the variant for drug discovery purposes Although the data presented here are based on a lim-ited number of compounds and only two helicase resi-dues, the experimental observations indicate that the helicase can influence protease inhibitors Therefore, the use of truncated NS3, containing only the protease domain, for the identification and optimization of HCV NS3 protease inhibitors is a procedure that may
be inappropriate The data also suggest that inhibitors can be designed to interact with residues in the helicase domain, potentially leading to more potent and selec-tive compounds
Conclusion
The strategy used in this study has demonstrated that specific amino acids in the helicase of full-length HCV NS3 can influence the inhibition of the protease As a consequence, anti-HCV protease drug discovery using full-length HCV NS3 as a model system for inhibition studies may be more appropriate than using the trun-cated protease In addition, protease inhibitor design can make use of the helicase domain as a novel anchor point for inhibitors
Fig 4 Effect of HCV NS3 helicase domain
substitutions Q526A, H528A and H528S on
the potency of NS3 protease inhibitors.
Error bars represent the standard deviations.
The scale on the y-axis ranges from 10 p M
(top) to 10 l M (bottom) on a logarithmic
scale Blue, wild-type; red, Q526A; yellow,
H528A; green, H528S.
Trang 6GCAGACCATCTTGAAT-3¢), H528A forward primer
(5¢-TCCCGTGTGTCAAGACGCTCTTGAAT-3¢) or H528S
forward primer (5¢-TCCCGTGTGTCAAGACTCTCTTG
AAT-3¢) and reverse primer (5¢-AGTCCCGGGGTGTT
CATGTATGCTC-3¢) Each PCR vial contained 50 ng of
template DNA, 0.2 mm dNTPs, 0.2 lm forward primer,
0.2 lm reverse primer (Thermo Electron GmbH, Ulm,
Ger-many) and 1.25 U Pfu Turbo DNA polymerase
(Strata-gene, La Jolla, CA, USA) in 50 lL Pfu Buffer [200 mm
Tris⁄ HCl pH 8.8, 20 mm MgSO4, 100 mm KCl, 100 mm
(NH4)2SO4, 1% Triton X-100, 1 mgÆmL)1 nuclease free
BSA (Stratagene)] The primers were phosphorylated at the
5¢-end to promote recircularization The vials were
sub-jected to 1 min of melting at 95C, 1 min of annealing at
73C and 15 min of extension at 72 C in 25 cycles using a
thermal cycler (GeneAmp PCR system 2400, Perkin-Elmer,
Boston, MA, USA) After this, 20 U DpnI restriction
enzyme (New England Biolabs, Beverly, MA, USA) was
added and the vials were left at 37C for 1 h The DNA
was then purified on a 1% Tris, borate, EDTA agarose gel,
and fragments corresponding to the PCR products were cut
out and purified using the GeneClean gel extraction kit
(Qbiogene, Illkirch Cedex, France) The purified PCR
prod-uct was then blunt-end ligated using the T4 rapid DNA
ligation kit (Roche Diagnostics Scandinavia AB, Bromma,
Sweden) and transformed to thermocompetent TOP10 cells
The DNA was sequenced using a Mega BACE 1000
sequencer (GE Healthcare, Uppsala, Sweden)
Expression and purification
The expression and purification of HCV NS3 variants were
performed essentially as described previously [9] That is,
6· 500 mL TOP10 cells with A600¼ 0.7 were induced
with 0.002% (w⁄ v) l-(+)-arabinose overnight at 21 C,
150 r.p.m The cells were then harvested and resuspended
in lysation buffer [25 mm Hepes pH 7.6, 0.3 m NaCl, 20%
glycerol, 10 mm b-mercaptoethanol and 0.1% Chaps
(Ana-trace, Maumee, OH, USA)] and 2 lgÆmL)1 DNAse I
(Roche Diagnostics Scandinavia AB) Lysation was
initi-ated by adding 1 mgÆmL)1 lysozyme (Sigma-Aldrich
Swe-den AB, Stockholm, SweSwe-den), and thereafter the cells were
sonicated The lysate was centrifuged at 25 000 g for
40 min, and the cleared supernatant was loaded on to a
10 mL chelating Sepharose fast flow gel (GE Healthcare)
loaded with Ni2+, and washed with buffer A (50 mm Hepes
pH 7.6, 0.3 m NaCl, 26% glycerol, 10 mm
b-mercaptoetha-nol and 0.1% Chaps), buffer A supplemented with 1 m
HiTrap desalting column (GE Healthcare) The sample was loaded on to a 2.5 mL PolyU gel (GE Healthcare), and the gel was washed with buffer B before purified enzyme was finally eluted with buffer B supplemented with 1 m NaCl All chromatographic steps were performed with an A¨KTA Explorer (GE Healthcare) The eluted fractions with highest absorbance at 280 nm were pooled and stored at ) 80 C The protein concentration was determined using a Brad-ford-based assay (Bio-Rad, Sundbyberg, Sweden), and the purity was estimated with SDS⁄ PAGE using the PHAST system and 8–25% precast PHAST gels and silver staining (GE Healthcare)
Enzymatic characterization
Protease activity was measured as described previously [9] That is, the hydrolysis of a depsipeptide substrate, Ac-Asp-Glu-Asp(EDANS)-Glu-Glu-Abu-w-[COO]Ala-Ser-Lys(DABCYL)-NH2 (AnaSpec, San Jose, CA, USA), was recorded continuously over time with a fluorescence plate reader (Fluoroskan Ascent Labsystems, Stockholm, Swe-den) Each sample well contained 276.5 lL assay buffer (50 mm Hepes pH 7.5, 10 mm dithiothreitol, 40% (w⁄ v) glycerol, 0.1% n-octyl-b-d-glucoside), 9.25 lL dimethylsulf-oxide, 0.75 lL of 10 mm peptide cofactor KKGSVVIV-GRIVLSGK in dimethylsulfoxide and 3.5 lL of 6 lgÆmL)1 NS3 (1 nm final concentration), and was incubated at 30C for 10 min before the reaction was started by the addition
of 10 lL substrate to a final concentration between 0.25 and 4 lm All measurements were performed in triplicate
Kmand kcatvalues were estimated by fitting the Michaelis– Menten equation to the data by simulated annealing (GOSA, Bio-Log, Ramonville, France) kcat⁄ Kmvalues were calculated from the estimated kcatand Kmvalues
Inhibitors and inhibition measurements
Eleven inhibitors were used in this study (Fig 2) They included the clinical compounds BILN 2061 [14] and
VX-950 [16], compounds 2, 3, 4, 5 (compounds 16, 4, 9 and 1, respectively, in [10]), compound 6 (compound 18a in [13]), compounds 7, 8, 9 (compound 20, 29 and 31, respectively,
in [12]) and the commercially available reference compound
1 (Product no N-1725, Bachem, Weil am Rein, Germany) (entry 14 in Table 4 in [18])
The inhibitors were dissolved in dimethylsulfoxide and preincubated with enzyme and NS4A cofactor at 30C for 10 min in the same buffer conditions as stated for the
Trang 7activity assay The reaction was started by the addition of
10 lL substrate to a final concentration of 0.5 lm Pilot
measurements over a large concentration range were
per-formed in order to estimate IC20 and IC80 Six inhibitor
concentrations between IC20and IC80with constant enzyme
and substrate amounts were used All measurements were
performed in triplicate The Ki and Vmax values were
esti-mated by fitting Eqn (1) (adapted from [19]) to the data
using simulated annealing (GOSA, Bio-Log)
VỬ
Vmax
[E]
ơS
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1
Km
Ki
ợđơI ơEỡ
đKmợ ơSỡ
0
@
1 A
2
ợ 4 ơE
đKmợ ơSỡ Km
Ki
v u t
2
6
K1
m
Ki
ợđơI ơEỡ
đKmợ ơSỡ
0
@
1 A
3
Acknowledgements
We would like to thank Gun Stenberg for her help in
primer design, and Robert Roẽnn and Pernilla Oẽrtqvist
(Department of Medicinal Chemistry, Pharmaceutical
Organic Chemistry, Uppsala University, Sweden) for
compounds 6, 7, 8 and 9 We would also like to thank
Boehringer Ingelheim for the kind gift of BILN 2061
and the VIRGIL DrugPharm Team for VX-950 This
work was conducted with support from the VIRGIL
European Union Network of Excellence
References
1 De Francesco R & Migliaccio G (2005) Challenges and
successes in developing new therapies for hepatitis C
Nature 436, 953Ờ960
2 Frick DN (2007) The hepatitis C virus NS3 protein: a
model RNA helicase and potential drug target Curr
Issues Mol Biol 9, 1Ờ20
3 Sali DL, Ingram R, Wendel M, Gupta D, McNemar C,
Tsarbopoulos A, Chen JW, Hong Z, Chase R, Risano
C et al (1998) Serine protease of hepatitis C virus
expressed in insect cells as the NS3⁄ 4A complex
Biochemistry 37, 3392Ờ3401
4 Frick DN, Rypma RS, Lam AM & Gu B (2004) The
nonstructural protein 3 protease⁄ helicase requires an
intact protease domain to unwind duplex RNA
effi-ciently J Biol Chem 279, 1269Ờ1280
5 Zhang C, Cai Z, Kim YC, Kumar R, Yuan F, Shi PY,
Kao C & Luo G (2005) Stimulation of hepatitis C virus
(HCV) nonstructural protein 3 (NS3) helicase activity
by the NS3 protease domain and by HCV
RNA-depen-dent RNA polymerase J Virol 79, 8687Ờ8697
6 Kim JL, Morgenstern KA, Lin C, Fox T, Dwyer
MD, Landro JA, Chambers SP, Markland W, Lepre
CA, OỖMalley ET et al (1996) Crystal structure of
the hepatitis C virus NS3 protease domain complexed with a synthetic NS4A cofactor peptide Cell 87, 343Ờ 355
7 Yao N, Reichert P, Taremi SS, Prosise WW & Weber
PC (1999) Molecular views of viral polyprotein process-ing revealed by the crystal structure of the hepatitis C virus bifunctional protease-helicase Structure 7, 1353Ờ 1363
8 Johansson A, Hubatsch I, A˚kerblom E, Lindeberg G, Winiwarter S, Danielson UH & Hallberg A (2001) Inhi-bition of hepatitis C virus NS3 protease activity by product-based peptides is dependent on helicase domain Bioorg Med Chem Lett 11, 203Ờ206
9 Poliakov A, Hubatsch I, Shuman CF, Stenberg G & Danielson UH (2002) Expression and purification of recombinant full-length NS3 protease-helicase from a new variant of hepatitis C virus Protein Expr Purif 25, 363Ờ371
10 Johansson A, Poliakov A, A˚kerblom E, Lindeberg G, Winiwarter S, Samuelsson B, Danielson UH & Hallberg
A (2002) Tetrapeptides as potent protease inhibitors of hepatitis C virus full-length NS3 (protease-helicase⁄ NTPase) Bioorg Med Chem 10, 3915Ờ3922
11 Johansson A, Poliakov A, A˚kerblom E, Wiklund K, Lindeberg G, Winiwarter S, Danielson UH, Samuelsson
B & Hallberg A (2003) Acyl sulfonamides as potent protease inhibitors of the hepatitis C virus full-length NS3 (protease-helicase⁄ NTPase): a comparative study
of different C-terminals Bioorg Med Chem 11, 2551Ờ 2568
12 Roẽnn R, Sabnis YA, Gossas T, A˚kerblom E, Danielson
UH, Hallberg A & Johansson A (2006) Exploration of acyl sulfonamides as carboxylic acid replacements in protease inhibitors of the hepatitis C virus full-length NS3 Bioorg Med Chem 14, 544Ờ559
13 Oẽrtqvist P, Peterson SD, A˚kerblom E, Gossas T, Sabnis
YA, Fransson R, Lindeberg G, Danielson UH, KarleƠn
A & Sandstroẽm A (2006) Phenylglycine as a novel P2 scaffold in hepatitis C virus NS3 protease inhibitors Bioorg Med Chem 15, 1448Ờ1474
14 Lamarre D, Anderson PC, Bailey M, Beaulieu P, Bolger
G, Bonneau P, Boẽs M, Cameron DR, Cartier M, Cord-ingley MG et al (2003) An NS3 protease inhibitor with antiviral effects in humans infected with hepatitis C virus Nature 426, 186Ờ189
15 Reiser M, Hinrichsen H, Benhamou Y, Reesink HW, Wedemeyer H, Avendano C, Riba N, Yong CL, Nehmiz G & Steinmann GG (2005) Antiviral efficacy of NS3-serine protease inhibitor BILN-2061 in patients with chronic genotype 2 and 3 hepatitis C Hepatology
41, 832Ờ835
16 Perni RB, Almquist SJ, Byrn RA, Chandorkar G, Chaturvedi PR, Courtney LF, Decker CJ, Dinehart K, Gates CA, Harbeson SL et al (2006) Preclinical profile
of VX-950, a potent, selective, and orally bioavailable
Trang 8Ingenito R, Cortese R, De Francesco R, Steinku¨hler
C & Pessi A (1998) Potent peptide inhibitors of
site in proteases I Papain Biochem Biophys Res Commun 27, 157–162