Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 3 Go ¨ ran Dahl 1 , Anja Sandstro ¨ m 2 , Eva A ˚ kerblom 2 and U. Helena Danielson 1 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 double-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 K m values and slightly lower k cat values compared to the wild-type. The K i 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. FEBS Journal 274 (2007) 5979–5986 ª 2007 The Authors Journal compilation ª 2007 FEBS 5979 two enzyme activities use truncated forms of NS3 for their studies. This has significant consequences. For example, in the crystal structure of the truncated pro- tease domain, the protease substrate binding site is located on the surface of the protein, and it appears to be shallow and featureless [6]. This has led researchers to believe that the design of potent and selective inhib- itors 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, we found that most of the contacts were with the pep- tide backbone, but that the side chains of residues Q526 and H528 appeared to interact directly with the P2 and P4 residues of the inhibitor [10]. Q526 and H528 are conserved between different strains of HCV [9], supporting the hypothesis that they may play an important role in the functionality of 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 AB 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. Hepatitis C virus NS3 protease ⁄ helicase G. Dahl et al. 5980 FEBS Journal 274 (2007) 5979–5986 ª 2007 The Authors Journal compilation ª 2007 FEBS Fig. 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]. G. Dahl et al. Hepatitis C virus NS3 protease ⁄ helicase FEBS Journal 274 (2007) 5979–5986 ª 2007 The Authors Journal compilation ª 2007 FEBS 5981 Results Strategy and enzyme production In order to determine the functional significance of the side chains of helicase residues hypothesized to interact directly with the substrate or inhibitors of the protease, Q526 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 K m values significantly, but k cat values were reduced (Table 1). Assuming a normal dis- tribution, the wild-type has a significantly higher k cat 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 (K i ) 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 K i 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 K i 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 K m 123456 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). Table 1. Kinetic parameters for all enzyme variants studied. The standard deviations from three determinations are provided. Kinetic parameter Enzyme variant Wild-type Q526A H528A H528S K m (lM) 0.27 ± 0.06 0.38 ± 0.17 0.29 ± 0.07 0.44 ± 0.30 k cat (s )1 ) 0.53 ± 0.03 0.30 ± 0.04 0.32 ± 0.02 0.37 ± 0.04 k cat ⁄ K m (lM )1 Æs )1 ) 1.96 ± 0.45 0.79 ± 0.37 1.10 ± 0.28 0.84 ± 0.58 Hepatitis C virus NS3 protease ⁄ helicase G. Dahl et al. 5982 FEBS Journal 274 (2007) 5979–5986 ª 2007 The Authors Journal compilation ª 2007 FEBS and k cat ⁄ K m values for the enzyme variants were indis- tinguishable from those of the wild-type, even though the k cat 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. Substitution 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 inhibition 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 K i 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 information 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 lM (bottom) on a logarithmic scale. Blue, wild-type; red, Q526A; yellow, H528A; green, H528S. G. Dahl et al. Hepatitis C virus NS3 protease ⁄ helicase FEBS Journal 274 (2007) 5979–5986 ª 2007 The Authors Journal compilation ª 2007 FEBS 5983 Experimental procedures PCR Mutations in the helicase domain of the previously used gene construct for full-length HCV NS3 [9] were introduced by PCR using Q526A forward primer (5¢-TCCCGTGTGT GCAGACCATCTTGAAT-3¢), H528A forward primer (5¢-TCCCGTGTGTCAAGACGCTCTTGAAT-3¢) or H 52 8S 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 MgSO 4 , 100 mm KCl, 100 mm (NH 4 ) 2 SO 4 , 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 95 °C, 1 min of annealing at 73 °C 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 37 °C 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 A 600 ¼ 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, Sweden), 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 Ni 2+ , 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 NaCl and, finally, buffer A supplemented with 50 mm imid- azole, before partially purified enzyme was eluted with buf- fer A supplemented with 250 mm imidazole. The eluted fractions with highest absorbance at 280 nm were pooled and the buffer was changed to buffer B [25 mm Hepes pH 7.6, 0.2 m NaCl, 20% glycerol, 10 mm b-mercaptoetha- nol and 0.1% n-octyl-b-d-glucoside (Anatrace)] using a 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]Al a-Ser- Lys(DABCYL)-NH 2 (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 lLof10mm peptide cofactor KKGSVVIV- GRIVLSGK in dimethylsulfoxide and 3.5 lLof6lgÆmL )1 NS3 (1 nm final concentration), and was incubated at 30 °C 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. K m and k cat values were estimated by fitting the Michaelis– Menten equation to the data by simulated annealing (GOSA, Bio-Log, Ramonville, France). k cat ⁄ K m values were calculated from the estimated k cat and K m values. 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 30 °C for 10 min in the same buffer conditions as stated for the Hepatitis C virus NS3 protease ⁄ helicase G. Dahl et al. 5984 FEBS Journal 274 (2007) 5979–5986 ª 2007 The Authors Journal compilation ª 2007 FEBS activity 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 IC 20 and IC 80 . Six inhibitor concentrations between IC 20 and IC 80 with constant enzyme and substrate amounts were used. All measurements were performed in triplicate. The K i and V max values were esti- mated by fitting Eqn (1) (adapted from [19]) to the data using simulated annealing (GOSA, Bio-Log). V ¼ V max [E]  ½S 2  ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi 1 K m K i þ ð½IÀ½EÞ ðK m þ½SÞ 0 @ 1 A 2 þ 4 ½E ðK m þ½SÞ K m K i v u u u t 2 6 4 À 1 K m K i þ ð½IÀ½EÞ ðK m þ½SÞ 0 @ 1 A 3 5 ð1Þ Acknowledgements We would like to thank Gun Stenberg for her help in primer design, and Robert Ro ¨ nn and Pernilla O ¨ rtqvist (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. 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Biochim Biophys Acta 185, 269–286. 20 Schechter I & Berger A (1967) On the size of the active site in proteases. I. Papain. Biochem Biophys Res Commun 27, 157–162. Hepatitis C virus NS3 protease ⁄ helicase G. Dahl et al. 5986 FEBS Journal 274 (2007) 5979–5986 ª 2007 The Authors Journal compilation ª 2007 FEBS . chain in uenced the charac- teristics of the enzyme. These results indicate that residues in the helicase domain of nonstructural protein 3 can in uence. Effects on protease inhibition by modifying of helicase residues in hepatitis C virus nonstructural protein 3 Go ¨ ran Dahl 1 , Anja