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A High-Throughput Radiometric Assay for Hepatitis C Virus NS3 Protease
Analytical Biochemistry 266, 192–197 (1999) Article ID abio.1998.2948, available online at http://www.idealibrary.com on
A High-Throughput Radiometric Assay for Hepatitis C Virus NS3 Protease Mauro Cerretani, Laura Di Renzo, Sergio Serafini, Alessandra Vitelli, Nadia Gennari, Elisabetta Bianchi, Antonello Pessi, Andrea Urbani, Stefano Colloca, Raffaele De Francesco, Christian Steinku¨hler, and Sergio Altamura 1 Istituto di Ricerche di Biologia Molecolare (IRBM) “P. Angeletti,” 00040 Pomezia, Rome, Italy
Received July 10, 1998
A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed. The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein. The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3–NS4A, NS4A–NS4B, NS4B–NS5A, and NS5A–NS5B junctions. To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A–NS4B junction. Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin. The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot. Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components. We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way. Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease. © 1999 Academic Press
Hepatitis C virus (HCV) 2 is considered the major causative agent of human posttransfusion and spo1
To whom correspondence should be addressed at IRBM, Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy. Fax: 1 39-691093225. E-mail: [email protected]. 2 Abbreviations used: HCV, hepatitus C virus; DTT, dithiothreitol; DMSO, dimethyl sulfoxide; TFA, trifluoroacetic acid. 192
radic non-A, non-B hepatitis (1, 2). The number of chronic carriers is estimated to be about 400 million and chronic infection may lead to development of liver cirrhosis and hepatocellular carcinoma (3). Protective vaccination is not available for HCV and the only established therapy is interferon treatment. However, interferon is successful only in a limited number of patients and has significant side effects (4, 5). For this reason, there is a considerable attention on understanding HCV replication and developing more effective anti-HCV therapies. HCV (6) is related to flavi- and pestiviruses in its genetic organization and is classified as a particular genus of the family Flaviviridae. HCV exists in many distinct variants: a total of 6 major genotypes and at least 11 subtypes have been recognized (7). The HCV virion has a positive-stranded RNA genome of 9.5 kb. This genome contains a single large open reading frame encoding for a polyprotein of 3010 –3030 residues with the gene order: 59-C-E1–E2–NS2–NS3– NS4A–NS4B–NS5A–NS5B-39 (1, 8 –10). This polyprotein is processed into at least 9 different mature gene products. Structural proteins, C (nucleocapsid protein) and E1 and E2 (envelope proteins), are cleaved by host cell signal peptidase. Processing events in the nonstructural region depend on the activity of virally encoded proteases (11, 12). Accordingly, cleavage at the NS2–NS3 junction is accomplished by a zinc-dependent enzymatic activity encoded between NS2 and NS3 (12). The N-terminal ;200 amino acids of the NS3 protein harbor a serine protease activity which is essential for all cleavages downstream of NS3 (11, 14 – 17). The C-terminal two-thirds of the NS3 protein contains an RNA helicase (18). The separately expressed helicase and protease domains of NS3 exhibit their respective activities in vitro (19 –22). The structure of NS3 protease was determined by X-ray crystallogra0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
HEPATITUS C VIRUS PROTEASE ASSAY
phy (23–25). According to the crystallographic data, the protease folds in a trypsin-like fold, consisting of two b-barrel domains and a catalytic triad of His-57, Asp-81, and Ser-139. Unexpectedly, the NS3 protease was found to also harbor a structural zinc-binding site (26). Many efforts have been made to characterize the mechanism by which the NS3 protease domain processes the downstream gene products. These studies have shown that NS3/NS4A cleavage occurs rapidly and in cis while the others occur in trans (NS4A/4B, NS4B/5A, NS5A/5B). To display its full activity, the NS3 protease must bind to the viral NS4A protein that acts as a cofactor, enhancing the proteolytic efficiency of NS3 (27–32). It has been shown that all the effects of NS4A can be efficiently mimicked by peptides encompassing residues 21–34 of the central region of NS4A (19, 21, 29 –32). The crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator domain reveals that the NS4A peptide intercalates within a b sheet of the enzyme core (24, 25). Because proteolytic processing of viral precursor protein is likely to be required for HCV replication, the viral protease is considered a primary target for antiviral drug discovery. Several NS3 protease assays have been described so far including a cell-based assay (33) and cell-free in vitro assays (34, 35). These assays are intrinsically unsuitable for mechanistic studies of inhibitors and have a low throughput. Recently, assays relying on the cleavage of peptidic substrates by a purified enzyme and chromatographic (21, 36), colorimetric (37) or fluorescence detection (38) of the cleavage products have been described. These assays permit a detailed characterization of enzyme inhibitors but suffer from low throughput, in the case of HPLC-based assays (21, 36), sensitivity to interferences, in the case of the fluorescence assay (38), or a very laborious assay protocol (37). We report here the development of a sensitive, discontinuous radiometric screening assay for NS3 proteolytic activity. The assay is entirely performed in microtiter plates, thus permitting facile and rapid high-throughput screening for NS3 protease inhibitors combined with the possibility to perform detailed mechanistic studies in the same assay format. Its sensitivity allows the accurate characterization of inhibitors with low-nanomolar affinities for the NS3 protease. Furthermore, the possibility of assaying a large number of samples was employed to screen for natural protease variants with improved buffer requirements. MATERIALS AND METHODS
NS3 protease. The serine protease domain of NS3 (amino acids 1–180, from HCV Bk, H, or J strain) was purified as previously described (26). The proteins were stored in aliquots at the concentration of 10 –30 mM in
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25 mM sodium phosphate, pH 7.5, 50% glycerol, 0.5% Chaps, 30 mM DTT at 280°C after shock–freezing in liquid nitrogen. 3 H-labeled peptide substrate ( 3H-Pep4AB). The peptide substrate 3H-Pep4AB (Ac-DEEMECASHLPYK-(eAc 3H)-NH 2), based on the sequence of the NS4AB cleavage site of the HCV polyprotein, was custom synthesized by Amersham. Dry powder of 3H-Pep4AB was dissolved in 100% DMSO and stored in aliquots at 280°C. The original stock had a specific activity of 17 Ci/mmol. Peptide substrate (Pep4AB). Peptide substrate Pep4AB (Ac-DEEMECASHLPYK-(e-Ac)-NH 2) was obtained from Peptides International Inc. Dry powder of Pep4AB was dissolved in deionized water at a concentration of 10 –15 mM. The concentration was confirmed by quantitative amino acid analysis. Aliquots were stored at 280°C. NS4A cofactor peptide (Pep4A). The NS4A cofactor peptide Pep4A, harboring residues 21–34 of NS4A and an N-terminal lysine tag, (H-KKKGSVVIVGRIILSGRNH 2) was purchased from Peptides International Inc. Dry powder was dissolved in water and concentration was confirmed by quantitative amino acid analysis. Aliquots were stored at 280°C. Chemicals. Fractogel TSK-650S DEAE resin was purchased from TOSOH (No. 07472). The original stock Fractogel TSK-650S DEAE resin, supplied as a suspension in 1 M NaCl, 20% ethanol, was washed with 10 mM Tris/HCl, pH 7.5, until the conductivity of the supernatant was the same as that of the washing buffer. After centrifugation at 2300 rpm for 2 min the resin was resuspended at a concentration of 20% (v/v) in 10 mM Tris/HCl, pH 7.5. The final concentration of resin in the assay was 10% (v/v). Microscint 40 was purchased from Packard (No. 6013646). All other chemicals were obtained from Sigma Chemical Co. HPLC assay. Cleavage assays were performed in 57 ml 50 mM Hepes, pH 7.5, 0.05% Triton X-100, 15% glycerol, 10 mM DTT. Buffer solutions containing 15 mM Pep4A were preincubated for 10 min with 10 nM NS3 protease and reactions were started by addition of 3 ml substrate peptide Pep4AB. Six duplicate data points at different substrate concentrations were used to calculate kinetic parameters. Incubation times were chosen to obtain ,7% substrate conversion and reactions were stopped by addition of 40 ml 1% TFA. Cleavage of peptide substrates was determined by HPLC using a Merck–Hitachi chromatograph equipped with an autosampler. Eighty-microliter samples were injected on a Lichrospher C18 reversed-phase cartridge column (4 3 75 mm, 5 mm, Merck) and fragments were separated using a 10 – 40% acetonitrile gradient at 5%/ min using a flow rate of 2.5 ml/min. Peak detection was
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accomplished by monitoring both the absorbance at 220 nm and tyrosine fluorescence (l ex 5 260 nm, l em 5 305 nm). Cleavage products were quantitated by integration of chromatograms with respect to appropriate standards. Kinetic parameters were calculated from nonlinear least-squares fit of initial rates as a function of substrate concentration with the help of a Kaleidagraph software, assuming Michaelis–Menten kinetics. Radiometric enzyme assay. The enzymatic reaction was performed in 96-well clear microtiter plates (Costar 96F polypropilene microplate). NS3 (10 nM) and 15 mM Pep4A in 50 mM Hepes, pH 7.5, 15% glycerol, 0.05% Triton X-100, 30 mM DTT, were preincubated with DMSO or inhibitors were dissolved in DMSO for 5 min at room temperature. The final concentration of DMSO in the assay was 10%. The reaction was started by the addition of 10 ml of substrate solution such that a final concentration of 10 mM Pep4AB and 80 nM 3H-Pep4AB (150,000 cpm/well) in 10 mM Hepes, pH 7.5, was obtained. The final volume of the reaction was 100 ml. Plates were sealed with an acetate tape and incubated for 20 min at room temperature under gentle shaking with an orbital shaker. The reaction was stopped by adding 100 ml of 20% (v/v) Fractogel TSK-650S DEAE resin and shaking was continued for 10 min at room temperature. The plate was than centrifuged at 2300 rpm for 2 min at room temperature. Thirty microliters of the reaction solution was transferred to a 96-well microtiter plate (Packard Picoplate). Two hundred fifty microliters of scintillation coktail (Microscint 40) was added and after vigorous shaking for at least 4 h at room temperature the radioactivity was measured in a Packard Top Count b-counter. Determination of K i and IC 50 values. K i values of inhibitors were calculated from substrate titration experiments performed in the presence of increasing amounts of inhibitor. Experimental data sets were simultaneously fitted to Eq. [1] using a multicurve fit macro with the help of a Sigmaplot software: V 5 ~V maxS!/~K m ~1 1 K i /I! 1 S!.
[1]
IC 50 values were calculated using a two-parameter logistic function, according to Eq. [2]: %activity 5 ~100/~1 1 ~@I#/IC 50 ! s !!,
[2]
where [I] is the inhibitor concentration and s is the slope factor of the curve. RESULTS AND DISCUSSION
Radiometric assay development for NS3 protease activity. We developed a simple and efficient radiometric assay for the proteolytic activity of NS3 protease,
monitoring the formation of an 3H-labeled product, generated from cleavage by the enzyme of the Pep4AB substrate. The Pep4AB substrate is a synthetic radiolabeled peptide that mimicks the NS4A/NS4B junction, having the sequence Ac-DEEMECASHLPYK-(e-Ac 3H)NH 2. It has been demonstrated that the protease cleaved this peptide with high efficiency between cysteine and alanine (21), yielding two products with different physicochemical properties. The noncleaved substrate and the N-terminal product (Ac-DEEMECOH) carry net negative charges, whereas the C-terminal fragment, being amidated, carries positive charges on the new N-terminus and the histidine residue. Addition of a TSK-650S DEAE ion exchange resin permitted the quantitative capture of the two negatively charged species in solution, which were then separated by centrifugation from the C-terminal proteolysis product. Detection of this product was rendered possible by previous introduction in the substrate of an 3H label via a C-terminal lysine residue, that was acetylated with 3H-labeled acetic anhydride (NH 2-ASHLPYK-(eAc 3H)-NH 2). It was thus possible to monitor the reaction by simple counting of the supernatant after addition of the resin. To define standard conditions for measuring proteolytic activity of NS3 protease in our assay, we took advantage of the possibility to perform the assay in a microplate format, thus permitting the rapid analysis of a very large number of samples. We used proteins encompassing the protease domain of three different isolates of HCV (Bk, J, and H) and determined the relative activities of these enzymes, in the presence of 15 mM Pep4A cofactor, by simultaneously varying the glycerol and detergent concentration of the buffer. This was done since both glycerol and detergent were previously shown to stabilize the NS3 protease domain of the Bk isolate (21). An example of a simultaneous titration of both Triton X-100 and glycerol for the Bk enzyme is shown in Fig. 1. This enzyme turned out to require 40% glycerol for optimal activity, leading to high viscosity of optimal activity buffers with concomitant difficulties in reproducible pipetting of buffer solutions. Analogous experiments were performed using different detergents and proteases. While both Bk- and H-strain proteases require glycerol concentrations exceeding 40% and Triton X-100 concentration of 0.5%, the J-strain enzyme has an activity optimum at 15% glycerol and 0.12% Triton X-100. The structural basis for this difference is presently being investigated. In the light of these findings we chose the J-strain enzyme for further characterization. Because detergents could potentially mask the effects of inhibitors on protease activity, we performed all subsequent assays in presence of only 0.05% Triton X-100. This led to a 10% decrease in specific activity. We next determined the dependence of the NS3-J
HEPATITUS C VIRUS PROTEASE ASSAY
FIG. 1. Combinatorial optimization of assay conditions using the radiometric assay in a microplate format. Multiparameter titrations using different detergents, glycerol concentrations and proteases from different HCV strains have been performed to define optimal enzymes and buffer composition. As an example, a simultaneous titration of glycerol and Triton X-100 is shown for the HCV BK-strain NS3 protease domain. The buffer contained 10 nM protease in 50 mM Hepes, pH 7.5, 30 mM DTT, 15 mM Pep4A cofactor and varying amounts of glycerol and detergent. Protease activity was determined as described under Materials and Methods.
protease activity on the NS4A cofactor peptide concentration under the optimized conditions. Using increasing amounts of Pep4A, we performed activity titration experiments. The reaction reached the maximal value of activity in the presence of more than 15 mM of Pep4A (not shown). From this titration experiment a dissociation constant for the NS3–Pep4A complex of 3 mM was calculated, which is in agreement with previously published values obtained with the Bk-strain enzyme (39). Based on these results, we defined the standard condition for the assay as being 50 mM Hepes pH 7.5, 15% glycerol, 0.05% Triton X-100, 30 mM DTT, and 15 mM Pep4A. We noticed that these optimized conditions differ from those found by Sali and co-workers (40) using a full-length NS3–NS4A complex. These authors however demonstrated that physicochemical conditions affected enzyme activity by altering the aggregation state of the complex. We did not observe similar effects on the aggregation state of the protease domain used in this study in complex with its cofactor peptide, hence explaining the differences in optimal buffer composition required for the maximum activity of the two different enzyme forms. The time dependence of proteolysis was also investigated. A deviation from linearity of the reaction was noted when 10% substrate conversion was exceeded
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(Fig. 2). This deviation was due to the inhibition of the enzyme by its N-terminal cleavage product Ac-DEMEEC-OH, which accumulated during the reaction (41). Below this threshold, a good linearity of the reaction was observed. We therefore performed the enzymatic reaction for 20 min. Under these conditions we obtained a signal to noise ratio of 10 with a good reproducibility (CV 5 6%) among different experiments. Addition of 150,000 cpm/well of labeled substrate in the absence of enzyme consistently gave a background of 250 6 50 cpm. Kinetic analysis of NS3 protease. To demonstrate the applicability of our assay to kinetic analysis, we determined the peptidolytic activity of the enzyme at increasing concentrations of the substrate. From substrate titration experiments, kinetic parameters of the cleavage reaction were determined, assuming Michaelis–Menten kinetics (data not shown). We obtained K m 5 10 6 2 mM and V max 5 125 6 10 cpm/min, from which k cat 5 5 6 0.4 min 21 could be calculated. These results were in excellent agreement with the data obtained by analyzing the cleavage reaction of the same substrate by HPLC (not shown). In this case we obtained K m 5 12 6 3 mM and k cat 5 4 6 0.5 min 21. Inhibition of protease activity by peptide inhibitors. We have previously found that the NS3 protease is inhibited by its N-terminal cleavage product, having the sequence Ac-DEMEEC-OH (41). This hexapeptide acted as a competitive inhibitor of the NS3 protease. In an HPLC assay performed at [S] 5 K m we obtained an IC 50 of 1.1 mM for this compound. An identical value was obtained using the radiometric assay (Fig. 3). Since during the cleavage reaction of the substrate peptide Pep4AB the same inhibitory product is generated, it can be predicted that the apparent IC 50 for
FIG. 2. Time course of substrate cleavage by NS3 J protease. 10 nM protease in 50 mM Hepes, pH 7.5, 30 mM DTT, 15% glycerol, 0.05% Triton X-100, 15 mM Pep4A was incubated in the presence of 10 mM Pep4AB (150,000 cpm). Reactions were stopped at timed intervals and the cleavage product was quantified by liquid scintillation counting.
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added Ac-DEMEEC-OH should be increased as a function of substrate conversion. This was verified experimentally. IC 50 values for Ac-DEMEEC-OH were determined after 10 min, 30 min, and 1 h of cleavage reaction. Under these conditions, IC 50s of 1, 1.4, and 2 mM were determined, indicating that overestimation of IC 50 values of added competitive inhibitors due to product inhibitor accumulation becomes significant only above 30 min reaction time. Optimization of the product inhibitor sequence has led to the hexapeptide Ac-DEDifEChaC-OH (42), which inhibited the enzyme with an IC 50 of 80 nM, determined at [S] 5 K m and a K i of 40 nM, calculated from a substrate competition experiment using an HPLC assay (not shown). To validate the reliability of the radiometric assay in accurately determining the potency and the inhibitory mechanism of compounds with nanomolar potencies, we measured the NS3 protease activity in the presence of increasing concentrations of Ac-DEDifEChaC-OH. Lineweaver–Burk analysis of the steady-state velocity of protease activity as a function of Pep4AB substrate at increasing Ac-DEDifEChaC-OH concentrations indicated that the inhibition was competitive with respect to substrate with K i 5 40.0 6 7.4 nM (Fig. 4), a value that is identical to the one obtained using the HPLC assay. These results confirmed that the radiometric assay is an accurate method for measuring NS3 protease activity and permits the mechanistic analysis of protease inhibitors with potencies in the nanomolar range. Because the entire assay was performed in a micro-
FIG. 4. Lineweaver–Burk plot of the inhibition of NS3 J protease by the product inhibitor DEDifEChaC-OH. 10 nM protease in 50 mM Hepes, pH 7.5, 30 mM DTT, 15% glycerol, 0.05% Triton X-100, 15 mM Pep4A was incubated in the presence of increasing concentrations of inhibitor. After 10 min preincubation, reactions were started by addition of substrate in a concentration range between 2.5 and 80 mM. Reactions were stopped after 30 min. Velocities were expressed as total counts (cpm) of cleaved substrate generated in 30 min. The following values were calculated from a fit of this experiment to Eq. [1] (Materials and Methods): K i 5 40.0 6 7.4 nM, K m 5 12.5 6 2.0 mM, and V max 5 120 6 6.2 cpm/min.
titer plate, it was possible to automate all steps of the reaction using robotic microplate manipulators. The assay is thus amenable for high-throughput screening of compound collections, natural product samples, or combinatorial libraries. REFERENCES
FIG. 3. Inhibition of NS3 J protease by the product inhibitor DEMEEC-OH. 10 nM protease in 50 mM Hepes, pH 7.5, 30 mM DTT, 15% glycerol, 0.05% Triton X-100, 15 mM Pep4A was incubated in the presence increasing concentrations of DEMEEC-OH. After 10 min preincubation, the reaction was started by addition of of 10 mM Pep4AB (150,000 cpm). Percentage residual activity was determined (squares). The same experiment was performed monitoring the cleavage of unlabeled Pep4AB by HPLC (circles). Activity data were fitted to Eq. [2]. From the fit we obtained IC 50 5 1.0 mM and s 5 1.0 (microplate assay) and IC 50 5 1.1 mM and s 5 0.9 (HPLC assay). Correlation coefficients for both fittings were R 5 0.99.
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A High-Throughput Radiometric Assay for Hepatitis C Virus NS3 Protease Mauro Cerretani, Laura Di Renzo, Sergio Serafini, Alessandra Vitelli, Nadia Gennari, Elisabetta Bianchi, Antonello Pessi, Andrea Urbani, Stefano Colloca, Raffaele De Francesco, Christian Steinku¨hler, and Sergio Altamura 1 Istituto di Ricerche di Biologia Molecolare (IRBM) “P. Angeletti,” 00040 Pomezia, Rome, Italy
Received July 10, 1998
A novel radiometric in vitro assay for discovery of inhibitors of hepatitis C viral protease activity, suitable for high-throughput screening, was developed. The NS3 protein of hepatitis C virus (HCV) contains a serine protease, whose function is to process the majority of the nonstructural proteins of the viral polyprotein. The viral NS4A protein is a cofactor of NS3 protease activity in the cleavage of NS3–NS4A, NS4A–NS4B, NS4B–NS5A, and NS5A–NS5B junctions. To establish an in vitro assay system we used NS3 proteases from different HCV strains, purified from Escherichia coli and a synthetic radiolabeled peptide substrate that mimics the NS4A–NS4B junction. Upon incubation with the enzyme the substrate was separated from the radiolabeled cleavage product by addition of an ion exchange resin. The assay was performed in a microtiter plate format and offered the potential for assaying numerous samples using a laboratory robot. Taking advantage of these features, we used the assay to optimize reaction conditions by simultaneously varying different buffer components. We showed that physicochemical conditions affect NS3 protease activity in a strain-specific way. Furthermore, the sensitivity of the assay makes it suitable for detection and detailed mechanistic characterization of inhibitors with low-nanomolar affinities for the HCV serine protease. © 1999 Academic Press
Hepatitis C virus (HCV) 2 is considered the major causative agent of human posttransfusion and spo1
To whom correspondence should be addressed at IRBM, Via Pontina Km 30,600, 00040 Pomezia, Rome, Italy. Fax: 1 39-691093225. E-mail: [email protected]. 2 Abbreviations used: HCV, hepatitus C virus; DTT, dithiothreitol; DMSO, dimethyl sulfoxide; TFA, trifluoroacetic acid. 192
radic non-A, non-B hepatitis (1, 2). The number of chronic carriers is estimated to be about 400 million and chronic infection may lead to development of liver cirrhosis and hepatocellular carcinoma (3). Protective vaccination is not available for HCV and the only established therapy is interferon treatment. However, interferon is successful only in a limited number of patients and has significant side effects (4, 5). For this reason, there is a considerable attention on understanding HCV replication and developing more effective anti-HCV therapies. HCV (6) is related to flavi- and pestiviruses in its genetic organization and is classified as a particular genus of the family Flaviviridae. HCV exists in many distinct variants: a total of 6 major genotypes and at least 11 subtypes have been recognized (7). The HCV virion has a positive-stranded RNA genome of 9.5 kb. This genome contains a single large open reading frame encoding for a polyprotein of 3010 –3030 residues with the gene order: 59-C-E1–E2–NS2–NS3– NS4A–NS4B–NS5A–NS5B-39 (1, 8 –10). This polyprotein is processed into at least 9 different mature gene products. Structural proteins, C (nucleocapsid protein) and E1 and E2 (envelope proteins), are cleaved by host cell signal peptidase. Processing events in the nonstructural region depend on the activity of virally encoded proteases (11, 12). Accordingly, cleavage at the NS2–NS3 junction is accomplished by a zinc-dependent enzymatic activity encoded between NS2 and NS3 (12). The N-terminal ;200 amino acids of the NS3 protein harbor a serine protease activity which is essential for all cleavages downstream of NS3 (11, 14 – 17). The C-terminal two-thirds of the NS3 protein contains an RNA helicase (18). The separately expressed helicase and protease domains of NS3 exhibit their respective activities in vitro (19 –22). The structure of NS3 protease was determined by X-ray crystallogra0003-2697/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
HEPATITUS C VIRUS PROTEASE ASSAY
phy (23–25). According to the crystallographic data, the protease folds in a trypsin-like fold, consisting of two b-barrel domains and a catalytic triad of His-57, Asp-81, and Ser-139. Unexpectedly, the NS3 protease was found to also harbor a structural zinc-binding site (26). Many efforts have been made to characterize the mechanism by which the NS3 protease domain processes the downstream gene products. These studies have shown that NS3/NS4A cleavage occurs rapidly and in cis while the others occur in trans (NS4A/4B, NS4B/5A, NS5A/5B). To display its full activity, the NS3 protease must bind to the viral NS4A protein that acts as a cofactor, enhancing the proteolytic efficiency of NS3 (27–32). It has been shown that all the effects of NS4A can be efficiently mimicked by peptides encompassing residues 21–34 of the central region of NS4A (19, 21, 29 –32). The crystal structure of the NS3 protease domain complexed with a synthetic NS4A activator domain reveals that the NS4A peptide intercalates within a b sheet of the enzyme core (24, 25). Because proteolytic processing of viral precursor protein is likely to be required for HCV replication, the viral protease is considered a primary target for antiviral drug discovery. Several NS3 protease assays have been described so far including a cell-based assay (33) and cell-free in vitro assays (34, 35). These assays are intrinsically unsuitable for mechanistic studies of inhibitors and have a low throughput. Recently, assays relying on the cleavage of peptidic substrates by a purified enzyme and chromatographic (21, 36), colorimetric (37) or fluorescence detection (38) of the cleavage products have been described. These assays permit a detailed characterization of enzyme inhibitors but suffer from low throughput, in the case of HPLC-based assays (21, 36), sensitivity to interferences, in the case of the fluorescence assay (38), or a very laborious assay protocol (37). We report here the development of a sensitive, discontinuous radiometric screening assay for NS3 proteolytic activity. The assay is entirely performed in microtiter plates, thus permitting facile and rapid high-throughput screening for NS3 protease inhibitors combined with the possibility to perform detailed mechanistic studies in the same assay format. Its sensitivity allows the accurate characterization of inhibitors with low-nanomolar affinities for the NS3 protease. Furthermore, the possibility of assaying a large number of samples was employed to screen for natural protease variants with improved buffer requirements. MATERIALS AND METHODS
NS3 protease. The serine protease domain of NS3 (amino acids 1–180, from HCV Bk, H, or J strain) was purified as previously described (26). The proteins were stored in aliquots at the concentration of 10 –30 mM in
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25 mM sodium phosphate, pH 7.5, 50% glycerol, 0.5% Chaps, 30 mM DTT at 280°C after shock–freezing in liquid nitrogen. 3 H-labeled peptide substrate ( 3H-Pep4AB). The peptide substrate 3H-Pep4AB (Ac-DEEMECASHLPYK-(eAc 3H)-NH 2), based on the sequence of the NS4AB cleavage site of the HCV polyprotein, was custom synthesized by Amersham. Dry powder of 3H-Pep4AB was dissolved in 100% DMSO and stored in aliquots at 280°C. The original stock had a specific activity of 17 Ci/mmol. Peptide substrate (Pep4AB). Peptide substrate Pep4AB (Ac-DEEMECASHLPYK-(e-Ac)-NH 2) was obtained from Peptides International Inc. Dry powder of Pep4AB was dissolved in deionized water at a concentration of 10 –15 mM. The concentration was confirmed by quantitative amino acid analysis. Aliquots were stored at 280°C. NS4A cofactor peptide (Pep4A). The NS4A cofactor peptide Pep4A, harboring residues 21–34 of NS4A and an N-terminal lysine tag, (H-KKKGSVVIVGRIILSGRNH 2) was purchased from Peptides International Inc. Dry powder was dissolved in water and concentration was confirmed by quantitative amino acid analysis. Aliquots were stored at 280°C. Chemicals. Fractogel TSK-650S DEAE resin was purchased from TOSOH (No. 07472). The original stock Fractogel TSK-650S DEAE resin, supplied as a suspension in 1 M NaCl, 20% ethanol, was washed with 10 mM Tris/HCl, pH 7.5, until the conductivity of the supernatant was the same as that of the washing buffer. After centrifugation at 2300 rpm for 2 min the resin was resuspended at a concentration of 20% (v/v) in 10 mM Tris/HCl, pH 7.5. The final concentration of resin in the assay was 10% (v/v). Microscint 40 was purchased from Packard (No. 6013646). All other chemicals were obtained from Sigma Chemical Co. HPLC assay. Cleavage assays were performed in 57 ml 50 mM Hepes, pH 7.5, 0.05% Triton X-100, 15% glycerol, 10 mM DTT. Buffer solutions containing 15 mM Pep4A were preincubated for 10 min with 10 nM NS3 protease and reactions were started by addition of 3 ml substrate peptide Pep4AB. Six duplicate data points at different substrate concentrations were used to calculate kinetic parameters. Incubation times were chosen to obtain ,7% substrate conversion and reactions were stopped by addition of 40 ml 1% TFA. Cleavage of peptide substrates was determined by HPLC using a Merck–Hitachi chromatograph equipped with an autosampler. Eighty-microliter samples were injected on a Lichrospher C18 reversed-phase cartridge column (4 3 75 mm, 5 mm, Merck) and fragments were separated using a 10 – 40% acetonitrile gradient at 5%/ min using a flow rate of 2.5 ml/min. Peak detection was
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accomplished by monitoring both the absorbance at 220 nm and tyrosine fluorescence (l ex 5 260 nm, l em 5 305 nm). Cleavage products were quantitated by integration of chromatograms with respect to appropriate standards. Kinetic parameters were calculated from nonlinear least-squares fit of initial rates as a function of substrate concentration with the help of a Kaleidagraph software, assuming Michaelis–Menten kinetics. Radiometric enzyme assay. The enzymatic reaction was performed in 96-well clear microtiter plates (Costar 96F polypropilene microplate). NS3 (10 nM) and 15 mM Pep4A in 50 mM Hepes, pH 7.5, 15% glycerol, 0.05% Triton X-100, 30 mM DTT, were preincubated with DMSO or inhibitors were dissolved in DMSO for 5 min at room temperature. The final concentration of DMSO in the assay was 10%. The reaction was started by the addition of 10 ml of substrate solution such that a final concentration of 10 mM Pep4AB and 80 nM 3H-Pep4AB (150,000 cpm/well) in 10 mM Hepes, pH 7.5, was obtained. The final volume of the reaction was 100 ml. Plates were sealed with an acetate tape and incubated for 20 min at room temperature under gentle shaking with an orbital shaker. The reaction was stopped by adding 100 ml of 20% (v/v) Fractogel TSK-650S DEAE resin and shaking was continued for 10 min at room temperature. The plate was than centrifuged at 2300 rpm for 2 min at room temperature. Thirty microliters of the reaction solution was transferred to a 96-well microtiter plate (Packard Picoplate). Two hundred fifty microliters of scintillation coktail (Microscint 40) was added and after vigorous shaking for at least 4 h at room temperature the radioactivity was measured in a Packard Top Count b-counter. Determination of K i and IC 50 values. K i values of inhibitors were calculated from substrate titration experiments performed in the presence of increasing amounts of inhibitor. Experimental data sets were simultaneously fitted to Eq. [1] using a multicurve fit macro with the help of a Sigmaplot software: V 5 ~V maxS!/~K m ~1 1 K i /I! 1 S!.
[1]
IC 50 values were calculated using a two-parameter logistic function, according to Eq. [2]: %activity 5 ~100/~1 1 ~@I#/IC 50 ! s !!,
[2]
where [I] is the inhibitor concentration and s is the slope factor of the curve. RESULTS AND DISCUSSION
Radiometric assay development for NS3 protease activity. We developed a simple and efficient radiometric assay for the proteolytic activity of NS3 protease,
monitoring the formation of an 3H-labeled product, generated from cleavage by the enzyme of the Pep4AB substrate. The Pep4AB substrate is a synthetic radiolabeled peptide that mimicks the NS4A/NS4B junction, having the sequence Ac-DEEMECASHLPYK-(e-Ac 3H)NH 2. It has been demonstrated that the protease cleaved this peptide with high efficiency between cysteine and alanine (21), yielding two products with different physicochemical properties. The noncleaved substrate and the N-terminal product (Ac-DEEMECOH) carry net negative charges, whereas the C-terminal fragment, being amidated, carries positive charges on the new N-terminus and the histidine residue. Addition of a TSK-650S DEAE ion exchange resin permitted the quantitative capture of the two negatively charged species in solution, which were then separated by centrifugation from the C-terminal proteolysis product. Detection of this product was rendered possible by previous introduction in the substrate of an 3H label via a C-terminal lysine residue, that was acetylated with 3H-labeled acetic anhydride (NH 2-ASHLPYK-(eAc 3H)-NH 2). It was thus possible to monitor the reaction by simple counting of the supernatant after addition of the resin. To define standard conditions for measuring proteolytic activity of NS3 protease in our assay, we took advantage of the possibility to perform the assay in a microplate format, thus permitting the rapid analysis of a very large number of samples. We used proteins encompassing the protease domain of three different isolates of HCV (Bk, J, and H) and determined the relative activities of these enzymes, in the presence of 15 mM Pep4A cofactor, by simultaneously varying the glycerol and detergent concentration of the buffer. This was done since both glycerol and detergent were previously shown to stabilize the NS3 protease domain of the Bk isolate (21). An example of a simultaneous titration of both Triton X-100 and glycerol for the Bk enzyme is shown in Fig. 1. This enzyme turned out to require 40% glycerol for optimal activity, leading to high viscosity of optimal activity buffers with concomitant difficulties in reproducible pipetting of buffer solutions. Analogous experiments were performed using different detergents and proteases. While both Bk- and H-strain proteases require glycerol concentrations exceeding 40% and Triton X-100 concentration of 0.5%, the J-strain enzyme has an activity optimum at 15% glycerol and 0.12% Triton X-100. The structural basis for this difference is presently being investigated. In the light of these findings we chose the J-strain enzyme for further characterization. Because detergents could potentially mask the effects of inhibitors on protease activity, we performed all subsequent assays in presence of only 0.05% Triton X-100. This led to a 10% decrease in specific activity. We next determined the dependence of the NS3-J
HEPATITUS C VIRUS PROTEASE ASSAY
FIG. 1. Combinatorial optimization of assay conditions using the radiometric assay in a microplate format. Multiparameter titrations using different detergents, glycerol concentrations and proteases from different HCV strains have been performed to define optimal enzymes and buffer composition. As an example, a simultaneous titration of glycerol and Triton X-100 is shown for the HCV BK-strain NS3 protease domain. The buffer contained 10 nM protease in 50 mM Hepes, pH 7.5, 30 mM DTT, 15 mM Pep4A cofactor and varying amounts of glycerol and detergent. Protease activity was determined as described under Materials and Methods.
protease activity on the NS4A cofactor peptide concentration under the optimized conditions. Using increasing amounts of Pep4A, we performed activity titration experiments. The reaction reached the maximal value of activity in the presence of more than 15 mM of Pep4A (not shown). From this titration experiment a dissociation constant for the NS3–Pep4A complex of 3 mM was calculated, which is in agreement with previously published values obtained with the Bk-strain enzyme (39). Based on these results, we defined the standard condition for the assay as being 50 mM Hepes pH 7.5, 15% glycerol, 0.05% Triton X-100, 30 mM DTT, and 15 mM Pep4A. We noticed that these optimized conditions differ from those found by Sali and co-workers (40) using a full-length NS3–NS4A complex. These authors however demonstrated that physicochemical conditions affected enzyme activity by altering the aggregation state of the complex. We did not observe similar effects on the aggregation state of the protease domain used in this study in complex with its cofactor peptide, hence explaining the differences in optimal buffer composition required for the maximum activity of the two different enzyme forms. The time dependence of proteolysis was also investigated. A deviation from linearity of the reaction was noted when 10% substrate conversion was exceeded
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(Fig. 2). This deviation was due to the inhibition of the enzyme by its N-terminal cleavage product Ac-DEMEEC-OH, which accumulated during the reaction (41). Below this threshold, a good linearity of the reaction was observed. We therefore performed the enzymatic reaction for 20 min. Under these conditions we obtained a signal to noise ratio of 10 with a good reproducibility (CV 5 6%) among different experiments. Addition of 150,000 cpm/well of labeled substrate in the absence of enzyme consistently gave a background of 250 6 50 cpm. Kinetic analysis of NS3 protease. To demonstrate the applicability of our assay to kinetic analysis, we determined the peptidolytic activity of the enzyme at increasing concentrations of the substrate. From substrate titration experiments, kinetic parameters of the cleavage reaction were determined, assuming Michaelis–Menten kinetics (data not shown). We obtained K m 5 10 6 2 mM and V max 5 125 6 10 cpm/min, from which k cat 5 5 6 0.4 min 21 could be calculated. These results were in excellent agreement with the data obtained by analyzing the cleavage reaction of the same substrate by HPLC (not shown). In this case we obtained K m 5 12 6 3 mM and k cat 5 4 6 0.5 min 21. Inhibition of protease activity by peptide inhibitors. We have previously found that the NS3 protease is inhibited by its N-terminal cleavage product, having the sequence Ac-DEMEEC-OH (41). This hexapeptide acted as a competitive inhibitor of the NS3 protease. In an HPLC assay performed at [S] 5 K m we obtained an IC 50 of 1.1 mM for this compound. An identical value was obtained using the radiometric assay (Fig. 3). Since during the cleavage reaction of the substrate peptide Pep4AB the same inhibitory product is generated, it can be predicted that the apparent IC 50 for
FIG. 2. Time course of substrate cleavage by NS3 J protease. 10 nM protease in 50 mM Hepes, pH 7.5, 30 mM DTT, 15% glycerol, 0.05% Triton X-100, 15 mM Pep4A was incubated in the presence of 10 mM Pep4AB (150,000 cpm). Reactions were stopped at timed intervals and the cleavage product was quantified by liquid scintillation counting.
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added Ac-DEMEEC-OH should be increased as a function of substrate conversion. This was verified experimentally. IC 50 values for Ac-DEMEEC-OH were determined after 10 min, 30 min, and 1 h of cleavage reaction. Under these conditions, IC 50s of 1, 1.4, and 2 mM were determined, indicating that overestimation of IC 50 values of added competitive inhibitors due to product inhibitor accumulation becomes significant only above 30 min reaction time. Optimization of the product inhibitor sequence has led to the hexapeptide Ac-DEDifEChaC-OH (42), which inhibited the enzyme with an IC 50 of 80 nM, determined at [S] 5 K m and a K i of 40 nM, calculated from a substrate competition experiment using an HPLC assay (not shown). To validate the reliability of the radiometric assay in accurately determining the potency and the inhibitory mechanism of compounds with nanomolar potencies, we measured the NS3 protease activity in the presence of increasing concentrations of Ac-DEDifEChaC-OH. Lineweaver–Burk analysis of the steady-state velocity of protease activity as a function of Pep4AB substrate at increasing Ac-DEDifEChaC-OH concentrations indicated that the inhibition was competitive with respect to substrate with K i 5 40.0 6 7.4 nM (Fig. 4), a value that is identical to the one obtained using the HPLC assay. These results confirmed that the radiometric assay is an accurate method for measuring NS3 protease activity and permits the mechanistic analysis of protease inhibitors with potencies in the nanomolar range. Because the entire assay was performed in a micro-
FIG. 4. Lineweaver–Burk plot of the inhibition of NS3 J protease by the product inhibitor DEDifEChaC-OH. 10 nM protease in 50 mM Hepes, pH 7.5, 30 mM DTT, 15% glycerol, 0.05% Triton X-100, 15 mM Pep4A was incubated in the presence of increasing concentrations of inhibitor. After 10 min preincubation, reactions were started by addition of substrate in a concentration range between 2.5 and 80 mM. Reactions were stopped after 30 min. Velocities were expressed as total counts (cpm) of cleaved substrate generated in 30 min. The following values were calculated from a fit of this experiment to Eq. [1] (Materials and Methods): K i 5 40.0 6 7.4 nM, K m 5 12.5 6 2.0 mM, and V max 5 120 6 6.2 cpm/min.
titer plate, it was possible to automate all steps of the reaction using robotic microplate manipulators. The assay is thus amenable for high-throughput screening of compound collections, natural product samples, or combinatorial libraries. REFERENCES
FIG. 3. Inhibition of NS3 J protease by the product inhibitor DEMEEC-OH. 10 nM protease in 50 mM Hepes, pH 7.5, 30 mM DTT, 15% glycerol, 0.05% Triton X-100, 15 mM Pep4A was incubated in the presence increasing concentrations of DEMEEC-OH. After 10 min preincubation, the reaction was started by addition of of 10 mM Pep4AB (150,000 cpm). Percentage residual activity was determined (squares). The same experiment was performed monitoring the cleavage of unlabeled Pep4AB by HPLC (circles). Activity data were fitted to Eq. [2]. From the fit we obtained IC 50 5 1.0 mM and s 5 1.0 (microplate assay) and IC 50 5 1.1 mM and s 5 0.9 (HPLC assay). Correlation coefficients for both fittings were R 5 0.99.
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