A cell-based β-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication

ANALYTICAL BIOCHEMISTRY Analytical Biochemistry 334 (2004) 344–355 www.elsevier.com/locate/yabio

A cell-based b-lactamase reporter gene assay for the identification of inhibitors of hepatitis C virus replication Paul Zucka,*,1, Edward M. Murrayb,1, Erica Steca, Jay A. Groblerb, Adam J. Simonb, Berta Strulovicia, James Inglesec, Osvaldo A. Floresb, Marc Ferrera a

Department of Automated Biotechnology, Merck and Co., 502 Louise Lane., North Wales, PA 19454, USA b Department of Biological Chemistry, Merck and Co., West Point, PA 19468, USA c NIH Chemical Genomics Center, 50 South Dr., Bethesda, MD 20892-8004, USA Received 14 June 2004 Available online 25 September 2004

Abstract This report describes the development, optimization, and implementation of a cell-based assay for high-throughput screening (HTS) to identify inhibitors to hepatitis C virus (HCV) replication. The assay is based on a HCV subgenomic RNA replicon that expresses b-lactamase as a reporter for viral replication in enhanced Huh-7 cells. The drug targets in this assay are viral and cellular enzymes required for HCV replication, which are monitored by fluorescence resonance energy transfer using cell-permeable CCF4AM as a b-lactamase substrate. Digital image processing was used to visualize cells that harbor viral RNA and to optimize key assay development parameters such as transfection and culturing conditions to obtain a cell line which produced a robust assay window. Formatting the assay for compound screening was problematic due to small signal-to-background ratio and reduced potency to known HCV inhibitors. These technical difficulties were solved by using clavulanic acid, an irreversible inhibitor of b-lactamase, to eliminate residual b-lactamase activity after HCV replication was terminated, thus resulting in an improved assay window. HTS was carried out in 384-well microplate format, and the signal-to-background ratio and Z factor for the assay plates during the screen were approximately 13-fold and 0.5, respectively.
2004 Elsevier Inc. All rights reserved.

There is a major health need to identify and develop novel, efficacious antiviral compounds to hepatitis C virus (HCV)2. Three percent of the human population is infected, making it the most common blood-borne pathogen in man [1,2]. Infection becomes chronic in about 85% of infected individuals [3] and can lead *

Corresponding author. Fax: +1 267 305 3625. E-mail address: [email protected] (P. Zuck). 1 These authors contributed equally to this work. 2 Abbreviations used: HCV, hepatitis C virus; UTR, untranslated region; IRES, internal ribosome entry sequences; HTS, high-throughput screening; FRET, fluorescence resonance energy transfer; DMEM, DulbeccoÕs modified EagleÕs medium; PBS, phosphate-buffered saline; DIP, digital image processing; 2 0 -C-Me-A, 2 0 -C-methyladenosine; SSC, standard saline citrate; EMC, encephalomyocarditis; BC, blue cells; DMSO, dimethyl sulfoxide. 0003-2697/$ - see front matter
2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ab.2004.07.031

to liver cirrhosis and hepatocellular carcinoma [4,5]. In the U.S., liver failure due to HCV infection is the major cause of liver transplantation and is estimated to cause 8000–10,000 deaths annually [2]. Currently, there is no HCV vaccine and approximately 46% of all infected individuals do not respond to ribavirin/interferon combination therapy [6]. HCV is a small, enveloped virus that has a 9.6-kb single-stranded, positive-sense, RNA genome (Fig. 1A). HCV genome has a 5 0 untranslated region (UTR), an open reading frame that encodes for a single polyprotein of 3010 amino acids, and a short 3 0 UTR that is required for replication. Cap-independent translation of the HCV genome is driven by the internal ribosome entry sequences (IRES) located in the 5 0 UTR. The resulting polyprotein is processed by viral and cellular proteinases

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Fig. 1. Organization of the complete HCV genome (A) and HCV replicon construct (B). To create a HCV subgenomic replicon, the HCV structural proteins are replaced with a reporter, which is translated by the HCV IRES (5 0 UTR). The EMC IRES is inserted to drive the translation of a single HCV polyprotein that encodes nonstructural proteins, NS3–NS5B. The HCV polyprotein is then processed by cellular and viral proteinases to generate individual nonstructural proteins, all of which are required for viral replication.

to produce four structural proteins (Core, E1, E2, and p7) and six nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). The structural proteins are components of the viral capsid and envelope and are dispensable for replication of the viral genome. In contrast to the structural proteins, the nonstructural proteins NS3–NS5B are essential components of the viral replicase (for HCV biology reviews see [7–9]). Even though HCV was identified as the viral cause of non-A non-B hepatitis in 1989 [10], it is still not possible to culture infectious virus in the laboratory. Lack of cell culture systems has severely hampered HCV antiviral drug discovery efforts, and the first cell culture system used to study HCV replication was not developed until 1999 [11]. This HCV replication system utilized neomycin phosphotransferase resistance (neor) to select for cell lines that harbored self-replicating HCV RNA molecules called replicons. The resulting cell lines could be used for low-throughput antiviral screens using assay formats based on detecting viral RNA or protein such as RPA or enzyme-linked immunosorbent assay. However, these two approaches are not amenable for highthroughput screening (HTS) because of the high background and the numerous steps in the assay protocol, such as processing of the samples, including extractions, washes, addition of reagents, etc. Developing a luciferase reporter gene assay was not appealing because it does not provide the option of FACS sorting of transfected cells, which would be necessary for expansion if only a low percentage of cells were able to support HCV replication. We previously described a quantitative HCV replication assay compatible with live-cell formats that employs subgenomic HCV replicons that express the

b-lactamase reporter [12] (Fig. 1B). This assay allows for the simultaneous quantitation of both the number of cells that harbor replicon and the average number of replicons per cell. The b-lactamase reporter gene system is amenable for HTS because it utilizes a fluorometric readout on live cells, so that cellular lysis or washes are not required to detect signal. An additional advantage of the b-lactamase reporter assay is that the detection is based on a self-quenched, fluorescence resonance energy transfer (FRET) substrate, CCF4-AM, which is cell permiant. This FRET substrate has dual emission at 460 nm (coumarin moiety, blue fluorescence) and 530 nm (fluorescein moiety, green fluorescence), which allows for a ratiometric readout. This ratiometric readout is useful for compound screening because the signal can be normalized to the cell number, thus reducing signal variation, and the assay is amenable to miniaturization [13]. This reporter gene assay was optimized into a sensitive, robust, high-throughput screening assay. The HTS assay was executed in 384-well microplate format and exhibited an average signal-to-background ratio of
13-fold, an average assay window with a Z 0 factor [14] of
0.6 (Z factor of 0.5), and IC50 values for known replication inhibitors in agreement with those reported previously.

Materials and methods Cell culture The enhanced Huh-7 cells used to generate the screening cell line are a special HCV-replicon-permissive

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cell line named MR-2 cells (which were derived from Huh-7 cells and are described in detail in [12]). For this work MR-2 cells will be referred to as Huh-7 cells. Huh7 cells were grown in CellGro DMEM (Mediatech Cat. No. 10-013-CM) supplemented with 10% fetal bovine serum, nonessential amino acids (Sigma), penicillin– streptomycin solution (Gibco), and glutamine (Gibco) (complete DMEM). Cells were cultured in a 37 C, 5% CO2-humidified incubator for all experiments. Black, clear-bottomed, tissue-culture-treated, 384-well microtiter plates were from Costar (Costar 3712). Clavulanic acid was purchased from United States Pharmacopeia (Catalog No. 134426) and was prepared by dissolving in 1· PBS (10 mM phosphate-buffered saline [138 mM NaCl, 2.7 mM KCl, pH 7.4]) to 300 mM. CCF4-AM/ background suppression mixture was purchased from Invitrogen Life Sciences (Cat. No. K1030) and prepared at a 6· stock solution according to the manufacturer. Transfected cells were aliquoted, frozen, and stored in liquid nitrogen. Frozen cells were thawed fresh on the day of the assay or the day before and diluted in prewarmed medium (37 C) to 40,000 cells/ml; 50 ll of the cell suspension was plated into 384-well microtiter plates. The cells were allowed to recover for at least 6 h at 37 C in the plates before beginning an assay. In vitro transcription of HCV b-lactamase replicon A plasmid DNA encoding the HCV subgenomic replicon sequence was previously reported [11]. Here this plasmid was used with the following modifications: the neomycin gene was replaced with b-lactamase (via PmeI AscI sites) and the amino acid at position 2204 was converted from Ser to Ile [15]. Plasmid DNA was linearized by restriction endonuclease digestion with ScaI for 2 h at 37 C. The digested plasmid DNA was purified using a PCR DNA purification kit (Qiagen) as per manufactureÕs instructions. HCV replicon RNA was synthesized using a T7 in vitro transcription MegaScript kit (Ambion). The T7 reaction was done on 1 lg of ScaI-digested template DNA at 37 C for 4 h. The reaction was then treated with DNase I (Ambion) for 15 min. The HCV RNA replicon was purified using an RNeasy kit (Qiagen) according to the manufacturerÕs instructions except that the elution was done twice instead of once. The RNA was quantified by absorbance at 260 nm. RNA purity and integrity were determined by absorbance at 260/280 nm and gel electrophoresis. The RNA was stored at 20 or 70 C. Transfection conditions For assay optimization experiments, enhanced Huh-7 cells were seeded at the indicated density of cells per well in a six-well plate 16–20 h before transfection. Transfection medium was prepared by adding 6 ll of DEMRIE-C

(Invitrogen) per 1 ml of OptiMEM I (Invitrogen) and vortexing. Then 2.7 lg of replicon RNA per 1 ml of transfection medium was added and the medium was mixed by inversion. The RNA transfection mixture was further diluted serially to 900 and 300 ng/ml using the transfection medium as the diluent. The Huh-7 cells were washed once with 2 ml/well of OpiMEM I just prior to adding the RNA transfection mixture. After the indicated transfection time, 2 ml of complete DMEM was added per well and the cells were incubated at 37 C, 5% CO2 humidified incubator (standard conditions). Digital image processing DIP was used to develop the HCV b-lactamase cell line for HTS. The system incorporates a digital charge-coupled device camera that is used to detect cells emitting fluorescence after they are treated with the FRET substrate CCF4-AM. Photographs of the cells are processed using the Image Pro Plus Software (Media Cybernetics, Version 4.1). This software analyzes images of fluorescent cellular samples by using software filters that can separate individual cells that are physically touching so that they can be counted. Individual cells are then segmented and counted based on the wavelength of the fluorescent light emitted (green for intact CCF4 and blue for hydrolyzed substrate). Kinetics experiment An equal number of b-lactamase cells from batch I and batch II (see text) were thawed at 37 C, mixed together, and seeded into T-75 flasks at 2 · 106 cells/flask. The cells were incubated overnight under standard conditions. Then 5 lM of HCV 2 0 -C-methyladenosine (2 0 C-Me-A) [16] was added to the cells and the cells were incubated for 0, 6, 12, 24, and 48 h. At the collection time the cells were washed with 10 ml of 1· PBS, trypsinized, counted, and pelleted (500g). Trizol (Invitrogen) was at 500 ll/3 · 106 cells and stored at 70 C. Northern blot Total RNA was purified from cells using Trizol (Invitrogen) as indicated by the manufacturer. RNA purity and quantity were determined by absorbance at 260 and 280 nm. Five micrograms of total RNA was electrophoresed in a glyoxal gel system (NorthernMax-Gly, Ambion) according to the manufacturers instructions. The resolved RNA was photographed and transferred to a Nytran membrane (Scheicher and Schuell) using NorthernMax-Gly buffers (Ambion) with TurboBlotter Rapid Downward Transfer System (Scheicher and Schuell). The RNA was cross-linked to the membrane using the Auto cross-link function on a UV

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Stratalinker 2400 (Stratagene). The membrane was prehybridized in 10 ml ExpressHyb hybridization solution (BD Biosciences) containing 100 lg/ml of sheared salmon sperm DNA (ssDNA) for 60 min at 68 C. A b-lactamase DNA template was used to generate a 32 P-labeled probe using Prime-It II random primed labeling kit (Stratagene). The 32P-labeled b-lactamase probe was purified through a ProbeQuant G-50 Micro column (Amersham–Pharmacia Biotech). Labeling efficiency was determined by liquid scintillation counting. The probe (4 · 107 CPM) was boiled at 100 C for 3 min in 1 ml ExpressHyb containing 100 lg/ml ssDNA prior to hybridizing in roller bottles for 16–20 h at 68 C in ExpressHyb containing 100 lg/ml ssDNA. The probe hybridization concentration was 4 · 106 CPM/ml. All washes were done in roller bottles at 65 C as follows: 100 ml 2· SSC and 1% SDS, 100 ml 1· SSC and 1% SDS, and 100 ml 0.1· SSC and 0.1% SDS. The Northern products were quantitated by phosphor autoradiography with Storm PhosphorImager and ImageQuant software (Molecular Dynamics). Replicon RNA signal was normalized to 28 s ribosomal RNA for analysis.

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sequentially to the wells using two separate CyBi Well 384/1536 (CyBio) pipettors. The cells were then incubated for 24 h at 37 C. Then 10 ll of 6· CCF4-AM/ background suppression mixture was added to the wells with a Multidrop (Labsystems), and the plates were incubated for 90 min at 25 C. Fluorescence was measured from the bottom of the wells with a Tecan Spectrafluor Plus (340 nm excitation, 460 and 530 nm emissions). Data from the 384-well assay plates were analyzed by subtracting the fluorescence emission at 460 and 530 nm from wells that contained medium and dye only (no cells) from the 460 and 530-nm values from all the test wells. A ratio of the emission at 460 nm/ 530 nm was then calculated for each well. Background levels were calculated from the ratio for wells that were inhibited by an IC100 dose of an HCV NS5B RNA polymerase inhibitor; 0% inhibition was defined as the median ratio for the wells in columns 3–22, which contained the test compounds. Percentage inhibition was calculated by the following formula: %Inh ¼ 100ð1  ððtest value  backgroundÞ =ð0%inhibition  backgroundÞÞÞ:

Western blot b-Lactamase cells were seeded at a density of 1.8 · 106 into T-175 flasks and incubated overnight. On the following day, the medium was replaced with 25 ml of fresh medium and appropriate inhibitors or controls were added. After 24 h, cells were harvested with trypsin and washed once with PBS, and cell pellets were then frozen following centrifugation. Cell pellets were then resuspended in 250 ll PBS, subjected to two additional freeze/thaw cycles, and centrifuged at 14,000 rpm for 5 min to remove cell debris. Protein concentrations, determined by the method of Bradford [17], were adjusted to 1.8 mg/ml with PBS. Then 4· SDS– PAGE running buffer (Invitrogen) was added to 1· final concentration and the samples were boiled for 10 min. Protein samples (50 ll) were electrophoresed on a 4–12% NuPage Gel (Invitrogen), transferred to a nitrocellulose membrane, blotted using a rabbit anti-b-lactamase antibody (Aurora Bioscience) at a 1:15,000 dilution overnight, and visualized using WesternBreeze chromogenic immunodetection system (Invitrogen) according to the manufacturerÕs instructions.

Miniaturization of the HCV replicon b-lactamase assay to 3456-well nanoplate format Methodology was slightly modified from that of the 384-well assay for nanoplate screening due to logistics of screening in such a miniaturized format and the nature of the dispensers used. All the non-test-compound reagents including the cell suspension were dispensed into 3456-well nanoplates using a flying reagent dispenser and fluorescence intensity was measured using a topology-compensating plate reader. Test compounds were preplated in the nanowells using a pieso-tip applicator (all instruments from Aurora Biosciences [San Diego, CA]), and the assay was initiated with the addition of 1.8 ll of medium containing 750 cells, cells with control inhibitor, or only medium for the basal signal control, all containing 0.5 lM clavulanic acid. The nanoplate was incubated at 37 C, 5% CO2 for 24 h. Then 0.4 ll of 6· CCF4-AM/background suppression mixture was added to the nanowells, and the plate was incubated at 25 C for 90 min prior to measuring fluorescence.

High-throughput screening The HTS was carried out in a fully automated mode on a linear track robotic system by Robocon, (Vienna, Austria). The assay was initiated by plating 50 ll of cell suspension into 384-well plates using a Multidrop (Labsystems) and letting the cells adhere for at least 6 h at 37 C. Then 1 ll of 25 lM clavulanic acid in PBS and 0.25 ll of 500 lM test compounds in DMSO were added

Results and discussion b-Lactamase HVC replicon A b-lactamase reporter HCV replicon was constructed by replacing the neomycin phosphotransferase gene from an existing replicon called a bicistrionic

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HCV replicon by a b-lactamase reporter gene, as described elsewhere [12]. This bicistrionic replicon was produced by deleting the structural proteins, inserting a neo reporter whose translation was driven by the HCV IRES, and introducing the encephalomyocarditis (EMC) IRES which drives the translation of HCV nonstructural proteins NS3 to NS5B (Fig. 1B) into the HCV genome [11]. Optimization of viral RNA lipotransfection conditions To create a cell line that would give a robust signal for high-throughput screening, transfection conditions that would yield the highest percentage of b-lactamasepositive cells (blue cells; BC) were optimized. Three major variables in the transfection experiments, viral RNA dose, cell density, and duration of the transfection, were optimized. The readouts for this experiment were visual cell toxicity (death) 24 h posttransfection and percentage of b-lactamase-positive cells 4 days posttransfection as measured by DIP. An arbitrary scale for cellular toxicity was applied by visualizing the cells under a light microscope 24 h after transfection (data not shown). In general, the results showed that there was a direct correlation between higher toxicity with higher dose of HCV replicon RNA and longer duration of the transfection. Viral RNA doses of 900–2700 ng/ml and 24-h transfection resulted in maximum toxicity. Lower RNA doses with short (4-h) transfection times resulted in no detectable cellular toxicity. Furthermore, high cell densities seemed to have a protective effect and toxicity was reduced at higher cell densities. This protective effect was observed for all RNA doses and transfection times assayed. Four days after transfection, the percentage of blue cells (%BC) was determined by DIP (Table 1). The range of %BC varied from 0 to 66%. An inverse correlation Table 1 Percentage blue cells under different transfection conditions RNA dose (ng/ml) 300

900

2700

Time (h)

1 · 105 cells/well

2 · 105 cells/well

3 · 105 cells/well

4 · 105 cells/well

4 8 24 4 8 24 4 8 24

30 16 23 54 49 0 49 0 0

32 16 23 44 47 20 46 34 16

23 14 17 27 48 33 41 61 33

26 16 24 28 49 35 39 66 47

Note. Optimization of HCV replicon transfection into Huh-7 cell. Cellular seeding density, HCV RNA concentration, and duration of transfection are varied to determine the best conditions for creating a b-lactamase cell for drug screening. Four days posttransfection the %BC is determined by DIP. The optimal transfection conditions are identified to be 300,000–400,000 cells/well transfected with 2.7 lg/ml replicon RNA for 8 h.

between the level of toxicity (cell death) at day 1 and the %BC at day 4 was observed: cells with the highest toxicity score at day 1 had the lowest %BC. However, some indication of toxicity was not necessarily detrimental for b-lactamase activity because the samples that had the highest %BC also had some toxicity at day 1. The optimal transfection conditions that gave the highest %BC at day 4 posttransfection were high RNA dose (2700 ng/ml), medium load time (8 h), and high cell density (300,000–400,000 cells/well). Cellular replication of the HCV replicon Because the viral RNA is of positive polarity and translation is driven by IRES, there is at least one round of viral RNA translation in the cell. Therefore, even in the absence of HCV RNA replication, b-lactamase activity can be detected 1 day after transfection. The ability of the HCV RNA to replicate in Huh-7 cells was tested by measuring both b-lactamase activity and amounts of viral RNA with time. A replication-defective HCV RNA (nul) was used a negative control for HCV RNA replication. This HCV RNA (nul) was engineered and constructed by introducing two amino acid changes (aspartic acids at amino acid positions 318 and 319 in NS5b that are essential for enzymatic polymerase activity were replaced with alanines) in the NS5b-RNA-dependent RNA polymerase so that the protein was not functional and replication of HCV RNA was disrupted. The residual b-lactamase activity from the transfected replicons can be detected 1 day after transfection in both replication-competent (wt) and replication-defective (nul) RNA constructs. However, with time, the nonreplicating RNA is degraded in the cell and b-lactamase activity is lost. In these experiments, b-lactamase activity is not detectable after 6 days in the nul control, while 70% of the cells transfected with wt replicon had b-lactamase activity after this time period (Fig. 2). To confirm that the RNA levels were also maintained (i.e., replicating) in wt cells and reduced in nul cells, the viral RNA was quantified from total cellular RNA extracted. The results showed that viral RNA was lost in cells that once harbored the nul replicon and that there were significant levels of HCV RNA in cells that were transfected with the functional wt replicon (data not shown). These results indicate that the HCV b-lactamase replicon was replication competent, that b-lactamase activity was lost in the absence of replication, and that b-lactamase activity is a true reporter for HCV subgenomic replication. In addition, these data demonstrate that a 6 day growth period posttransfection is needed to detect b-lactamase activity due solely to replicated viral RNA. Next, we sought to assess the time of compound incubation to be able to detect inhibition of replication activity. This was tested by measuring the

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Fig. 2. Enhanced Huh-7 cells stained with CCF-4AM after transfection with wild type (wt) or replication-defective (nul) HCV replicon RNA. At day 1 posttransfection, >95% of the cells stain positive for b-lactamase activity, indicating that the transfection efficiency is identical for both samples. At day 6 posttransfection,
65% of the cells harboring wt RNA are positive for b-lactamase activity, whereas none of the cells transfected with nul replicon RNA stain positive.

Fig. 3. HCV RNA b-lactamase protein and activity in the presence of NS5B inhibitor or controls. (A) Northern blot analysis showing the decay of HCV RNA. In the absence of replication, replicon RNA degrades with time until no HCV RNA is detected at 48 h 28 s ribosomal RNA is shown as a control for RNA quality and gel loading. (B) Quantitation of HCV replicon RNA normalized to 28 s ribosomal RNA and plotted as a percentage of RNA at time zero. In 9 h
50% of HCV replicon RNA is degraded and
82% is gone by 24 h. However, there is still
18% HCV RNA to translate protein and generate a b-lactamase signal, causing background. (C) Western blot analysis of b-lactamase levels in the presence of clavulanic acid and an NS5B inhibitor. Lane 1, Multimark prestained standard (31 kDa); lane 2, DMSO control; lane 3, DMSO + 0.5 lM clavulanic acid, lane 4, IC100 dose of NS5B inhibitor. Cells were treated for 24 h with the indicated treatments. The top band is b-lactamase and the bottom band is a nonspecific band which shows uniformity of gel loading. We hypothesize that it is this residual b-lactamase activity that is being inhibited by clavulanic acid. (D) The decay rates of b-lactamase activity were measured in the presence of an NS5B inhibitor at 100% inhibitory dose (open symbols) either with (circles) or without (triangles) 0.5 lM clavulanic acid. Closed symbols represent cells without NS5B inhibitor. In the presence of only NS5B inhibitor, the half-life of b-lactamase activity was 15 h. With the addition of 0.5 lM clavulanic acid, the half-life was reduced to 5 h.

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Fig. 4. Schematic representation of replicon HTS assay format. (A) Preparation of frozen replication-harboring cells for HTS. Transfected cells were cultured until b-lactamase signal decayed to a steady state level, expanded to produce enough cells for the complete HTS, and frozen. The lag time between transfection and freezing was
6 days. (B) During HTS, cells were thawed on the day before the assay, plated, and let adhere for at least 6 h. Compounds were added, and the b-lactamase activity was measured after 24 h.

HCV RNA levels as a function of time [incubation time after 6 days posttransfection (see Materials and methods)] in the presence of an HCV-NS5b-RNA-dependent RNA polymerase inhibitor. The data from this experiment indicate that, ins the presence of a HCV replication inhibitor, the amount of HCV RNA was reduced by 50% in
9 h and by 82% after 24 h (Figs. 3A and B). Based on the results above, an assay for inhibitors of HCV RNA replication was established using the following protocol: Cells are transfected with HCV b-lactamase replicon RNA. To ensure that the assay was indeed monitoring replicating HCV mRNA expression, cells are incubated for 4–6 days before freezing. To test for inhibitory activity, cells were thawed and seeded on plates for 6–24 h and compounds were added for 24 h before b-lactamase activity was measured (Fig. 4). Conversion of HCV replicon b-lactamase assay from DIP to plate reader format To convert the HCV b-lactamase assay from DIP to a plate reader format, b-lactamase activity was measured at increasing cell numbers to determine the dynamic range of the assay and the optimal cell seeding. b-Lactamase activity was measured both as absolute fluorescence intensity at 460 nm (b-lactamase-positive cells, blue) and as a ratio of fluorescence intensity at 460 nm/530 nm. The absolute blue signal at 460 nm increased linearly (R = 0.92) with cell number from 625 to 20,000 cells/well, indicating that the signal correlated directly with the number of blue cells per well (Fig. 5A). When the same data shown in Fig. 5A are analyzed by the ratiometric method, the average blue/green ratio generated is
1.7 and no longer changes relative to cell number plated per well (Fig. 5B). This result confirms that the blue/green ratio method normalizes the signal so that it is no longer dependent on cell number plated per well. This means that using blue/green ratio analysis can eliminate the potential variable of having different cell numbers plated per well.

Fig. 5. Linearity of HCV replicon b-Lactamase assay. (A) b-Lactamase cells (d), b-lactamase cells mixed with Huh-7 cells (s), or Huh-7 cells (.) were seeded at 313 cells/well, increased by twofold to 20,000 cells/well, stained with CCF-4 AM, and read on a CytoFluor 4000 plate reader. Counts detected at 460 nm are plotted as a function of cell number and a linear relationship is shown. b-Lactamase cells alone and the b-lactamase Huh-7 cell mixture yield very similar graphs, indicating that there is very little quenching of blue signal from green cells. (B) The blue/green ratio method shows that for b-lactamase cells only the signal generated is independent of cell number, but when blactamase cells are mixed with Huh-7 cells (total cells per well constant at 20,000; b-lactamase cells increased by 2· for each well), the ratio drops as a function of blue cells per total cells.

To mimic the effect of a potential inhibitor in the screen, an experiment in which the signal from cells containing constitutively active b-lactamase was measured

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in the presence of green cells (no b-lactamase) was conducted. b-Lactamase-expressing cells were serially diluted into parental Huh-7 cells so that the total number of cells plated was fixed at 20,000 but the number of b-lactamase cells were reduced by half for each data point. When the cells were analyzed by the blue signal only the result was a linear relationship (R = 0.99) between the number of blue cells in the sample and the blue signal generated, as was the case when the blue cells only were plated (Fig. 5B). This result suggests that green cells do not quench the blue signal of the b-lactamase cells as the percentage of blue cells decreases in the sample. A linear relationship (R = 0.99) is also observed between signal and blue cell percentage when analysis is done by the blue/green ratio method (Fig. 5B). Residual b-lactamase activity in the HCV replicon transfected cells Initial experiments showed high, reproducible levels of b-lactamase activity after the 24 h incubation period. However, there remained significant levels of residual blactamase activity when known inhibitors of HCV NS5b RNA polymerase were used to inhibit replication (HCV b-lactamase replicon cells in the presence of inhibitor EC95 gave a blue/green ratio of 0.4, much higher than that of the untransfected parental cells, which gave a blue/green ratio of 0.05). More critically, IC50 values obtained for known inhibitors in the b-lactamase replication appeared 3- to 10-fold less potent than values obtained by assays that directly quantitated viral RNA in the same cell line (data not shown). While minimally acceptable screening data could be obtained using the assay in this format (threefold signal-to-background ratio and a Z factor of 0.5), a larger signal-to-background ratio and improved sensitivity to known inhibitors without increasing compound screening concentrations or incubation time were desirable. The high background and reduction in the apparent potency of inhibitors appeared to be related to high levels of residual b-lactamase activity. This activity results from several competing cellular processes, including viral RNA replication by viral enzymes, RNA degradation by cellular RNases, b-lactamase expression from replicon RNA, and b-lactamase degradation in the cells. After viral RNA replication is halted by inhibitors, blactamase will continue to be translated by viral IRES until all replicon RNA is completely degraded by the cellular RNases. The half-life of the replicon RNA in Huh-7 cells has been determined to be
9 h (Fig. 3B) in the presence of replication inhibitors. Measurements of the persistence of the b-lactamase activity in the presence of a 100% inhibitory dose of a viral replication inhibitor revealed that the half-lifetime for b-lactamase activity is
15 h (Fig. 3D, open circles), requiring incubation of nearly 3 days to completely eliminate the activ-

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ity. With a 9 h half-life of the replicon RNA, a 24 h assay incubation would leave
15% of the starting RNA to continue de novo b-lactamase synthesis. A 3- to 4 h b-lactamase half-life would result in the persistence of an additional 0.4–1.5% of the starting b-lactamase plus enzyme molecules that remain from de novo synthesis during the 24 h assay. We reasoned that, since there was still residual HCV RNA in the cells after 24 h inhibition of replication, b-lactamase was still being produced at low levels after this time period. Despite the low levels of b-lactamase, there was still a background resulting in a reduced assay window. Several approaches could be taken to increase the assay window: the time that the cells are exposed to inhibitor could be extended beyond 24 h to allow for greater reporter decay or the addition of a b-lactamase inhibitor could decrease the residual b-lactamase activity without significantly affecting the actual signal of the assay. As increasing incubation time would have a negative impact on throughput during the screening and would raise concerns of increasing apparent compound toxicity with prolonged incubation time, the use of a b-lactamase inhibitor seemed a more attractive approach. Clavulanic acid improves assay parameters Clavulanic acid is an irreversible inhibitor of b-lactamase that has been previously used as a means of suppressing enzymatic activity in constitutively active b-lactamase reporter gene systems [18]. In these applications, clavulanic acid is added at a concentration that completely inactivates all b-lactamase molecules in the cell. Excess clavulanic acid is then removed by washing the cells, and the assay then begins with the detection of the activity of only newly synthesized b-lactamase molecules. This approach, however, requires wash steps that create significant technical difficulties for robotics automation in an HTS environment, such as first to last plate timing issues for bulk washes or clogging of the washer. In addition, such assay formats are not conducive to miniaturization beyond the 384-well microplate format. To evaluate the effect of clavulanic acid in a homogeneous assay format on the signal-to-background ratio of the replicon assay, the replicon-expressing cells were treated with an increasing concentration of clavulanic acid while in the presence of an IC100 dose of NS5B inhibitor 2 0 -C-Met-A (Fig. 6) or DMSO-only control. Signal-to-background ratio was calculated by dividing the b-lactamase activity obtained from each clavulanic acid concentration with the activity from replication-inhibited wells (by an IC100 dose of 2 0 -C-Me-A) which contained an equivalent clavulanic acid concentration. Clavulanic acid concentrations from 20 nM to 2 lM resulted in a marked increase in the signal-to-background ratio of the assay (Fig. 6). This can be attributed to a 100-fold potency increase of clavulanic acid in the repli-

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Fig. 6. Clavulanic-acid-dependent signal-to-background ratio for the HCV replicon b-lactamase assay. In a 384-well plate containing HCVreplicon-b-lactamase-expressing cells, a dose response of clavulanic acid was made in the presence of either a 100% 28 inhibitory concentration of an NS5B inhibitor (closed symbols) or a buffer control (open symbols). The bars represent the ratio of signal (control) to background (inhibitor) with peak signal-to-background ratios at 0.1–0.5 lM clavulanic acid. Each data point is the median of 4 replicate wells.

cation-inhibited cells versus cells without replication inhibition. This shift in potency can be explained by the observations of Fisher et al. [19] that the potency of clavulanic acid for inactivation of b-lactamase was a function of the fold molar excess of clavulanic acid over b-lactamase. Indeed, in the presence of a replication inhibitor, the amount of b-lactamase in the cell decreases as intracellular enzymes degrade the replicon RNA and b-lactamase, and thus the fold molar excess of clavulanic acid over b-lactamase increases at a fixed concentration of clavulanic acid. At concentrations of clavulanic acid that exhibit improved signal-to-background ratio (20 nM–2 lM), the fold molar excess of clavulanic acid over b-lactamase is apparently not sufficient to significantly inhibit the larger amounts of b-lactamase present in the absence of replication inhibitors. When HCV replication is inhibited, viral RNA levels will decrease, causing b-lactamase protein levels to also decrease in the cell. Meanwhile, the number of cellular clavulanic molecules stays fixed and at some point the

number of clavulanic acid molecules will become sufficient to inhibit the enzymatic activity of all b-lactamase molecules at which time no blue signal is generated and background is reduced. Based on our optimization experiments, it was determined that 0.5 lM clavulanic acid gives the best signal-to-background ratio (Fig. 6). Additionally, we quantified cellular b-lactamase levels by Western blot after 24 h of 0.5 lM clavulanic acid treatment to confirm that the expression of b-lactamase was not affected (Fig. 3C). To assess the sensitivity of the assay to replication inhibitors at increasing concentrations of clavulanic acid, 2 0 -C-Me-A was titrated in the presence of increasing concentrations of clavulanic acid (Fig. 7A). At all concentrations of clavulanic acid tested, there was a marked reduction in the background signal achieved at a 100% inhibitory concentration of NS5B inhibitor, compared to the signal obtained from cells that had not been exposed to clavulanic acid. As the concentration of clavulanic acid increased, the apparent potency of the NS5B inhibitor increased. As explained above, this might be due to the fact that the inhibitory effect of clavulanic acid on b-lactamase activity is proportional to its fold molar excess over the enzyme. Based on these data, 0.5 and 0.25 lM concentrations of clavulanic acid were chosen to evaluate the high-throughput screening based on their signal-to-background ratios and the improved apparent potency of the control replication inhibitor. The final concentration of clavulanic acid used during the HTS was determined by testing a limited set of compounds from the screening collection for HCV replication activity, in the presence of 0.5 M and 0.25 lM clavulanic acid. These results (Figs. 7B and C) show that, by using higher concentrations of clavulanic acid, the apparent potency of some compounds is improved and they can then be readily identified as ‘‘hits’’ in the plates. Based on the result of this experiment, 0.5 lM was chosen as the concentration to use in the highthroughput screen. A reevaluation of the half-life of

Fig. 7. HCV replicon b-lactamase assay is more sensitive to inhibitors of replication at increasing concentrations of clavulanic acid present in the assay. (A) Dose responses of 2 0 -C-Me-A, a HCV NS5BA polymerase inhibitor, at increasing concentrations of clavulanic acid. The potency of the compounds increased at higher amounts of clavulanic acid in the assay: IC50s of 2 0 -C-Me-A were 1.8, 1.3, 0.9, 0.6, and 0.5 lM at clavulanic acids concentrations of 0, 0.125, 0.25, 0.5, and 1 lM, respectively. A plate with compounds was tested in the presence of two concentrations of clavulanic acid. A set of compound plates was screened using either 0.25 lM (B) or 0.5 lM (C) clavulanic acid. Shown are representative plates from this test which show losses of apparent potency of active compounds (circled) with decreased clavulanic acid concentration.

P. Zuck et al. / Analytical Biochemistry 334 (2004) 344–355

the b-lactamase activity in the presence of 0.5 lM clavulanic acid revealed a much improved signal decay profile over that of the initial experiments. In the presence of NS5B inhibitor and 0.5 lM clavulanic acid the halflife was reduced to 5 h (from 15 h) while in the presence of clavulanic acid alone it showed no reduction in signal compared to that of control cells through 48 h (Fig. 3D, open triangles). HTS of the HCV replicon b-lactamase assay Once the HCV b-lactamase replication system was optimized, a protocol for the production of large amounts of transfected cells was developed to support the primary HTS of
270,000 compounds and for follow-up assays. To decrease assay day-to-day variability, a large homogenous population of cells (over 2.8 billion cells) was produced so that the same lot of cells could be used throughout the drug discovery process. The same population of cells was used for robotics protocol validation, primary screening, confirmation of hits, and EC50 determination of selected compounds. The large population of transfected cells was prepared in two batches (over 1.4 billion cells per batch) that were frozen prior to assaying. The cells were frozen at 1 · 107 cells/vial. This enabled thawing and mixing equal numbers of cells from each batch. Over 90% of the cells were viable after thawing with no loss of b-lactamase activity. This strategy allowed us to produce high-quality, uniform data throughout the screen, with very stable assay statistics. Fig. 8 shows a summary chart with the median signal and basal b-lactamase activity for each of the first 1000 plates of the HTS, illustrating the stability of the assay window throughout the screen. The average %CV was 13, Z factor was 0.5, and signal-to-background ratio was
13. Using an inhibition cutoff of 40% (which corresponds to approximately the mean + 3standard deviation for all the data points

Fig. 8. Summary of the first 1000 plates of the HTS. The highthroughput screening was carried out and ratiometric readout calculated as described under Materials and methods. Closed circles represent the medians of the center 320 wells of a 384-well plate (test-compound-containing wells). Open circles are the median values of control wells containing an IC100 dose of an HCV replication inhibitor. The median signal-to-background ratio for the 1000 plates was 13-fold, and the median %CV was 13%. Included in the graph are all plates including plates which failed quality control due to pipettor errors, robotic failures, or other technical problems.

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from the screen), the hit rate was 1.5%, and the false positive rate was 0.1% (estimated by counting hits from empty wells). At the conclusion of the primary HTS, hits were selected for further confirmation and follow-up assays (Fig. 9). The selection process included analysis of the fluorescence intensity for each hit in the green fluorescence channel, as an indication of cellular dye uptake, which is an indirect measure of cellular toxicity. Therefore, compounds that were hits because of low dye uptake were not selected for follow-up. In addition, compounds with reactive functional groups were discarded. After applying these selection criteria,
1000 compounds were selected and tested in confirmatory assays, in triplicate, under the same assay conditions as used in the primary screen, and in the presence and absence of clavulanic acid. The results of the confirmatory assay show that 70% of the original hits were reproduced when using a 35% inhibition cutoff. This inhibition cutoff was selected because it was close to the average mean + 3SD of the screen (
45% inhibition) and produced a manageable number of hits for follow-up. The median value for these confirmed hits was 66% inhibition. The concurrent screen without clavulanic acid yielded a 38% confirmation rate using the same cutoff criteria and a median inhibition of 39% (Fig. 9). These results indicate that 42% of the confirmed actives in the clavulanic-acid-containing screen would not have been identified had the clavulanic acid been omitted, thus reaffirming the validity of the approach used herein. Miniaturization of the HCV replicon assay to 3456-well nanoplate format Recent advances in ultra-high-throughput screening technology have allowed the HCV b-lactamase assay

Fig. 9. HTS and follow-up strategy for HCV replicon b-lactamase assay. Shown is the funneling down of the 270,000-member screening collection to a manageable number of compounds that show desirable properties in the HCV replicon b-lactamase and cytotoxicity assays.

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Fig. 10. HCV replicon b-lactamase assay in 3456-well plate. The HCV replicon assay was validated in a 3456-well plate format using a flying reagent dispenser; 750 cells/well were added in a final volume of 1.8 ll. Wells on the left (100% inhibition) are plated with an IC100 dose of an HCV replication inhibitor. Wells on the right contain an IC50 dose of inhibitor. The center wells are where test compounds would be during an HTS campaign. This plate gave a 28-fold signal-to-background ratio, with a %CV of 14%.

to be miniaturized to a 3456-well nanoplate format [20,21]. Although the technology was not available at the time that the primary screen was implemented, the HCV replicon assay was tested in the nanoplate format to analyze its miniaturization potential for future use. As shown in the scatter plot of the assay in a nanoplate format (Fig. 10) this assay scales very well to 3456-well format and produced plates with a 14% CV and a signal-to-background ratio of
25-fold. Had the assay been tested in the 3456-well format, there would have been a savings of 1250 cells/well, and the screen could have been accomplished in 2 days instead of 6 weeks.

Conclusions A robust screening assay using a b-lactamase reporter gene format has been developed to identify inhibitors of HCV replication. The use of b-lactamase as a reporter, coupled to the use of a cell-permeable FRET b-lactamase substrate, enables the measurement of biological responses in live cells, without the need of a cell lysis step. As described in this work, by using the b-lactamase reporter system, we were able to quantify the number of cells harboring viral RNA in a population, in contrast to other reporter techniques, where an average signal is measured. The FRET b-lactamase substrate used also allows for a ratiometric readout which is used to normalize the signal to the number of cells per well in the assay, making the assay very robust for the purpose of HTS. The ratiometric readout allows for miniaturization of these assays to small-volume microplate formats, and these are now routinely miniaturized to a volume of <2 ll for ultra-high-throughput screening in the 3456well format. Several technical challenges needed to be solved to produce a b-lactamase HCV reporter cell line that was suitable for HTS. A robust assay window and good sen-

sitivity to known inhibitors of replication are required to identify novel inhibitors to HCV replication. Viral RNA transfection conditions that maximized the percentage of cells that would be permissive for HCV replication were identified. It was determined that b-lactamase activity measured 6 days after transfection was due solely to HCV replication and not to residual activity from the transfected RNA and that viral replication could be blocked by a known HCV RNA polymerase inhibitor. The initial assay was further optimized for HTS to increase the assay window and the sensitivity of the assay to HCV replication inhibitors. When known HCV replication inhibitors were used to further characterize the blactamase cell line, high background (relative to EC95 of HCV inhibitor) and a loss of inhibitor potency were observed. It was determined that the source of this problem was residual HCV RNA and b-lactamase activity present even after adding a HCV inhibitor for as long as 24 h. The inclusion of an optimized concentration of clavulanic acid, a b-lactamase inhibitor, in the assay significantly increased the signal-to-background ratio and the sensitivity of the assay to replication inhibitors. In addition, the IC50s of known inhibitors of HCV replication appeared more potent by three- to fivefold. We propose that some constitutively active b-lactamase reporter assays could potentially benefit from optimizing the concentration of clavulanic acid during the length of the assay in a nonwash protocol. An optimal amount of clavulanic acid could improve assay quality by both increasing signal-to-background ratio and aiding in the identification of weaker inhibitors. A novel approach to intact cell robotics HTS was applied by creating a large homogenous population of cells for the screening campaign, thus greatly improving the overall performance of the screen. The assay proved to be robust in a fully automated robotics 384-well microplate format, and it was successful in finding known inhibitors of HCV replication that existed in the screening library in addition to potentially novel inhibitors. The robustness of the assay also allowed for miniaturization to a 3456-well format for ultra-high-throughput screening.

Acknowledgments We thank Peter Hodder for support during the screen and Rick Peltier and Ira Hoffman for valuable software assistance on the screening data analysis.

References [1] N.C.o.H. Statistics, in: Third National Health and Nutrition Examination Survey, 1988– 1994, Hyattsville, MD: Center for Disease Control and Prevention, (1996).

P. Zuck et al. / Analytical Biochemistry 334 (2004) 344–355 [2] World Health Organization., Hepatitis C: global prevalence, Wkly. Epidemiol. Rec. 72 (1997) pp. 341–344. [3] D.M. Knipe, P.M. Howley, in: Fields Virology, fourth Ed., vol. 1 (2001) p. 1147. [4] M. Takahashi, G. Yamada, R. Miyamoto, T. Doi, H. Endo, T. Tsuji, Natural course of Hepatitis C, Am. J. Gastroenetrol. 88 (1993) 240–243. [5] M.J. Tong, N.S. El-Farra, A.R. Reikes, R.L. Co, Clinical outcomes after transfusion-associated Hepatitis C, N. Engl. J. Med. 332 (1995) 1463–1466. [6] M.W. Fried, M.L. Shiffman, K.R. Reddy, C. Smith, G. Marinos, F.L. Goncales, D. Haussinger, M. Diago, G. Carosi, D. Dhumeaux, A. Craxi, A. Lin, J. Hoffman, J. Yu, Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection, N. Engl. J. Med. 347 (2002) 975–982. [7] S. Rosenberg, Recent advances in the molecular biology of Hepatitis C virus, J. Mol. Biol. 313 (2001) 451–464. [8] K.E. Reed, C.M. Rice, in: H.W. Reesink (Ed.), Hepatitis C virus, Karger, Base, Switzerland, 1998, pp. 1–37. [9] R. Bartenschlager, V. Lohmann, Replication of hepatitis C virus, J. Gen. Virol. 81 (2000) 1631–1648. [10] Q.L. Choo, G. Kuo, A.J. Weiner, L.R. Overby, D.W. Bradley, M. Houghton, Isolation of a cDNA clone derived from blood-borne non-A, non-B viral hepatitis genome, Science 244 (1989) 359–362. [11] V. Lohmann, F. Korner, J.-O. Koch, U. Herian, L. Theilmann, R. Bartenschlager, Replication of subgenomic hepatitis C virus RNAs in hepatoma cell line, Science 285 (1999) 1437–1449. [12] E.M. Murray, J.A. Grobler, E.J. Markel, M.F. Pagnoni, G. Paonessa, A.J. Simon, O.A. Flores, Persistent replication of hepatitis C virus replicons expressing the b-lactamase reporter in subpopulations of highly permissive huh7 cells, J. Virol. 77 (2003) 2928–2935. [13] G. Zlokarnik, P.A. Negulescu, T.E. Knapp, L. Mere, N. Burres, L. Feng, M. Whitney, K. Roemer, R.Y. Tsien, Quantitation of

[14]

[15]

[16]

[17]

[18]

[19]

[20]

[21]

355

transcription and clonal selection of single living cells with blactamase as reporter, Science 279 (1998) 84–88. J.-H. Zhang, T.D.Y. Chung, K.R. Oldenburg, A simple statistical parameter for use in evaluation and validation of high throughput screening assays, J. Biomol. Screen. 4 (1999) 67–73. K.J. Blight, A.A. Kolykhalov, C.M. Rice, Efficient initiation of HCV RNA replication in cell culture, Science 290 (2000) 1972– 1974. S.S. Carroll, J.E. Tomassini, M. Bosserman, K. Getty, M.W. Stahlhut, A.B. Eldrup, B. Bhat, D. Hall, L. Simcoe, R. LaFemina, C.A. Rutkowski, B. Wolanski, Z. Yang, G. Migliaccio, R. De Francesco, L.C. Kuo, M. MacCoss, D.B. Olsen, Inhibition of Hepatitis C virus RNA replication by 2 0 -modified nucleosides analogs, J. Biol. Chem. 278 (2003) 11979–11984. M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye staining, Anal. Biochem. 72 (1976) 248–254. G. Zlokarnik, Fusions of b-Lactamase as a reporter for gene expression in live mammalian cells, Methods Enzymol. 326 (2000) 220–241. J. Fisher, R.L. Charnas, J.R. Knowles, Kinetic Studies on the inactivation of Escherichia coli RTEM b-lactamase by clavulanic acid, Biochemistry 17 (1978) 2180–2184. O. Kornienko, R. Lacson, P. Kunapuli, J. Schneeweis, I. Hoffman, T. Smith, M. Alberts, J. Inglese, B. Strulovici, Miniaturization of whole live cell-based GPCR assays using microdispensing and detection systems, J. Biomol. Screen. 9 (2004) 186– 195. P. Kunapuli, R. Ransom, K.L. Murphy, D. Pettibone, J. Kerby, S. Grimwood, P. Zuck, P. Hodder, R. Lacson, I. Hoffman, J. Inglese, B. Strulovici, Development of an intact cell reporter gene beta-lactamase assay for G protein-coupled receptors for high-throughput screening, Anal. Biochem. 314 (2003) 16–29.