High-throughput screening of low molecular weight NS3-NS4A ...

Antiviral Chemistry & Chemotherapy 16:385–392

High-throughput screening of low molecular weight NS3-NS4A protease inhibitors using a fluorescence resonance energy transfer substrate Kenji Sudo2, Kayo Yamaji1, Kouich Kawamura1, Tomoko Nishijima1, Naoko Kojima1, Kazuhiko Aibe1, Kunitada Shimotohno3† and Yasuaki Shimizu1* 1

Institute for Drug Discovery Research, Yamanouchi Pharmaceutical Co. Ltd., Ibaraki, Japan Rational Drug Design Laboratories, Fukushima, Japan 3 Virology Division, National Cancer Center Research Institute, Tokyo, Japan 2

*Corresponding author: Tel: +81 6 6390 1153, Fax: +81 6 6304 5367, E-mail: [email protected] †

Present address: Institute for Virus Research, Kyoto University, Kyoto, Japan

Hepatitis C virus (HCV) NS3-NS4A protease is an attractive target for anti-HCV agents because of its important role in replication. An optimized fluorescence resonance energy transfer (FRET) substrate for NS3-NS4A protease, based on the sequence of the NS5A-5B cleavage site, was designed and synthesized. High-throughput screening of in-house compound libraries was performed using a FRET substrate FS10 (MOCAcDKIVPC-SMSYK-Dnp) and MBP-NS3-NS4A fusion

protein. Several hit compounds were found, including YZ-9577 (2-oxido-1,2,5-oxadiazole-3,4diyl) bis (phenylmethanone) with potent inhibitory activity (IC50=1.6 µM) and good selectivity against other human serine proteases. Keywords: HCV, NS3-NS4A protease, fluorescence resonance energy transfer, serine protease, inhibitor

Introduction Human hepatitis C virus (HCV) is the major aetiologic agent of post-transfusion and sporadic non-A, non-B hepatitis. Chronic infection with HCV has also been linked to the development of liver cirrhosis and hepatocellular carcinoma (reviewed in Houghton et al., 1991). The HCV genome encodes a large polyprotein precursor of about 3000 amino acids, processed by host-derived, virusencoded, or both, proteases into 10 different polypeptides, encoded on the viral genome as follows: 5′-C-E1-E2-p7NS2-NS3-NS4A-NS4B-NS5A-NS5B (Kato et al., 1990; Choo et al., 1991; Houghton et al., 1991; Takamizawa et al., 1991). The N-terminal third of the NS3 protein, the NS3 protease (Cpro-2) domain, encodes a chymotrypsinlike serine protease (Grakoui et al., 1993; Hijikata et al., 1993). NS4A, a 54-residue protein expressed immediately downstream of NS3 in the viral polyprotein, is an effector or cofactor of NS3 protease activity which is necessary for the efficient cleavage of the NS3/4A, NS4A/4B, NS4B/5A and NS5A/5B junction sites (Bartenschlager et al., 1994, Failla et al., 1994). As the maturational proteolytic process is an essential step in replication of HCV, inhibition of the viral protease responsible for this event has become an important focus for the development of anti-HCV agents.

©2005 International Medical Press

The validity of this strategy has been confirmed by proofof-concept clinical trials of BILN-2061, an investigational NS3 protease inhibitor, in the treatment of HCV-infected patients (Lamarre et al., 2003). To facilitate the identification of potential NS3-NS4A protease inhibitors, a wide range of techniques has been used to measure NS3-NS4A protease activity in vitro. These include: i) an in vitro translation assay using viral polyprotein fragments as substrate (Lin & Rice, 1995); ii) high performance liquid chromatography (HPLC) methods with small synthetic peptides as substrate (Kakiuchi et al., 1995; Steinkuhler et al., 1996; Sudo et al., 1996) and iii) an ELISA assay using anti-substrate-specific antibodies (Kakiuchi et al., 1998). Owing to the labour intensive and time-consuming nature of these procedures, however, none are suitable for high-throughput screening. Although a C-terminal anilide-type substrate that releases para-nitroaniline (pNA) or (7-methoxycoumarine-4-yl) acetyl (MOCAc) is commonly used for some serine and cysteine proteases, this method cannot be adapted to NS3NS4A protease because of its strict recognition of the residues of P′ positions in their cleavage sites. An alternative approach in cases such as this is the use of internally

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quenched fluorogenic substrates. A number of quenched substrates based on the fluorescence resonance energy transfer (FRET) mechanism have been described for several viral proteases (Matayoshi et al., 1990; Handa et al., 1995; Holskin et al., 1995; Wang et al., 1997), including HCV NS3 protease (Yamaji et al., 1995; Taliani et al., 1996; Kakiuchi et al., 1999). In this study, the core sequence required for efficient cleavage of the NS5A-NS5B junction was determined and this information was used to design new FRET substrates. Using one of the substrates and MBP-NS3NS4A protein, a rapid and convenient high-throughput screening (HTS) system for NS3-NS4A protease inhibitors was constructed, and HTS of in-house compound libraries was performed. One hit compound, YZ-9577, showed potent inhibitory activity against NS3-NS4A protease and good selectivity against other human serine proteases.

Materials and methods Preparation of MBP-NS3-NS4A fusion protein MBP-NS3-NS4A, a fusion protein containing an HCV polypeptide spanning amino acids 1027 to 1711 (which cover the full length of NS3 and NS4A proteins) fused with Escherichia coli MBP at the N terminus was used as the source of NS3 protease activity. Expression and purification of MBP-NS3-NS4A and MBP-NS3 have been described previously (Shimizu et al., 1996; Sudo et al., 1996). The enzyme was stored at –80°C in 20% glycerol until use.

Peptide synthesis All peptides used were synthesized using an automated multiple peptide synthesizer (model PSSM-8; Shimadzu Corporation, Kyoto, Japan). For the synthesis of FS09 [MOCAc-DKIVPC-SMSK(Dnp)-NH2], FS10 [MOCAc-DKIVPC-SMSYK(Dnp)-NH2] and FS11 [MOCAc-KDKIVPC-SMSYK(Dnp)-NH2], FmocLys(Dnp)-DMPAMP resin was prepared as described previously (Knight et al., 1992; Nagase et al., 1994). Amino acids for DKIVPC-SMS (for FS09), DKIVPC-SMSY (for FS10) and KDKIVPC-SMSY (for FS11) were incorporated into the Fmoc-Lys(Dnp)-DMPAMP resin using the above synthesizer. N-termini peptide resins were acylated with 7-methoxycoumarin-4-acetic acid using standard synthesis cycles (Fields et al., 1992). Cleavage from resins and deprotection of side chains were performed in 89% trifluoroacetic acid (TFA) and 11% scavenger cocktail (ethanedithiol:thioanisole, 1:1) at 25°C for 1 h. Peptides were purified by preparative reverse-phase HPLC (ODS-80Tm, 2.15×30 cm; Tosoh Corporation, Tokyo, Japan) using 0.1% aqueous TFA/acetonitrile-based

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mobile phases. Peptide validation by analysis of amino acid composition and electron-spray (ESI) mass spectrometry confirmed that purities exceeded 95%. All peptides were stored frozen at –20°C as 10 mM stock solutions in 100% dimethyl sulfoxide (DMSO).

Design of peptide substrate Relative rates of hydrolysis for peptide substrates were measured using HPLC. A standard assay was performed in a total volume of 200 µl assay buffer (50 mM TrisHCl, 30 mM NaCl, 1 mM MgCl2 and 2 mM DTT; pH7.5) containing 1.4 µg MBP-NS3-NS4A (0.06 µM), 26 µM peptide substrate and 5 mM of internal standard peptide, IS5 (NH2-SMSYKDK-COOH). The reaction was initiated by the addition of synthetic substrates and incubated for 3 h at 37°C, then terminated by the addition of 0.1% TFA. Analytical HPLC was performed using a Shimadzu LC-10A liquid chromatography system (Kyoto, Japan). Each sample (20 µl) was analysed by reverse-phase HPLC (ODS80Ts, 0.46×15 cm; Tosoh; Tokyo), eluting with a linear gradient from 0.1% TFA in water to 0.1% TFA in 60% acetonitrile over 30 min at a flow rate of 1.0 ml/min. Peptides were detected by UV absorbance at 210 nm and cleavage products were quantified by calculating peak areas. Kinetic constants for proteolytic cleavage of S13 by MBP-NS3-NS4A or MBP-NS3 were determined by a Lineweaver–Burke plot in which the initial reaction rates versus peptide concentrations were plotted over a range of 50 to 400 µM, as described previously (Shimizu et al., 1996).

Continuous fluorescence assay of NS3-NS4A protease activity For fluorometric NS3-NS4A protease assay, 200 µl assay buffer (100 mM Tris-HCl, 30 mM NaCl, 2 mM DTT; pH8.0) containing 0.5, 1 or 2 µg of MBP-NS3-NS4A were dispensed into 96-well plates (MS-8596, Sumitomo Bakelite Co., Ltd., Tokyo, Japan). Reactions were started by the addition of 2 µl of 0.1 mM FS09, FS10 or FS11 substrate (final 1 µM), then incubated at 37°C. Progress of the enzyme reaction was monitored using a Fluostar spectrofluorometer (Tecan Japan Co., Ltd., Tokyo, Japan) at an excitation wavelength of 320 nm and emission wavelength of 405 nm.

HTS for NS3-NS4A protease inhibitors HTS of in-house low molecular weight (less than 500 daltons) compound libraries was performed in a stopped time format using 96-well plates. Test compounds (2 µl each; 1 mM in DMSO) were added to a 96-well plate, then mixed with 200 µl assay buffer containing 2 µg MBP-NS3-NS4A per well. Reactions were started by the


NS3-NS4A protease inhibitors

addition of 2 µl of 0.1 mM FS10 substrate, incubated for 1 h at 37°C, then terminated by the addition of 2 µl of acetic acid. Compounds that reduced fluorescence intensity obtained from the control sample (DMSO) by more than 50% were identified as HTS hit compounds and used for the next IC50 measurement. IC50 values for inhibitors were calculated as the concentration of inhibitor required to reduce fluorescence to 50% of the control value.

inhibitors were calculated as the concentration of inhibitor required to reduce fluorescence to 50% of the control value.

Results Determination of minimum sequence for NS3NS4A protease substrate As NS3 protease cleaves the NS5A-NS5B junction more efficiently than NS3-NS4A, NS4A-NS4B, NS4B-NS5A junctions (Steinkuhler et al., 1996), the amino acid sequence derived from the NS5A-NS5B junction was used to design a sensitive fluorogenic substrate for the NS3 protease. This group previously used a 20 mer synthetic peptide substrate (S1) that mimics the NS5A-5B junction for an in vitro HPLC assay (Kakiuchi et al., 1995; Sudo et al., 1996); however, this was too long to modify as a FRET substrate. As FRET efficiency is critically dependent on the distance between the donor and acceptor, plus efficient fluorescence quenching is important for a low fluorescence background and a large dynamic range, the S1 substrate was systematically truncated from both ends, then relative rates of peptide hydrolysis were examined (Table 1). Some amino acids were substituted to K (Lys) to increase substrate solubility. All tested substrates were cleaved more effectively by MBP-NS3-NS4A than MBP-NS3. An 18 mer peptide substrate, S2 (P9–P9′), was cleaved as efficiently as the original S1 substrate, but S3 (P8–P8′), S4 (P7–P7′) and S5 (P7–P6′) showed gradual reductions in hydrolysis rate to 44%. In contrast, S6 (P9–P5′) and S7 (P9–P4′), with further C-terminal truncation and Nterminal extension by two K residues next to the P7 G (Gly), recovered their hydrolysis rate to the original level.

Human protease selectivity assay Inhibitory activity of HTS hit compounds against several human serine proteases was measured using a fluorogenic assay in a total volume of 200 µl of phosphate-buffered saline (PBS) in 96-well microtitre trays (MS-8596, Sumitomo Bakelite Co. Ltd., Tokyo, Japan). Serial fivefold dilutions of the inhibitors and peptidyl-MCA (4-methyl-coumaryl-7-amide) substrates at a final concentration of 0.1 mM were mixed with trypsin (0.1 mg/ml), chymotrypsin (0.001 mg/ml), plasmin (0.1 mg/ml) or elastase (1.0 mg/ml) and incubated for 1 h at 37°C. Trypsin and chymotrypsin were purchased from Funakoshi Co. Ltd. (Tokyo, Japan). Plasmin and elastase were purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Substrates used in this assay were benzoyl-Arg-MCA for trypsin, succinyl-Ala-Ala-ProPhe-MCA for chymotrypsin, t-butyloxycarbonyl-GlnLys-Lys-MCA for plasmin and succinyl-Ala-ProAla-MCA for elastase, which were purchased from Peptide Institute Inc., (Osaka, Japan). Enzymatic activity was measured by fluorophotometric analysis at an excitation wavelength of 380 nm and emission wavelength of 460 nm using Fluostar (Tecan Japan, Tokyo, Japan). IC50 values for

Table 1. Minimum sequence determination for NS3-NS4A substitute Peptide Sequence Substrate No. S-1 S-2 S-3 S-4 S-5 S-6 S-7 S-8 S-9 S-10 S-11 S-12 S-13

P- 10 9

8

7

G E E

A G A G A G G G K K G K K G K K G K G G

K

6 5

Hydrolysis rate (%)

4

3

2

1

–

1′ 2′ 3′ 4′ 5′ 6′ 7′ 8′ 9′ 10′

NS3-NS4A

NS3

I I I I I I I I I I I I D K I

V V V V V V V V V V V V V

P P P P P P P P P P P P P

C C C C C C C C C C C C C

– – – – – – – – – – – – –

S S S S S S S S S S S S S

85 92 75 59 44 88 89 9 11 22 62 49 85

19 14 8

D D D D D D D D D D D

D D D D D D D D D D D

M M M M M M M M M M M M M

S S S S S S S S S S S S S

Y Y Y Y Y Y Y

T T T T T T

W W W W W

T T T T

Y K D K Y K D K Y

G A L G A G

NT

4 NT

7 0 0 2 5 9 8

Various peptide substrates were incubated with MBP-NS3-NS4A or MBP-NS3 at 37˚C for 3 h in a total volume of 200 µl. Sample was then analysed using HPLC as described in materials and methods; Substituted Ks are indicated by underlining; NT, not tested.

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Design and characterization of FRET substrates An optimized FRET substrate was designed based on the core sequence of S13. Fluorescent donor MOCAc and quenching acceptor Dpn groups were introduced at the extremities of S13 (FS11). Two other FRET substrates, FS10, in which K at P7 was removed, and FS09, in which both K at P7 and Y at P4′ of FS11 were removed, were also synthesized (see materials and methods). Continuous proteolysis of three substrates of different length by MBP-NS3NS4A was monitored by fluorescence intensity change using a fluorometer. The hydrolysis rate of FS10 was fastest and the fluorescence intensity of FS10 after 30 min of incubation was about twofold higher than FS11 (Figure 1). In contrast, the hydrolysis rate of FS09 was reduced compared to FS11 (Figure 1). FS10 was therefore selected for all further experiments as an optimized FRET substrate. The signal-tonoise ratio of FS10 was more than 20, sufficient for the detection of NS3-NS4A protease activity. When FS10 was incubated with various amounts of MBP-NS3-NS4A (0.5, 1 and 2 µg per well, corresponding

Table 2. Kinetic constants for proteolytic cleavage of S13 Enzyme

Km (µM)

Kcat (min–1)

Kcat/Km (min–1/µM)

MBP3-NS3 MBP-NS3-NS4A

456 104

0.28 6.06

6.23×10–4 5.82×10–2

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Figure 1. Cleavage of FRET substrate, FS09 , FS10 and FS11 by NS3-NS4A protease

FS10 FS11

FS09 Blank

Fluorescence intensity

1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

50

100

150

200

Time (min) Reactions were carried out in a 96-well plate at 37˚C and monitored continuously with a fluorometer as described in materials and methods.

to 0.022, 0.043 and 0.086 µM, respectively), the initial rate of cleavage was proportional to the concentration of enzyme added (Figure 2). When 1 or 2 µg of enzyme was used, the rate of cleavage decreased with longer incubation times because of substrate depletion.

Figure 2. Cleavage of FS10 as a function of NS3-NS4A enzyme concentration 2 µg protein 1 µg protein 0.5 µg protein 1.4 1.2


S8 (P7–P3′) with further C-terminal truncation of the P4′ Y (Tyr) residue, however, caused a dramatic decrease in hydrolysis rate (9%). These results suggest that the Y at P4′ is critical for substrate recognition. Although N-terminal truncation beyond the P8 residue caused a moderate decrease in hydrolysis rate (S4, S5), substitutions of G at P7 and D (Asp) at P5 with both K residues (S13) produced a recovery in hydrolysis rate to the level of the original S1 substrate. When the N-terminal was truncated further, the hydrolysis rate (S11, S12) was gradually reduced. P7 was therefore defined as necessary for substrate recognition and consequently it was presumed that S13 is a suitable basic sequence for FRET substrates. The steady-state kinetic constant for the proteolytic activities of MBP-NS3-NS4A and MBP-NS3 using S13 substrate was investigated. Table 2 shows that for MBPNS3, the Km value was 456 µM and kcat was 0.28 min–1 for S-13. In contrast, in MBP-NS3-NS4A, Km (104 µM) was about four times lower than for MBP-NS3 whereas kcat (6.06 min–1) was about 22 times higher. Since the coefficient of proteolytic efficiency, kcat/Km, was increased about 93 times in the presence of NS4A, the following HTS system was utilized with MBP-NS3-NS4A.

1 0.8 0.6 0.4 0.2 0 0

50

100

150

200

Time (min) FS10 was incubated with various amounts of MBP-NS3-NS4A protease at 37˚C, then monitored as described in materials and methods.


NS3-NS4A protease inhibitors

Figure 3. Optimum pH for NS3-NS4 protease activity

1 Fluorescence intensity

To determine the optimum pH, cleavage reactions were conducted at various pH levels. As shown in Figure 3, pH8.0 was found to be the optimum pH for this enzymatic reaction. Optimum temperature for the assay was also investigated. Although cleavage proceeded sufficiently at 25°C, the most efficient cleavage was observed at 37°C and 42°C (data not shown). In addition, DMSO, in which the substrate and inhibitors were dissolved, had no significant effect on NS3-NS4A protease activity when used at a final concentration of 2% or less (data not shown). Comparison of the time-course of proteolytic cleavage of FS10 between MBP-NS3 and MBP-NS3-NS4A as expected showed MBP-NS3 hydrolyzed FS10 very slowly, but the rate of substrate cleavage was significantly augmented by the use of MBP-NS3-NS4A (Figure 4).

0.8 0.6 0.4 0.2 0 6.5

7

7.5

8

8.5

pH

HTS screening of in-house compound libraries

Discussion Although a combination of pegylated IFN-α and ribavirin produces a sustained virological response in 72–82% of patients infected with HCV genotypes 2 and 3, eradication failure still occurs in 54–58% of patients infected with genotype 1 (Pawlotsky, 2003). In addition, the adverse effects of IFN-α and ribavirin are a major problem and sometimes lead to the discontinuation of treatment in

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Figure 4. Kinetic analysis of cleavage reaction of FS10 substrate by either MBP-NS3-NS4A or MBP-NS3 alone

MBP-NS3 MBP-NS3-NS4A

1.6


In an attempt to identify novel low molecular weight NS3-NS4A protease inhibitors, HTS of in-house chemical libraries using FS10 and MBP-NS3-NS4A found four compounds with IC50 values less than 10 µM (Table 3). These IC50 values obtained from fluorometric assay were similar to those obtained previously by HPLC assay (Sudo et al., 1996). When the enzyme selectivity of these compounds was examined against four human serine proteases (trypsin, chymotrypsin, plasmin and elastase), YZ-3750 [1,8-dihydroxy-2-naphthaldehyde, molecular weight (MW) 188.18], YZ-9527 [5-(3,4-dihydroxybenzylidene)-1-(4-ethoxyphenyl)pyrimidine-2,4,6(1H,3H,5H) -trione, MW 368.34] and YZ-9553 [(2,5-dioxopiperazine-1,4-diyl)bis{(5-nitro-2-furyl)methylene} diacetate, MW 480.34] displayed a broad range of inhibitory effects on these human serine proteases. This lack of selectivity rendered them inappropriate as lead compounds for antiHCV agents. In contrast, YZ-9577 [(2-oxido-1,2,5oxadiazole-3,4-diyl)bis(phenylmethanone), MW 294.26] selectively inhibited NS3-NS4A protease and had selectivity indices (IC50 value for human serine protease/IC50 value for HCV protease) against trypsin, chymotrypsin, plasmin and elastase of 20, 7.5, >31 and >31, respectively. On this basis, YZ-9577, a potent and highly selective protease inhibitor, was selected as a candidate lead compound for development as a novel anti-HCV agent.

1.4 1.2 1 0.8 0.6 0.4 0.2 0 0

20

40

60

80

100

120

Time (min) FS10 was incubated with 2 µg per well of MBP-NS3-NS4A or MBP-NS3 in 200 µl of assay buffer at 37˚C, then monitored as described in materials and methods.

certain patient populations. Together, the limited efficacy and side effects of IFN-α-based therapies highlight the unmet need for new therapies. A promising alternative approach to the control of HCV infection is NS3-NS4A serine protease inhibitors. Recently, proof-of-concept clinical trials of BILN 2061 (MW 774.94) have confirmed the usefulness of NS3-NS4A protease inhibitors in the reduction of HCV RNA plasma levels (Lamarre et al., 2003). Nevertheless, further clinical trials are on hold pending the resolution of animal toxicity issues (Hinrichsen et al., 2004)

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Table 3. Inhibitory activity of HTS hit compounds against NS3-NS4 protease and other human serine proteases

N

OH

HN

O

O +

O–

N

O

O

N

OH

O

N

N O O

YZ-3750

O

O

O

N O

O

O

OH

+

O

YZ-9577 YZ-9527

YZ-9553

O

N+ O–

O

OH

O–

O

O

Compound

HCV protease IC50 (µM) Plate HPLC

Trypsin IC50 (µM)

Chymotrypsin IC50 (µM)

Plasmin IC50 (µM)

Elastase IC50 (µM)

YZ-3750 YZ-9527 YZ-9553 YZ-9577

0.5 4.4 1.4 1.6