EP2057278A2 - Procédé pour identifier des inhibiteurs de protéases - Google Patents

Procédé pour identifier des inhibiteurs de protéases

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Publication number
EP2057278A2
EP2057278A2 EP07811572A EP07811572A EP2057278A2 EP 2057278 A2 EP2057278 A2 EP 2057278A2 EP 07811572 A EP07811572 A EP 07811572A EP 07811572 A EP07811572 A EP 07811572A EP 2057278 A2 EP2057278 A2 EP 2057278A2
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EP
European Patent Office
Prior art keywords
seq
peptide
protease
substrate
hepatitis
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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EP07811572A
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German (de)
English (en)
Inventor
William P. Taylor
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Vertex Pharmaceuticals Inc
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Vertex Pharmaceuticals Inc
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Publication of EP2057278A2 publication Critical patent/EP2057278A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase

Definitions

  • the invention relates to methods for assaying the activity of Hepatitis C NS3 protease and for methods of screening for inhibitors of HCV NS3 protease.
  • HCV hepatitis C virus
  • the HCV genome encodes a polyprotein of 3010-3033 amino acids, which has the structure NH 2 -C-E l-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH [Q. L. Choo, et. al., "Genetic Organization and Diversity of the Hepatitis C Virus," Proc. Natl. Acad. Sci. USA, 88, pp. 2451-2455 (1991); N. Kato et al., "Molecular Cloning of the Human Hepatitis C Virus Genome From Japanese Patients with Non-A, Non-B Hepatitis,” Proc. Natl. Acad. Sci. USA, 87, pp.
  • the HCV nonstructural (NS) proteins are presumed to provide the essential catalytic machinery for viral replication.
  • the NS proteins are derived by proteolytic cleavage of the polyprotein [R. Bartenschlager et. al., "Nonstructural Protein 3 of the Hepatitis C Virus Encodes a Serine-Type Proteinase Required for Cleavage at the NS3/4 and NS4/5 Junctions," J. Virol., 67, pp.
  • the HCV NS protein 3 contains a serine protease activity that helps process the majority of the viral enzymes, and is thus considered essential for viral replication and infectivity.
  • the first 181 amino acids of NS3 (residues 1027-1207 of the viral polyprotein) have been shown to contain the serine protease domain of NS3 that processes all four downstream sites of the HCV polyprotein [C. Lin et al., "Hepatitis C Virus NS3 Serine Proteinase: Trans-Cleavage Requirements and Processing Kinetics", J. Virol., 68, pp. 8147- 8157 (1994)].
  • the NS3 protein also possesses helicase and NTPase domains.
  • HCV NS3 serine protease and its associated cofactor, NS4A help process all of the viral enzymes, and are thus considered essential for viral replication. This processing appears to be analogous to that carried out by the human immunodeficiency virus aspartyl protease, which is also involved in viral enzyme processing. HIV protease inhibitors inhibit viral protein processing and are potent antiviral agents in humans, indicating that interrupting this stage of the viral life cycle results in therapeutically active agents. Consequently the analogous HCV NS3 protease and NS4A are attractive targets for drug discovery. [07] Several potential HCV protease inhibitors have been described [PCT Publication Nos.
  • inhibitors would have therapeutic potential as protease inhibitors, particularly as serine protease inhibitors, and more particularly as HCV NS3 protease inhibitors.
  • Such compounds may be useful as antiviral agents, particularly as anti-HCV agents. Consequently, an assay that can identify more effective HCV NS3 protease inhibitors is needed.
  • the present invention provides a method for assaying HCV NS3 protease activity using an NS3 «4A protease molecule.
  • the invention also provides a method for screening and identifying modulators of NS3 protease.
  • the invention provides a method for measuring the activity of HCV NS3 protease by adding to a sample containing an isolated protein comprising SEQ ID NO:1 and a peptide substrate based on the'NS5A/5B cleavage site for HCV genotype Ia. The substrate and products are separated by HPLC and the product peaks are quantitated.
  • the method further comprises adding to the sample an NS4A cofactor peptide comprising SEQ ID NO:4.
  • the peptide substrate can comprise the sequence of SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:6.
  • the substrate comprises SEQ ID NO:2 and the product peak is quantitated using absorbance data collected at 210 ⁇ m.
  • the C-terminal product peak can also be quantified using fluorescence data collected at about 350 ⁇ m excitation /about 490 ⁇ m emission when the substrate comprises SEQ ID NO:5. If the substrate is SEQ ID NO:6, the N-terminal product peak can be quantitated using fluorescent data collected at about 440 ⁇ m excitation /about 520 ⁇ m emission.
  • the invention provides a method for identifying modulators of the Hepatitis C NS3»4A protease.
  • a test compound is added to a sample that includes a protein comprising SEQ ID NO: 1.
  • a peptide substrate based on the NS5 A/NS5B cleavage site for Hepatitis C genotype 1 a is added and the substrate and products are separated using HPLC and the product peaks are quantitated.
  • the method further comprises adding to the sample an NS4A cofactor peptide comprising SEQ ID NO:4.
  • the invention provides a method for determining whether a test compound modifies the activity of the NS3 protease or the activity of the NS4A cofactor.
  • a test compound added to a sample that includes a protein comprising SEQ ID NO: 1.
  • An NS4A cofactor peptide is added and a peptide substrate based on the NS5A/NS5B cleavage site for Hepatitis C genotype 1 a is also added.
  • First amounts of products are measured by separating the substrate and products on a reverse phase HPLC column; and quantifying the product peaks.
  • First amounts of products are compared to second amounts of products, wherein the second amounts of products are measured in the absence of a NS4A cofactor peptide.
  • the NS4A cofactor peptide comprises SEQ ID NO:4.
  • the peptide substrate is SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:6.
  • the invention provides a kit comprising a sample that includes a protein containing SEQ ID NO: 1 and a peptide substrate based on the NS5A/NS5B cleavage site for Hepatitis C genotype 1 a.
  • the kit further comprises an NS4A cofactor peptide comprising SEQ ID NO:4.
  • the peptide substrate is SEQ ID NO:2, SEQ ID NO:5 or SEQ ID NO:6, or a combination thereof.
  • the kit further comprises one or more known modulators of NS4A cofactor and one or modulators of NS3 protease.
  • analog refers to derivatives of the reference molecule, such as substrates or cofactors, which retain desired activity, such as ability to be cleaved by the NS3 protease or to act as a cofactor for the protease.
  • analog refers to compounds having a native peptide sequence and structure with one or more amino acid additions, substitutions and/or deletions. The amino acid changes include non-naturally occurring amino acids.
  • analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into four families: (1) acidic— aspartate and glutamate; (2) basic—lysine, arginine, histidine; (3) non- polar—alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar— glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine.
  • Phenylalanine, tryptophan and tyrosine are sometimes classified as aromatic amino acids.
  • an isolated replacement of leucine with isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar conservative replacement of an amino acid with a structurally related amino acid will not have a major effect on the biological activity.
  • the polypeptide of interest may include up to about 5-10 conservative or non-conservative amino acid substitutions, or even up to about 15-25 conservative or non-conservative amino acid substitutions, or any integer between 5 and 25, so long as the desired function of the molecule remains intact.
  • polypeptide or "protein” refers to a polymer in which the monomers are amino acid residues which are joined together through amide bonds.
  • polypeptide As used herein is intended to encompass any amino acid sequence and includes modified sequences such as glycoproteins.
  • polypeptide is specifically intended to cover naturally occurring proteins, as well as those which are recombinantly or synthetically synthesized, which occur in at least two different conformations wherein both conformations have the same or substantially the same amino acid sequence but have different three dimensional structures.
  • “Fragments” are a portion of a naturally occurring protein. Fragments can have the same or substantially the same amino acid sequence as the naturally occurring protein.
  • Substantially the same or substantially similar means that an amino acid sequence is largely, but not entirely, the same, but retains a functional activity of the sequence to which it is related.
  • two amino acid sequences are “substantially the same” or “substantially homologous” if they are at least 85% identical.
  • the method of the present invention identifies modulators of the Hepatitis C NS3 protease and/or modulators of the NS3 « 4A protease and cofactor by measuring the activity of the Hepatitis C NS3 protease in vitro.
  • the method includes (a) providing a sample that includes an isolated protein with NS3 protease activity, (b) adding a peptide substrate, (c) optionally adding an NS4A cofactor peptide, (d) separating the substrate and products, and quantifying the product peaks.
  • the method can be used to detect modulators of NS3 protease and/or NS3"4A protease and its cofactor, including activators and inhibitors.
  • the protease protein can comprise the entire NS3 polypeptide of HCV, the NS3 and NS4A polypeptide, and longer polypeptides that include NS3, NS4A and NS4B. Shorter protease polypeptides can also be used in the assay, including, for example, the NS3 polypeptide and a fragment comprising from 1 to 53 amino acids of the NS4A polypeptide.
  • the protease protein can also comprise shorter polypeptides that have protease activity, for example, the first 181 amino acids of theNS3 protein or longer fragments of the NS3 protein.
  • the NS3-4A polypeptides and the NS3-4A-4B polypeptides can be continuous as in the HCV polyprotein, or the N S3 and NS4A polypeptides can be interrupted by a linker or other peptide sequence. Likewise, the NS3 and NS4A-4B polypeptides can be continuous or interrupted.
  • the cleavage site between NS3 and NS4A can be mutated in the nucleic acid sequence, before it is expressed, so that upon expression, NS4A remains covalently bound to NS3.
  • the protease proteins can be fusion proteins containing a heterologous protein attached.
  • an N- or C-terminal histidine or GST tag can be added to the protease proteins to aid in purification.
  • Other peptides or components can also be added, for example to immobilize the protease protein on a solid support.
  • Polynucleic acids encoding the protease proteins can inserted into expression vectors using recombinant DNA techniques and expressed in a number of cells or organisms including, without limitation, bacteria, such as E. coli; yeast, such as Saccharomyces cerevisiae, other yeasts and fungi, insect cells using a baculovirus vector or other expression system and mammalian systems, including cultured cells.
  • bacteria such as E. coli
  • yeast such as Saccharomyces cerevisiae, other yeasts and fungi
  • insect cells using a baculovirus vector or other expression system and mammalian systems, including cultured cells.
  • the vectors and expression systems for the above are well known to those skilled in the art and can be found in Current Protocols in Molecular. Biology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, 1988; with supplements 2007, which is incorporated herein by reference.
  • the substrate can be a peptide or a modified peptide that mimics one of the natural cleavage sites of the NS3 protease, sequences of HCV protease sites NS5A/5B, NS4A/4B and NS4B/5A (Grakoui et al., Virology, 67:2832-43, 1993).
  • the substrate comprises a recognition site flanked on both sides by at least 3 amino acids, which can be those amino acids naturally adjacent to the protease cleavable peptide bond.
  • the substrate peptides can be as short as 8 amino acids or can be longer than 20 amino acids.
  • One or more copies of a protease recognition site can be present in the substrate peptide, and they can be present in series.
  • Depsipeptide substrates, peptides with at least one ester bond in the peptide can also be used.
  • Other residues, such as non-naturally occurring amino acids can be added or substituted for amino acids in the peptide substrate.
  • the substrate peptides can also contain modifications, including for example, rare amino acids and dextra-amino acids. The modified peptides can be chemically synthesized.
  • the substrate polypeptide can be used without a signal used to detect cleavage of the substrate (detectable signal), for example, the NS5AB peptide of SEQ ID NO:2.
  • the substrate can also have a fluorescent label, such as EDANS (1-naphthalenesulfonic acid-5(2- aminoethylamide), as in the NS5AB-EDANS peptide of SEQ ID NO:5, FITC (fluorescein isothiocyanate), as in FITC-NS5AB-1 of SEQ ID NO:6, or other fluorescent labels.
  • the label can be attached to the N-terminal or C-terminal end of the substrate, or can be attached to an internal residue. Other fluorescent labels known to the skilled artisan can also be used.
  • the substrate can also be labeled with a chromogenic substrate that can be detected upon cleavage.
  • a chromogenic substrate that can be detected upon cleavage.
  • the C-terminal carboxyl groups can be esterified with a chromophoric alcohol, including 3- or 4-nitrophenol, 7-hydroxy-4-methyl-coumarin and 4- phenylazophenol, as described in Zhang et al., Analytical Biochemistry, 270:268-75, 1999.
  • the cleaved substrate is detected spectrophotometrically.
  • 3-nitrophenol and 7- hydroxy-4-methyl-coumarin can be detected at 340 ⁇ m, 4-phenylazophenol at 370 ⁇ m, and 4- nitrophenol at 400 ⁇ m.
  • Other chromogenic substrates can also be used.
  • the substrate can also labeled with two labels that form a Resonance Energy Transfer (RET) pair.
  • the substrate can contain a chromophore and a fluorophore placed on opposites of the scissile bond so that the chromophore quenches the fluorescence of the fluorophore.
  • RET Resonance Energy Transfer
  • the chromophore and the fluorophore are separated and fluorescence can be detected.
  • EDANS can be on one side of the scissile bond and a DABCYL (4-dimethylaminophenylazobenzoyl) group attached to an amino acid residue on the other side.
  • DABCYL must be close enough to quench the fluorescence of EDANS, for example, DABCYL and EDANS can be separated by approximately six residues or more.
  • An NS3 protease assay using a substrate containing a RET pair is described in the PCT publication WO 05/0431 18, which is incorporated herein by reference.
  • DABCYL can also be used to quench the fluorescence of MCA (methoxycoumarin acetic acid), TET (tetrachlorofiuorescein), JOE (carboxy-4'-5'-dichloro-2',7'-dimethoxyfluorescein) FAM (carboxyfluorescein) and other fluorophores and chromophores.
  • MCA methoxycoumarin acetic acid
  • TET tetrachlorofiuorescein
  • JOE carboxy-4'-5'-dichloro-2',7'-dimethoxyfluorescein
  • FAM fluorophores
  • a detectable signal on the substrate can also comprise an epitope, an antibody binding region, an enzyme, a protein binding domain or a nucleic acid binding domain.
  • the substrate can also contain a radioactive label.
  • the substrate can be attached to a solid support.
  • the substrate can be attached to a solid support coated with antibodies that recognize an epitope on the N- or C- terminal end of the peptide substrate.
  • a FLAG tag can be attached to the N-terminal part of the substrate and attached to a microtiter plate whose wells have been coated with anti-FLAG antibodies.
  • the solid support can also be latex particles, paramagnetic particles, paramagnetic latex particles, membranes, polystyrene microbeads and glass beads.
  • the substrate can also be attached to a gel chromatography matrix, such as aff ⁇ gel, via crosslinking.
  • a substrate attached to a solid support can be labelled as described above.
  • An NS4A cofactor can be added to the assay to increase the activity of the NS3 protease.
  • the cofactor can be added to any of the NS3 protease polypeptides described above, including those that include the NS4A polypeptide, e.g., SEQ ID NO:1.
  • the cofactor can be the entire 54 amino acid NS4A peptide or can be shorter versions of it. If shorter versions are used, the NS4A cofactor includes the central part of NS4A, amino acids 21-34 of NSA4.
  • the cofactor can be the peptide with SEQ ID NO:4, which is 23 amino acids long and includes the central part of NSA4.
  • the cofactor can also consist of amino acids 21-34 of NSA4. Longer peptides from 14 amino acids up to the entire 54 amino acids of the NS4A peptide can also be used. D. Separation and quantitation of the products.
  • the invention provides a method for identifying a compound which modulates NS3 protease activity including incubating components comprising the test compound and the polypeptide containing NS3 protease, under conditions sufficient to allow the components to interact and determining the affect of the compound on the activity of the protease.
  • the term "affect" as used herein encompasses any means by which NS3 protease activity can be modulated.
  • Such compounds include, for example, polypeptides, peptidomimetics, chemical compounds and biologic agents as described below.
  • Incubating includes conditions that allow contact between the test compound and NS3 protease. Contacting includes in solution and in solid phase.
  • the test ligand(s)/compound(s) can be a combinatorial library for screening a plurality of compounds. Compounds identified in the method of the invention can be further evaluated.
  • the method of the invention includes combinatorial chemistry methods for identifying chemical compounds that affect NS3 protease activity. Using the methods of the present invention, one can identify molecules and compounds that modulate, activate or inhibit the activity of NS3 protease.
  • the assay can also identify compounds that modulate the function of NS4A as a cofactor for NS3 protease.
  • the assay is carried out in the presence and absence of an NS4A cofactor, the products are quantified and the amounts of products are compared.
  • a wide variety of assays can be used to screen for molecules and compounds that modulate NS3 protease activity, including labeled in vitro protein-protein binding assays, in vitro and in vivo assays that measure NS3 protease activity.
  • assays a plurality of assay mixtures are run in parallel with different candidate molecule or compound concentrations to obtain a differential response to the various concentrations.
  • one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection.
  • Candidate compounds and molecules encompass numerous chemical classes, including organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate compounds and molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and in one embodiment include at least an amine, carbonyl, hydroxyl or carboxyl group. In another embodiment, at least two of the functional chemical groups are included in the candidate compound.
  • the candidate molecules and compounds comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • Candidate molecules and compounds are also found among biomolecules including, but not limited to peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.
  • Candidate molecules and compounds are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides.
  • libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced.
  • natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and can be used to produce combinatorial libraries.
  • Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification and amidification to produce structural analogs.
  • a variety of other reagents can be included in the screening assay. These include reagents like salts, neutral proteins, e.g., albumin, detergents, etc. that are used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Reagents that improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors and anti-microbial agents may be used. The mixtures of components are added in any order that provides for the requisite binding. Incubations are performed at any suitable temperature, typically between 4° C and 40° C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high-throughput screening. The details of some assay conditions are included in the examples below.
  • kits may comprise a carrier means being compartmentalized to receive one or more container means such as vials, tubes, and the like, each of the container means comprising one of the separate elements to be used in the method.
  • container means such as vials, tubes, and the like
  • each of the container means comprising one of the separate elements to be used in the method.
  • one of the container means may comprise a sample containing NS3 protease polypeptide.
  • a second container may comprise a peptide substrate for NS3 protease.
  • the constituents may be present in liquid or lyophilized form, as desired.
  • a third container means may comprise an NS4A cofactor peptide.
  • a kit can also comprise a fourth and fifth container means comprising a known compound that inhibits NS3 protease and a second compound that activates NS3 protease, respectively.
  • the kit can also comprise a sixth and seventh container means comprising a known compound that inhibits NS4A cofactor and a second compound that activates NS4A cofactor, respectively.
  • EXAMPLE 1 Construction and Expression of the HCV NS3 Serine Protease Domain
  • a DNA fragment encoding residues Ala'-Ser 181 of the HCV NS3 protease was obtained by PCR from the HCV Conl replicon plasmid, I 377 neo/NS3-3'/wt (re-named as pBR322-HCV-Neo in this study) [V. Lohmann et al., Science, 285, pp. 1 10-113 (1999)] and inserted into pBEVl 1 (S. Chamber, et al., personal communication) for expression of the HCV proteins with a C-terminal hexa-histidine tag in E. coli. All constructs were confirmed by sequencing.
  • the expression construct for the HCV NS3 serine protease domain was transformed into BL21/DE3 pLysS E. coli cells (Stratagene). Freshly transformed cells were grown at 37° C in a BHI medium (Difco Laboratories) supplemented with 100 ⁇ g/ml carbenicillin and 35 ⁇ g/mL chloramphenicol to an optical density of 0.75 at 600 ⁇ m. Induction with 1 mM IPTG was performed for four hours at 24° C. The cell paste was harvested by centrifugation and flash frozen at -80° C prior to protein purification. All purification steps were performed at 4° C.
  • cell paste was lysed in 1.5 L of buffer A (50 mM HEPES (pH 8.0), 300 mM NaCl, 0.1% n-octyl- ⁇ -D-glucopyranoside, 5 mM ⁇ -mercaptoethanol, 10% (v/v) glycerol) and stirred for 30 minutes.
  • the lysate was homogenized using a Microfluidizer (Microfluidics, Newton, Mass.), followed by ultra-centrifugation at 54,000 x g for 45 minutes.
  • Imidazole was added to the supernatant to a final concentration of 5 mM along with 2 mL of Ni-NTA resin pre-equilibrated with buffer A containing 5 mM imidazole. The mixture was rocked for three hours and washed with 20 column volumes of buffer A plus 5 mM imidazole. The HCV NS3 protein was eluted in buffer A containing 300 mM imidazole. The eluate was concentrated and loaded onto a Hi-Load 16/60 Superdex 200 column, pre- equilibrated with buffer A. The appropriate fractions of the purified HCV protein were pooled and stored at -80° C.
  • This assay is a modification of that described by Landro, et al. (Landro JA, Raybuck SA, Luong YC, O'Malley ET, Harbeson SL, Morgenstern KA, Rao G and Livingston DL. Biochemistry 1997, 36, 9340-9348), and uses a peptide substrate (NS5AB), based on the NS5A/NS5B cleavage site for genotype Ia HCV.
  • the substrate stock solution 25 mM
  • a synthetic peptide cofactor (KK4A) was used as a substitute for the central core region of NS4A.
  • Peptide sequences are shown in Table 1.
  • reaction was performed in a 96-well microtiter plate format using 25 ⁇ M to 50 ⁇ M HCV NS3 protease domain in buffer containing 50 mM HEPES pH 7.8, 100 mM NaCl, 20% glycerol, 5 mM DTT and 25 ⁇ M KK4A.
  • the final DMSO concentration was no greater than 2% v/v.
  • Reactions were quenched by addition of trifluoroacetic acid (TFA) to yield a final concentration of 2.5%.
  • TFA trifluoroacetic acid
  • the SMSY product was separated from substrate and KK4A using a microbore separation method.
  • the instrument used was a Agilent 1100 with a G1322A degasser, either a G1312A binary pump or a G131 1 A quaternary pump, a G1313A autosampler, a G1316A column thermostated chamber and a G1315A diode array detector.
  • the column was a Phenomenex Jupiter, 5 ⁇ m C 18, 300 A, 150 x 2 mm, P/O 00F-4053-B0, with a flow-rate of 0.2 mL/min.
  • the column thermostat was at 40° C.
  • Mobile phases were HPLC grade H 2 O/0.1% TFA (solvent A) and HPLC grade CH 3 CN/0.1% TFA (solvent B).
  • the SMSY product peak was quantified using the data collected at 210 ⁇ M.
  • Recombinant baculovirus containing NS3*4A was produced by co-transfection of pVL1392-His-NS3»4A with linearized Autographa califomica nuclear polyhedrosis virus (AcMNPV) DNA into Spodoptera frugoperda (Sf9) insect cells.
  • the transfected insect cells containing recombinant baculovirus clones were subsequently isolated by plaque purification.
  • High-titer clonal baculovirus was routinely used to infect Sf9 insect cells for protein production. In production, Sf9 cells were grown at 27° C until they reached a density of 2.0 x 10 6 cells/mL. At this point, the insect cells were infected with virus. After 72 hours or when the cell viability was between 70-80% the culture was harvested and the cells were ready for purification.
  • the NS3 « 4A protein (SEQ ID NO:1) was purified as follows. Cell paste was thawed in at least five volumes of Lysis Buffer (50 mM Na 2 HPO 4 pH 8.0, 10% Glycerol, 300 mM NaCl, 5 mM ⁇ -mercaptoethanol, 0.2 mM PMSF, 2.5 ⁇ g/mL Leupeptin, 1.0 ⁇ g/mL E64, 2.0 ⁇ g/mL Pepstatin) per gram of cell paste. The cell paste was then homogenized on ice using a Dounce homogenizer.
  • Lysis Buffer 50 mM Na 2 HPO 4 pH 8.0, 10% Glycerol, 300 mM NaCl, 5 mM ⁇ -mercaptoethanol, 0.2 mM PMSF, 2.5 ⁇ g/mL Leupeptin, 1.0 ⁇ g/mL E64, 2.0 ⁇ g/mL Pepstatin
  • the cells were mechanically disrupted by passing once through a microfiuidizer (Microfluidics Corporation, Newton, MA), and the cell lysate was collected on ice.
  • the cell lysate was centrifuged at 100,000 x g for 30 minutes at 4° C and the supematants were decanted.
  • the pellet was resuspended in wash buffer (Lysis Buffer + 0.1% ⁇ -octyl glucopyranoside), homogenized using a Dounce homogenizer and centrifuged at 100,000 x g for 30 minutes at 4° C.
  • Insoluble NS3»4A was extracted from the pellets by resuspending in Extraction Buffer (Lysis Buffer + 0.5% lauryl maltoside) using 2.5 mL/g cell paste.
  • the mixture was homogenized using a Dounce homogenizer and mixed at 4° C for three hours or more. The mixture was centrifuged at 100,000 x g for 30 minutes at 4° C. The supernatants were decanted and pooled.
  • the NS3»4A protein was further purified using Nickel-NTA metal affinity chromatography. Imidazole from a 2 M stock, pH 8.0, solution was added to the pooled supernatants so that the final concentration of imidazole was 10 raM. The supernatants were incubated batchwise overnight at 4° C with Nickel-NTA affinity resin that had been pre- equilibrated with Lysis Buffer + 10 mM imidazole. 1 mL of resin per 5 ⁇ g of expected NS3- 4A was used. The resin was next settled by gravity or by centrifiigation at 500 x g for five minutes.
  • the resin was poured into a gravity flow column and washed with 10 or more column volumes of Nickel Wash Buffer (Lysis Buffer + 0.1% lauryl maltoside + 10 mM imidazole).
  • the column was eluted with three to four column volumes of Nickel Elution Buffer (Nickel Wash Buffer + 300 mM imidazole).
  • the elution fractions were collected on ice and evaluated using SDS-PAGE.
  • 100 ⁇ M DFP protease inhibitor was added to gel samples before adding SDS sample buffer and boiling. The peak fractions were pooled and protein concentration was determined by measuring absorbance at 280 ⁇ m and by dividing by the extinction coefficient (e), which for NS3»4A is 1.01.
  • the NS3*4A protein was purified further using gel filtration chromatography.
  • a Superdex. 20026/60 column was equilibrated with Superdex Buffer (20 mM HEPES pH 8.0, 10% glycerol, 300 mM NaCl 5 10 mM ⁇ -mercaptoethanol, 0.05% lauryl maltoside) at a rate of 3 mL/min.
  • the nickel purified NS3 » 4A was concentrated in a Centriprep 30 to greater than 2 mg/mL, if necessary, and was filtered through a 0.2 ⁇ m syringe filter and up to 10 mL was loaded onto the Superdex 200 column. After 0.3 column volumes passed through, 4-5 mL fractions were collected.
  • NS3»4A protein elutes in two peaks. Peak 1 contains aggregated NS3 # 4A and peak 2 contains active protein. The fractions of peak 2 were pooled, aliquoted and frozen at -70° C.
  • This assay follows the cleavage of a peptide substrate by full-length hepatitis C viral protein NS3-4A.
  • a synthetic peptide cofactor (NS4A Peptide) was used to supplement NS4A. Peptide sequences are shown in Table 3.
  • the hydrolysis reaction was performed in a 96-well microtiter plate format using 100 ⁇ M to 125 ⁇ M HCV NS3*4A in buffer containing 50 mM HEPES pH 7.8, 100 mM NaCl, 20% glycerol, 5 mM DTT and 25 ⁇ M NS4A peptide.
  • the final DMSO concentration was no greater than 2% v/v.
  • Reactions using NS5AB or NS5AB- EDANS as substrate were quenched by the addition of 10% trifluoroacetic acid (TFA) to yield a final TFA concentration of 2.5%.
  • TFA trifluoroacetic acid
  • the column was a Phenomenex Jupiter, 5 ⁇ m C 18, 300 A, 150 x 2 mm, P/O 00F-4O53-BO, with a flow-rate of 0.2 mL/min using HPLC grade H 2 O/0.1% TFA (solvent A) and HPLC grade CH 3 CN/0.1% TFA (solvent B) as mobile phases.
  • the C-terminal product peak (NH 2 -SMSY-COOH) was quantified using the absorbance data collected at 210 ⁇ m.
  • the column was a Phenomenex Aqua, 5 ⁇ m C 18, 125 A, 50 x 4.6 mm, P/O 00B-4299-EO, with a flow-rate of 1.0 mL/min using HPLC grade H 2 O/0.1% TFA (solvent A) and HPLC grade CH 3 CN/0.1% TFA (solvent B) as mobile phases.
  • the C-terminal product peak (NH 2 - SMSYT-Asp(EDANS)-KKK-COOH) was quantified using the fluorescence data collected at 350 ⁇ m excitation / 490 ⁇ m emission.
  • the column was a Phenomenex Prodigy, 5 ⁇ m ODS(2), 125 A, 50 x 4.6 mm, P/O 0OB-330O-EO, with a flow- rate of 1.0 mL/min using 10 mM sodium phosphate pH 7.0 in HPLC grade H 2 O (solvent A) and 65% HPLC Grade CH 3 CN / 35% 10 mM sodium phosphate pH 7.0 in HPLC grade H 2 O (solvent B) as mobile phases.
  • the N-terminal product peak (FITC-Ahx-EDVV-(alpha)Abu- C-COOH) was quantified using the fluorescence data collected at 440 ⁇ m excitation / 520 ⁇ m emission.
  • the ratio of N-terminal product to unreacted FITC-NS5AB-1 substrate was determined using a Caliper LabChip 3000 with detection at 488 ⁇ m excitation / 530 ⁇ m emission, using a chip buffer of 100 mM Tris pH 7.0, 10 niM EDTA, 0.01% (v/v) Brij-35, and 0.1% (v/v) CR-3.
  • test compound dissolved in DMSO
  • test compound dissolved in DMSO
  • Neat DMSO was included as a no inhibitor control.
  • the cleavage reaction was initiated by the addition of peptide substrate at a concentration either equal to Km or equal to one-half times K 1n , and allowed to proceed at 30° C for 20 minutes.
  • the mixture was quenched, and the extent of reaction was determined as described above. Eleven concentrations of compound were used to titrate enzyme activity for inhibition. Activity vs.

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Abstract

L'invention concerne un procédé pour doser l'activité HCV NS3 protéase à l'aide d'une molécule de NS3 4A protéase. L'invention concerne également un procédé pour cribler et identifier des modulateurs de la NS3 protéase.
EP07811572A 2006-08-28 2007-08-28 Procédé pour identifier des inhibiteurs de protéases Withdrawn EP2057278A2 (fr)

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AU2007290535A1 (en) 2008-03-06
JP2010502184A (ja) 2010-01-28

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