EP1214454A1 - Inhibiteurs de replication du virus de l'hepatite c - Google Patents

Inhibiteurs de replication du virus de l'hepatite c

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Publication number
EP1214454A1
EP1214454A1 EP00957832A EP00957832A EP1214454A1 EP 1214454 A1 EP1214454 A1 EP 1214454A1 EP 00957832 A EP00957832 A EP 00957832A EP 00957832 A EP00957832 A EP 00957832A EP 1214454 A1 EP1214454 A1 EP 1214454A1
Authority
EP
European Patent Office
Prior art keywords
valine
isoleucine
leucine
polypeptide
hcv
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.)
Withdrawn
Application number
EP00957832A
Other languages
German (de)
English (en)
Inventor
Paul L. Darke
Amanda R. Jacobs
Lawrence C. Kuo
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Merck and Co Inc
Original Assignee
Merck and Co Inc
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by Merck and Co Inc filed Critical Merck and Co Inc
Publication of EP1214454A1 publication Critical patent/EP1214454A1/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/162—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/576—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis
    • G01N33/5767—Immunoassay; Biospecific binding assay; Materials therefor for hepatitis non-A, non-B hepatitis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/24011—Flaviviridae
    • C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
    • C12N2770/24222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • Hepatitis C virus is a positive strand RNA virus that is the major cause of non-A, non-B hepatitis.
  • the HCV genome encodes a single polyprotein of approximately 3000 amino acids, containing the viral proteins in the order: C-El-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B.
  • the NS proteins are thought to be non-structural and are involved with the enzymatic functions of viral replication and processing of the viral polyprotein.
  • proteolytic release of mature NS4A, NS4B, NS5A and NS5B is catalyzed by the chymotrypsin-like serine protease contained within the N-terminal domain of NS3 (termed "NS3 protease"), while host cell proteases release C, El, E2, and p7, and create the N-terminus of NS2 at amino acid 810.
  • NS3 protease chymotrypsin-like serine protease contained within the N-terminal domain of NS3
  • host cell proteases release C, El, E2, and p7, and create the N-terminus of NS2 at amino acid 810.
  • the cleavage between amino acids 1026 and 1027 of the HCV polypeptide which separates NS2 from NS3 is dependent upon protein regions of both NS2 and NS3 flanking the cleaved site, and this autocleaving moiety is termed the NS2/3 protease.
  • the cleavage is independent of the catalytic activity of the NS3 protease, as demonstrated with mutational studies. (Grakoui, et al, (1993) Proc. Natl. Acad. Sci. USA 90, 10583-10587; and Hijikata, et al, (1993) J. Virol. 67, 4665-4675.)
  • the NS2/3 cleavage reaction has been studied in bacterial, mammalian and insect cells, and following in-vitro translation of the protein. (Grakoui, et al, (1993) Proc. Natl Acad. Sci. USA 90, 10583-10587; Selby, et al, (1993) J. Gen.
  • NS4A is a cofactor for NS3 activity.
  • the NS3 N-terminus that is formed by NS2/3 cleavage is markedly affected by association with the NS4A.
  • Stable NS3-NS4 complex formation involves the N-terminal amino acid residues of NS3.
  • the NS4A amino acid region primarily responsible for cofactor activity is located at about amino acids 22 to 31. (Shimizu, et al, (1996) J. Virol. 70, 127-132; Butkiewicz, et al, (1996) Virology 225, 328-338; and Lin, et al, (1995) J. Virol. 69, 4373-4380.)
  • the present application identifies the NS4A binding site present on NS2/3 as a target site for inhibiting NS2/3 protease activity.
  • Methods inhibiting NS2/3 activity by targeting the identified target site are described along with examples of compounds useful in such methods and guidance for obtaining additional useful compounds. Such compounds and methods are preferably employed to inhibit HCV replication or processing.
  • the "NS4A target site” refers to the NS4A binding site present on NS2/3.
  • Proteinaceous and non-proteinaceous compounds can be used to target the NS4A target site.
  • Preferred proteinaceous compounds are those containing a NS4A polypeptide region of about 11 contiguous amino acids that bind to the NS4A target site, variants of such compounds, prodrugs of such compounds, and pharmaceutical salts thereof.
  • the region of NS4A, present in different HCV isolates, that binds to the target site is located at about amino acids 22-32.
  • Polypeptide regions of about 11 contiguous amino acids of NS4A that bind to the NS4A target site can readily be identified based upon the known NS4A amino acid sequences of different HCV isolates. Variations of such polypeptide regions can be obtained by substituting amino acids.
  • Preferred substitutions are conservative substitutions and substitutions in those amino acids not essential for exerting an inhibitory effect on NS2/3 autocleavage.
  • Structure I provides a generic structure for polypeptides containing a region targeting the NS4A target site that can inhibit NS2/3 autocleavage. Structure I is as follows:
  • Xl is either serine, cysteine, or threonine
  • ⁇ 2 is either valine, leucine, or isoleucine
  • ⁇ is either valine, leucine, isoleucine, serine, cysteine or threonine
  • ⁇ 4 is either valine, leucine, or isoleucine
  • ⁇ 5 is either valine, leucine, or isoleucine
  • ⁇ 6 is either lysine, arginine, or histidine
  • ⁇ 7 is either valine, leucine, or isoleucine
  • ⁇ 8 is either aspartic acid, glutamic acid, valine, leucine, isoleucine, lysine, arginine, or histidine
  • ⁇ 9 is either valine, leucine, or isoleucine
  • XlO is either serine, cysteine, threonine, asparagine, glutamine, aspartic acid, or glutamic acid; each Yl is an independently selected amino acid, each ⁇ 2 is an independently selected amino acid,
  • Zl is an optionally present protecting group covalently joined to Y 1
  • Z2 is an optionally present protecting group covalently joined to ⁇ 2
  • m is from 0 to 300
  • n is from 0 to 300.
  • Preferred compounds can inhibit NS2/3 autocleavage at least about
  • IC 50 50%, at least about 75%, at least about 85%, or at least about 95%; and/or have an IC 50 of at least about 5 ⁇ M.
  • Reference to "at least" with respect to IC 50 indicates potency.
  • the ability of a compound to inhibit NS2/3 autocleavage is preferably measured using techniques such as those described in the Example section provided below.
  • a first aspect of the present invention features a method of inhibiting HCV replication in an HCV infected cell using an effective amount of a compound that inhibits NS2/3 autocleavage.
  • An effective amount to inhibit NS2/3 autocleavage is an amount that will cause a detectable reduction in NS2/3 autocleavage.
  • Another aspect of the present invention features a method of inhibiting HCV replication in an HCV infected cell using an effective amount of a nucleic acid comprising a nucleotide sequence encoding for (a) a polypeptide comprising an NS4A fragment at least about 11 amino acids in length or (b) a Structure I polypeptide.
  • the NS4A fragment is targeted to the NS4A target site and inhibits autocleavage of
  • An effective amount to inhibit HCV replication is an amount that will cause a detectable reduction in HCV replication.
  • Nucleic acid comprising a nucleotide sequence encoding for a polypeptide can express the polypeptide inside a cell. Such nucleic acid can also contain additional nucleotide sequences that may, for example, encode for other proteins.
  • a nucleotide sequence encoding for a polypeptide comprising an NS4A fragment at least about 11 amino acids in length encodes for at least 11 consecutive amino acids of an NS4A fragment.
  • the polypeptide can contain additional amino acid sequence regions present, or not present, in NS4A. Such additional regions should be selected so as not to reduce the ability of the polypeptide to exert its effect on HCV NS2/3 autocleavage. Examples of additional regions include those that remain part of the active polypeptide and those that are cleaved inside a cell.
  • Another aspect of the present invention describes a method of treating a patient for HCV comprising the step of inhibiting NS2/3 autocleavage. Inhibiting HCV autocleavage is preferably performed using an effective amount of a compound that binds to the NS4A target site and reduces NS2/3 autocleavage.
  • a patient refers to a mammal undergoing treatment.
  • a patient includes an individual being treated for an HCV infection or being treated prophylactically.
  • the patient is a human.
  • Another aspect of the present invention describes a method of inhibiting or preventing HCV replication in a patient comprising the step of treating the patient with an effective amount of a compound containing a NS4A fragment at least about 11 amino acids in length or a Structure I polypeptide, a pharmaceutically acceptable salt of such a compound, or a prodrug thereof.
  • An effective amount to inhibit or prevent HCV replication is an amount that produces a detectable reduction in HCV replication in a patient infected with HCV or confers to a patient the ability to resist HCV infection.
  • Another aspect of the present invention describes a method of inhibiting or preventing HCV replication in a patient comprising the step of administering to the patient an effective amount of a nucleic acid comprising a nucleotide sequence encoding for (a) a polypeptide comprising an NS4A fragment at least about 11 amino acids in length or (b) a Structure I polypeptide.
  • the NS4A fragment is targeted to the NS4A target site and inhibits autocleavage of NS2/3.
  • Another aspect of the present invention describes a compound that is either (1) an HCV inhibitor polypeptide comprising an NS4A fragment at least about 11 amino acids in length that can inhibit autocleavage of NS2/3; (2) a Structure I polypeptide; (3) a pharmaceutically acceptable salt of (1) or (2); or (4) a prodrug of (1), (2), or (3); provided that if the compound is (1) or (2) then the compound contains either, or both, an amino protecting group or a carboxy protecting group.
  • nucleic acid comprising a nucleotide sequence encoding for a HCV inhibitor polypeptide comprising either (a) an NS4A fragment at least about 11 amino acids in length that can inhibit autocleavage of NS2/3 or (b) a Structure I polypeptide.
  • Another aspect of the present invention describes a pharmaceutical composition for inhibiting HCV replication comprising a pharmaceutically acceptable carrier; and an effective amount of a compound that is either (1) an HCV inhibitor polypeptide comprising an NS4A fragment at least about 11 amino acids in length that can inhibit autocleavage of NS2/3; (2) a Structure I polypeptide; (3) a pharmaceutically acceptable salt of (1) or (2); or (4) a prodrug of (1), (2), or (3).
  • a pharmaceutically acceptable carrier refers to a carrier suitable for therapeutic administration that is combined with an active ingredient.
  • the carrier itself is generally not active in treating or preventing a disease, but rather facilitates administration of the active ingredient.
  • pharmaceutically acceptable carriers include calcium carbonate, calcium phosphate, lactose, glucose, sucrose, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents. Additional examples, some of which are described below under "Administration", are well known in the art.
  • Another aspect of the present invention features a pharmaceutical composition for inhibiting HCV replication comprising a pharmaceutically acceptable carrier; and an effective amount of a nucleic acid encoding for a polypeptide that either (a) comprises a fragment of NS4A at least about 11 amino acids in length, wherein the fragment can inhibit autocleavage of NS2/3; or (b) is a Structure I polypeptide.
  • Another aspect of the present invention features a method for inhibiting HCV polyprotein processing comprising the step of contacting a cell expressing an HCV polypeptide that contains at least NS2/3 with an effective amount of a compound that is either (1) an HCV inhibitor polypeptide comprising an NS4A fragment at least about 11 amino acids in length that can inhibit autocleavage of NS2/3; (2) a Structure I polypeptide; (3) a pharmaceutically acceptable salt of (1) or (2); or (4) a prodrug of (1), (2), or (3).
  • Another aspect of the present invention features a method of screening for a compound that inhibits HCV replication or HCV polyprotein processing.
  • the method is performed by (a) selecting for a compound that binds to the NS4A target site using a polypeptide comprising NS2/3 or a binding portion thereof, and (b) measuring the ability of the compound to inhibit HCV replication or HCV polyprotein processing.
  • “Comprising NS2/3 or a binding portion thereof indicates that polypeptide regions from NS2 and NS3 needed for binding to NS4A are both present.
  • HCV polyprotein processing refers to the formation of one or more HCV peptides.
  • HCV polyprotein processing can be measured using different techniques such as by measuring the presence of an individual protein or the activity associated with an individual protein.
  • HCV processing is performed by measuring the activity or formation of NS2 or NS3.
  • Another aspect of the present invention features a method of screening for a compound that inhibits HCV replication or HCV polyprotein processing in the presence of a non-saturating amount of a NS4A agonist.
  • a "NS4A agonist” is a compound that competes with NS4A for binding to NS2/3. The NS4A agonist also inhibits, to some extent, NS2/3 autocleavage.
  • Figure 1 illustrates HCV NS2/3 processing reactions where different fragments are separated on SDS-PAGE.
  • Translated proteins were incubated 60 minutes at 20°C and are identified by arrows adjacent to the lanes. The migration position of molecular weight markers are shown in kDa.
  • Panel A The NS2/3 reactions shown are: 849-1240J at the start of a 20°C incubation (lane 1), and after 1 hour (lane 2); Mal849-1240J at the start of incubation (lane 3), and after 1 hour (lane 4).
  • Panel B A representative gel image is shown for the testing of peptides against the 810-1615BK autocleavage. The samples analyzed are: No added peptide (lane 1); peptides of SEQ. ID. NO.
  • Table 2 infra, provides the sequences for the SEQ. ID. NOs.
  • Figure 2 illustrates titration of NS4A peptide inhibition of NS2/3. Data shown are for peptides of SEQ. ID. NO. 11 (circles) and SEQ. ID. NO. 12 (triangles).
  • IC 50 curves are shown after optimization of the adjustable parameters which produced slope coefficients (d) of 1.0 - 1.5 for all fits. IC 50 values are in Table 2.
  • the present application identifies the NS4A binding site present on NS2/3 as a target site for inhibiting NS2/3 protease activity.
  • inhibition of NS2/3 by NS4A peptides is believed to be brought about by NS4A acting at the N-terminus of NS3 (the NS2/3 cleavage point).
  • Compounds targeting the HCV target site can be produced independent of such a theory based upon the structure of polypeptides identified herein inhibiting NS2/3 autocleavage and using the guidance provided herein to obtain proteinaceuous or non- proteinaceous compounds inhibiting NS2/3 autocleavage.
  • Non-therapeutic applications include research related applications, such as providing a tool for stabilizing NS2/3 and studying the effects of NS2/3 on HCV polyprotein processing, and for studying the cellular effects of inhibiting NS2/3 autocleavage.
  • Therapeutic applications include treating a patient infected with HCV and prophylactically treating a patient.
  • patients that can be infected with HCV include chimpanzees and humans.
  • Prophylactic treatment is preferably performed on patients having a higher risk of being infected with HCV such as those undergoing a blood transfusion.
  • COMPOUNDS TARGETING THE NS4A TARGET SITE Using the present application as guide proteinaceous and non- proteinaceous compounds targeting the NS4A target site can be obtained.
  • the provided guidance includes the identification of a target site, examples of compounds directed to the target site, examples of compound modification, and a description of techniques that can be used to obtain additional compounds.
  • Preferred proteinaceous compounds are those containing a polypeptide region of about 11 contiguous amino acids that bind to the NS4A target site, variants of such compounds, prodrugs of such compounds, and pharmaceutically acceptable salts thereof.
  • Polypeptide regions of about 11 contiguous amino acids binding to the NS4A target site include amino acid sequences that may, or may not, be present in a naturally occurring NS4A polypeptide.
  • a variant of a polypeptide refers to a proteinaceous compound containing one or more non-peptide groups.
  • variants include cyclized peptide analogs, altered amino acid side chains, altered peptide linkages, and the presence of non-amino acid groups.
  • a prodrug is a substance that is acted on in vivo or inside a cell to produce an active compound. The prodrug itself may be active or inactive.
  • prodrugs are used to achieve a particular purpose such as facilitating intracellular transport of a compound targeting the NS4A target site.
  • a particular purpose such as facilitating intracellular transport of a compound targeting the NS4A target site.
  • the production of prodrugs facilitating compound intracellular transport is well known in the art, and an example of the production of prodrugs is described by Janmey, et al, U.S. Patent No. 5,846,743, hereby incorporated by reference herein.
  • Compounds of the present invention include those having one or more chiral centers.
  • the present invention is meant to comprehend diastereomers as well as their racemic and resolved, enantiomerically pure forms and pharmaceutically acceptable salts thereof.
  • Proteinaceuous compounds can contain D-amino acids, L- amino acids or a combination thereof.
  • amino acids within a chiral center are L-amino acids.
  • Proteinaceuous compounds targeting the NS4A target site contain a polypeptide region targeting the target site. Polypeptide regions targeting the NS4A target site are present at approximately amino acids 22-32 of NS4A. Examples of proteinaceous compounds targeting the NS4A target site are provided for by polypeptide regions found in different NS4A isolates located at amino acids 22-32 of NS4A.
  • amino acids sequences located at amino acids 22-32 of NS4A from different isolates of NS4A are well known in the art and can be found in different sources including publications and Gen-Bank. Table 1 provides an example of several NS4A sequences present in different HCV isolates. Table 1
  • the provided amino acid sequence starts at amino acid 21 of NS4A unless a "-" is present, in which case the amino acid sequence starts at amino acid 22. Butkiewicz, et al., (1996) Virology 225, 328-338, hereby inco ⁇ orated by reference herein.
  • Proteinaceous compounds targeting the NS4A target site can be produced to contain a region corresponding to about amino acids 22-32 of NS4A and can contain additional polypeptide and non-polypeptide regions.
  • the NS4A polypeptide regions are at least about 11 amino acids in length. In different embodiments the NS4A region is at least about 12, 14, 20, 40 amino acids in length. Additional polypeptide regions that can be present include additional
  • NS4A regions and polypeptide regions or amino acids not related to NS4A are not greater than about 650 amino acids, not greater than about 200 amino acids, not greater than about 100 amino acids, or not greater than about 50 amino acids.
  • Additional non-polypeptide regions include the presence of amino- and/or carboxy-terminal groups that facilitate cellular uptake and/or facilitate survival of the polypeptide. Possible groups include those cleaved inside a cell and those remaining part of the active compound.
  • a large number of additional NS4A regions can be selected based upon the known structures of NS4A from different isolates and can be selected independent of the known structures of NS4A. Additional regions selected independent of the known structures of NS4A could be chosen, for example, randomly or to achieve a particular pu ⁇ ose such as producing a prodrug. The affect of additional sequences on NS2/3 autocleavage can readily be tested using techniques exemplified in the examples provided below.
  • Polypeptide regions targeting the NS4A target site can also be produced based upon a comparison of NS4A occurring in different isolates and the use of conservative amino acid substitutions.
  • Conservative amino acid substitutions generally involve exchanging amino acids within the same group (e.g., neutral and hydrophobic, neutral and polar, basic, and acidic). Additional amino acid substitutions can readily be identified by testing the effect of different amino acid substitutions on the ability to inhibit NS2/3 autocleavage.
  • Structure I provides a generic structure of a polypeptide encompassing a region targeting the NS4A target site. Structure I is as follows:
  • ⁇ l is either serine, cysteine, or threonine, preferably serine or cysteine
  • ⁇ 2 is either valine, leucine, or isoleucine, preferably valine or isoleucine, more preferably valine
  • ⁇ is either valine, leucine, isoleucine, serine, cysteine or threonine, preferably valine or isoleucine, more preferably valine
  • X4 is either valine, leucine, or isoleucine, preferably valine or isoleucine, more preferably isoleucine
  • ⁇ 5 is either valine, leucine, or isoleucine, preferably valine or isoleucine, more preferably valine
  • ⁇ 6 is either lysine, arginine, or histidine, preferably arginine or histidine, more preferably arginine
  • ⁇ 7 is either valine, leucine, or isoleucine, preferably iso
  • XlO is either serine, cysteine, threonine, asparagine, glutamine, aspartic acid, or glutamic acid, preferably serine, asparagine, or glutamic acid; each Yl is an independently selected amino acid, each ⁇ 2 is an independently selected amino acid, Zl is an optionally present protecting group covalently joined to ⁇ l, preferably, Zl is either an optionally substituted -C MO alkyl, optionally substituted -C 2-10 alkenyl, optionally substituted aryl, -C ⁇ _ 6 alkyl optionally substituted aryl, -C(O)-(CH 2 ) ⁇ _ 6 - COOH, -C(O)- C ⁇ -6 alkyl, -C(O)-optionally substituted aryl, -C(O)-O-C ]-6 alkyl, or C(O)-O-optionally substituted aryl, more preferably acetyl, propyl, succinyl,
  • Z2 is an optionally present protecting group covalently joined to ⁇ , preferably amide, methylamide, or ethylamide; m is from 0 to 300, in different embodiments m is 0 to 100, 0 to 50, 0 to 25, 0 to 10, and 0 to 5; and n is from 0 to 300, in different embodiments m is 0 to 100, 0 to 50, 0 to 25, 0 to 10, and 0 to 5.
  • An “optionally present protecting group covalently joined to Yl” refers to the presence of a group joined to the amino terminus which reduces the reactivity of the amino terminus under in vivo conditions. In the absence of the protecting group -NH is present at the amino terminus.
  • an “optionally present protecting group covalently joined to Y2" refers to the presence of a group joined to the carboxy terminus which reduces the reactivity of the carboxy terminus under in vivo conditions.
  • the carboxy terminus protecting group is preferably attached to the ⁇ -carbonyl group of the last amino acid. In the absence of the protecting group -COOH is present at the carboxy terminus.
  • Alkyl refers to carbon atoms joined by carbon-carbon single bonds.
  • the alkyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups.
  • the alkyl group is 1 to 4 carbons in length. Examples of alkyl include methyl, ethyl, propyl, isopropyl, cyciopropyl, butyl, and t-butyl.
  • Alkyl substituents are selected from the group consisting of halogen (preferably -F or -Cl) -OH, -CN, -SH, -NH 2 , -NO 2 , -C 1-2 alkyl substituted with 1 to 6 halogens (preferably -F or -Cl, more preferably -F), -CF 3 , -OCH 3 , or -OCF 3 .
  • Alkenyl refers to a hydrocarbon group containing one or more carbon-carbon double bonds.
  • the alkenyl hydrocarbon group may be straight-chain or contain one or more branches or cyclic groups.
  • the alkenyl group is 2 to 4 carbons in length.
  • Alkenyl substituents are selected from the group consisting of halogen (preferably -F or -Cl), -OH, -CN, -SH, -NH 2 , -NO 2 , -C ⁇ -2 alkyl substituted with 1 to 5 halogens (preferably -F or -Cl, more preferably -F), -CF 3 , -OCH 3 , or
  • Aryl refers to an optionally substituted aromatic group with at least one ring having a conjugated pi-electron system, containing up to two conjugated or fused ring systems.
  • Aryl includes carbocyclic aryl, heterocyclic aryl and biaryl groups.
  • the aryl is a 5 or 6 membered ring, more preferably benzyl.
  • Aryl substituents are selected from the group consisting of -C]. alkyl, -C ⁇ _ alkoxy, halogen
  • Proteinaceous compounds can be produced using standard techniques.
  • polypeptide region of a proteinaceuous compound can be chemically or biochemically synthesized.
  • Techniques for chemical synthesis of polypeptides are well known in the art. (See e.g., Vincent, in Peptide and Protein Drug Delivery, New York, N.Y., Dekker, 1990.)
  • Biochemical production of polypeptides can be performed using cells as biological factories to produce nucleic acid encoding for the polypeptide.
  • Nucleic acid sequences encoding for polypeptides targeting the NS4A target site can be produced by taking into account the genetic code.
  • the genetic code providing the sequences of nucleic acid triplets coding for particular amino acids is well known in the art. Amino acids are encoded for by codons as follows:
  • a desired polypeptide may be recombinantly expressed using an expression vector encoding for the desired polypeptide and containing a promoter and other appropriate regulatory elements suitable for transcription and translation of the nucleic acid in a desired host.
  • Expression vectors may be introduced into host cells using standard techniques. Examples of techniques for introducing nucleic acid into a cell and expression of nucleic acids are provided in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, and Sambrook, et al, in Molecular Cloning, A Laboratory Manual, 2 nd Edition, Cold Spring Harbor Laboratory Press, 1989.
  • expression is achieved in a host cell using an expression vector.
  • An expression vector contains nucleic acid encoding for a desired polypeptide along with regulatory elements for proper transcription and processing.
  • the regulatory elements that are present include a transcriptional promoter, a ribosome binding site, a terminator, and an optionally present operator.
  • Another preferred element is a polyadenylation signal providing for processing in eukaryotic cells.
  • GENE-SWITCHTM Wang, et al, Gene Ther. (1997) 4, 432-41, U.S. Patent No. 5,874,534 and International Publication WO 93/23431, each of which are hereby inco ⁇ orated by reference herein
  • tetracycline operator U.S. Patent Nos. 5,464,758 and 5,650,298, both of which are hereby inco ⁇ orated by reference herein.
  • a variety of mammalian expression vectors are well known in the art including pcDNA3 (Invitrogen), pMClneo (Stratagene), pXTl (Stratagene), pSG5 (Stratagene), EBO-pSV2-neo (ATCC 37593), pBPV-l(8-2) (ATCC 37110), pdBPV-MMTneo(342-12) (ATCC 37224), pRSVgpt (ATCC 37199), pRSVneo (ATCC 37198), pSV2-dhfr (ATCC 37146), pUCTag (ATCC 37460), and .lambda.ZD35 (ATCC 37565).
  • a variety of bacterial expression vectors are well known in the art including pETl la (Novagen), lambda gtll (Invitrogen), pcDNAII (Invitrogen), pKK223-3 (Pharmacia).
  • a variety of fungal cell expression vectors are well known in the art including pYES2 (Invitrogen), and Pichia expression vector (Invitrogen).
  • a variety of insect cell expression vectors are well known in the art including Blue Bac m (Invitrogen).
  • Recombinant host cells may be prokaryotic or eukaryotic. Examples of recombinant host cells include the following: bacteria such as E.
  • mammalian cell lines include L cells L-M(TK.sup.-) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-K1 (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).
  • a desired polypeptide can be purified by standard techniques such as those using antibodies binding to the polypeptide.
  • Antibodies specifically recognizing a polypeptide can be produced using the polypeptide as an immunogen.
  • the polypeptide used as an immunogen should be at least 9 amino acids in length. Examples of techniques for producing and using antibodies are described in Ausubel, Current Protocols in Molecular Biology, John Wiley, 1987-1998, Harlow, et al,
  • Non-Proteinaceuous compounds targeting the NS4A target site include compounds that are designed based on the structure of polypeptides targeting the NS4A target site and compounds that are selected based on the ability to bind to the NS4A target site.
  • peptidomimetic compounds Compounds designed based on the structure of polypeptides targeting the NS4A target site are peptidomimetic compounds.
  • Preferred peptidomimetic compounds have additional characteristics, compared to polypeptides, that enhance their therapeutic applications. Such additional characteristics may include increased cell permeability and prolonged biological half-time.
  • Techniques for designing and synthesizing peptidomimetics are well known in the art. (See, Gilon, et al, U.S. Patent 5,874,529, and Gante, Angew. Chem. Int. Ed. Engl. (1994) 33, 1699-1720, both of which are hereby inco ⁇ orated by reference herein.)
  • GENE THERAPY Gene therapy using a nucleic acid encoding for a polypeptide targeting the NS4A target site can be performed taking into account the present disclosure and general gene therapy techniques well known in the art. Preferably, gene therapy is performed using an expression vector.
  • Expression vectors useful in gene therapy include those serving as delivery vehicles and those that are introduced into a cell by a delivery vehicle or appropriate technique.
  • Expression vectors that can act as delivery vehicles are well known in the art, examples of which include retrovirus vectors, adenovirus vectors, and adeno-assoicated virus vectors.
  • Nonviral gene delivery methods are also well known in the art, examples of which include the use of liposomes, direct injection of DNA and polymers.
  • Gene therapy can be performed in vivo or ex vivo.
  • In vivo gene therapy is performed by directly administering nucleic acid to a patient.
  • Ex vivo gene therapy is performed by administering nucleic acid to cells outside of a patient and then introducing the treated cells into a patient.
  • the guidance provided herein can be used in methods screening for compounds that inhibit HCV replication or HCV polyprotein processing. Such methods include those identifying HCV inhibitory compounds targeting the NS4A target site and those using NS4A agonists.
  • HCV polyprotein processing can be tested for by measuring the ability of the compound to alter the formation or activity of products normally produced by HCV polypeptide processing.
  • HCV processing is tested for by measuring the activity or formation of NS2 or NS3. Targeting the NS4A Target Site
  • HCV inhibitory compounds targeting the NS4A target site can be screened for by first identifying a compound that binds to the NS4A target site using a polypeptide comprising NS2/3 or a binding portion thereof. The identified compound is then tested for its ability to inhibit HCV replication or HCV polyprotein processing.
  • the NS2/3 portion used in the screening contains a sufficient amount of a NS2 region and a NS3 region to bind NS4A.
  • the NS2 region preferably contains at least about 70 amino acids from the NS2 carboxy terminus; and in different embodiments contains at least about 100 or 200 amino acids of NS2.
  • the NS3 region preferably contains at least about 150 amino acids from the amino terminus of NS3; and in different embodiments contains at least about 200 or 300 amino acids.
  • Compounds binding to the NS4A target site are preferably identified using a competitive assay involving a compound known to bind to the NS4A target site. Such identification may be performed starting with a compound present in a test preparation containing a plurality of different compounds or on a compound by compound basis. Examples of plurality of different compounds include a preparation containing 2 or more, 5 or more, 10 or more, or 20 or more compounds.
  • Non-saturating levels of an NS4A agonist can be employed in assays screening for HCV inhibitory compounds.
  • NS4A agonists may alter NS2/3 conformation thereby increasing the accessibility of the NS2/3 active site to HCV inhibitory compounds.
  • HCV inhibitory compounds can be identified independent of such a theory including HCV inhibitory compounds that bind to an allosteric site in the presence of NS4A.
  • the NS4A agonist can compete with NS4A for binding to NS2/3 under the conditions used in the screening method.
  • NS4A agonists include proteinaceous compounds such as NS4A itself, and the peptides described in the examples below. Additional NS4A agonists, including non-proteinaceous compounds, can be identified using the procedures described herein.
  • the NS4A agonist employed in the assay is present at a level sufficient to cause a detectable inhibition of NS2/3 autocleavage.
  • the NS4A agonist is present at a concentration no more than 2X or IX its Kd or IC50; or is present at a concentration about equal to its K ⁇ j or IC50.
  • ADMINISTRATION Compounds targeting the NS4A target site can be formulated and administered to a patient using the guidance provided herein along with techniques well known in the art. The preferred route of administration ensures that an effective amount of compound reaches the target. Guidelines for pharmaceutical administration in general are provided in, for example, Remington's Pharmaceutical Sciences 18 Edition, Ed. Gennaro, Mack Publishing, 1990, and Modern Pharmaceutics 2" Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990, both of which are hereby inco ⁇ orated by reference herein.
  • Compounds targeting the NS4A target site having appropriate functional groups can be prepared as acidic or base salts.
  • Pharmaceutically acceptable salts in the form of water- or oil-soluble or dispersible products) include conventional non-toxic salts or the quaternary ammonium salts that are formed, e.g., from inorganic or organic acids or bases.
  • salts include acid addition salts such as acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2- hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thio
  • Compounds targeting the NS4A target site can be administered using different routes including oral, nasal, by injection, transdermal, and transmucosally.
  • Active ingredients to be administered orally as a suspension can be prepared according to techniques well known in the art of pharmaceutical formulation and may contain microcrystalline cellulose for imparting bulk, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosity enhancer, and sweeteners/flavoring agents.
  • these compositions may contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium stearate and lactose and/or other excipients, binders, extenders, disintegrants, diluents and lubricants.
  • compositions When administered by nasal aerosol or inhalation, compositions can be prepared according to techniques well known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, abso ⁇ tion promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents.
  • the compounds may also be administered in intravenous (both bolus and infusion), intraperitoneal, subcutaneous, topical with or without occlusion, or intramuscular form, all using forms well known to those of ordinary skill in the pharmaceutical arts.
  • the injectable solutions or suspensions may be formulated using suitable non-toxic, parenterally-acceptable diluents or solvents, such as Ringer's solution or isotonic sodium chloride solution, or suitable dispersing or wetting and suspending agents, such as sterile, bland, fixed oils, including synthetic mono- or diglycerides, and fatty acids, including oleic acid.
  • these compositions When rectally administered in the form of suppositories, these compositions may be prepared by mixing the drug with a suitable non-irritating excipient, such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
  • a suitable non-irritating excipient such as cocoa butter, synthetic glyceride esters or polyethylene glycols, which are solid at ordinary temperatures, but liquidify and/or dissolve in the rectal cavity to release the drug.
  • Suitable dosing regimens for the therapeutic applications of the present invention are selected taking into factors well known in the art including age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal and hepatic function of the patient; and the particular compound employed.
  • NS2/3 autocleavage reactions were thawed on ice and cleavage was initiated by incubation at 20°C, either in the original translation mixture or following a 10-fold dilution into a 10,000 molecular weight filtrate of reticulocyte lysate produced with Amicon Microcon-10 filters. Samples taken at the times indicated were combined with SDS gel sample buffer and frozen on dry ice. NS2/3 cleavage reactions with the 810-1615BK were performed with 1% Triton X- 100 present, as described by Pieroni, et al, (1997) J. Virol. 71, 6373-6380.
  • the product NS2 from 810-1615 BK was used to generate data shown for screening of peptides and IC 5 0 calculations, due to its migration on gels in a region with less background than the higher molecular weight products, and due to the ability to initiate the 810-1615BK reaction with Triton X-100 (Santolini, et al, (1995) J. Virol. 69, 7461-7471).
  • the IC 50 values were determined by first expressing the product level found as a fraction of the no-inhibitor control product level, then fitting the equation
  • a is the minimal level of fractional activity (tending to 0)
  • a+b is the maximal level (tending to 1)
  • x is the concentration of inhibitor
  • c is the IC 50
  • d is a slope coefficient.
  • Typical NS2/3 processing reactions are shown in Figure 1. The reaction occurred on a time scale of minutes, with the rate and final extent of reaction varying somewhat with the sequence expressed. NS2/3 810-1615BK was cleaved as much as 60% with a 3 hour incubation, and the maltose binding protein fusion, Mal849-1240J, to nearly 100%. In all constructs, the mutations His952Ala or Cys993Ala prevented the appearance of products, as previously reported (Hijikata, et al, (1993) 7. Virol. 67, 4665-4675; and Grakoui, et al, (1993) Proc. Natl. Acad. Sci. USA 90, 10583-7).
  • Peptide inhibition of NS2/3 processing was measured using peptides containing the NS2/3 cleavage sequence, peptides targeted to the NS4 target site and peptides not related to the target site. Peptides targeted to the NS4A target site were designed based on the region of NS4A binding to the target site. The results are shown in Table 2.
  • IC 50 values were determined as described in Example 1, and are an average of two determinations.
  • Percent inhibitions shown for cleavage site and unrelated peptides were obtained with a final peptide concentration of 1 mg/ml, which when expressed as a molar concentration corresponds to a minimum of 500 ⁇ M for the group of cleavage site peptides.
  • c Percent inhibitions shown for NS4A peptides were obtained with a final peptide concentration of 50 ⁇ M.
  • '"Ac refers to acetyl.
  • NS2/3 reactions with 810-1615BK were performed for 30 minutes, as described in Example 1.
  • SEQ. ID. NO. 9 represents NS4A residues 21-34, and has lysine residues appended to each end to enhance solubility.
  • SEQ. ID. NO. 10 has the same amino acids as SEQ. LD. NO. 9, but in a random order. Similar results were obtained with NS2/3 Mal849-1240J.
  • NS4A peptides were examined for their effect upon NS2/3 autocleavage. Significant inhibition was observed, as shown for a peptide of SEQ. ID. NO. 9 in Figure IB. The inhibition appeared to occur immediately, since no pre- incubation of NS2/3 with peptide was performed before initiation of the reaction. Also, changes in inhibitor potency were not observed using 20 minute or 45 minute incubations.

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Abstract

La présente invention concerne le site de liaison de NS4A présent sur NS2/3 en tant que site cible pour inhiber l'activité protéase NS2/3. Elle concerne également des procédés permettant d'inhiber l'activité NS2/3 par ciblage du site cible identifié, ainsi que des exemples de composés utiles dans ces procédés, et une marche à suivre pour obtenir des composés utiles supplémentaires. Ces composés et procédés sont de préférence utilisés pour inhiber la réplication du virus de l'hépatite C.
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