Note: Descriptions are shown in the official language in which they were submitted.
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TITLE OF THE INVENTION
HEPATITIS C VIRUS REPLICATION INHIBITORS
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims priority to U.S. Serial No. 60/151,395,
filed August 30, 1999, hereby incorporated by reference herein.
BACKGROUND OF THE INVENTION
The references cited herein are not admitted to be prior art to the
claimed invention.
Hepatitis C virus (HCV) is a positive strand RNA. virus that is the
major cause of non-A, non-B hepatitis. (Choo, et al., (1989) Science 244, 362-
364;
and Choo, et al., (1989) Science 244, 359-362.) The HCV genome encodes a
single
polyprotein of approximately 3000 amino acids, containing the viral proteins
in the
order: C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NSSA-NSSB. 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. Release of the individual
proteins
from the polyprotein precursor is mediated by both cellular and viral
proteases.
(Choo, et al., (1991) Proc. Natl. Acad. Sci. USA 88, 2451-2455; Takamizawa, et
al.,
(1991) J. Virol. 65, 1105-1113; Neddermann, et al., (1997) Biol. Chem. 378,
469-476;
Lohmann, et al., (1996) J. Hepatol. 24, 11-19; and Houghton, et al., (1991)
Hepatology 14, 381-388.)
The proteolytic release of mature NS4A, NS4B, NSSA and NSSB 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,
E1, E2,
and p7, and create the N-terminus of NS2 at amino acid 810. (Mizushima, et
al.,
(1994) J. Virol. 68, 2731-2734, and Hijikata, et al., (1993) Proc. Natl. Acad.
Sci. USA
90, 10773-10777.)
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. (Grakoui, et al., (1993) Proc. Natl. Acad. Sci. USA 90, 10583-
10587;
and Komoda, et al., (1994) Gene 145, 221-226.) The cleavage is independent of
the
catalytic activity of the NS3 protease, as demonstrated with mutational
studies.
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(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.
Virol. 74, 1103-1113; Hijikata, et al., (1993) J. Virol. 67, 4665-4675;
Santolini, et al.,
(1995) J. Virol. 69, 7461-7471; D'Souza, et al., (1994) J. Gen. Virol. 75,
3469-3476;
and Pieroni, et al., (1997) J. Virol. 71, 6373-6380.) The protein region
essential for
NS2/3 cleavage activity has been approximately mapped to amino acids 898 to
1207
of the HCV open reading frame. (Grakoui, et al., (1993) Proc. Natl. Acad. Sci.
USA
90, 10583-10587; Hijikata, et al., (1993) J. Virol. 67, 4665-4675;, and
Santolini, et al.,
(1995) J. Virol. 69, 7461-7471.) The catalytic mechanism of NS2/3 cleavage is
speculated to be either a metalloprotease or cysteine protease. (Wu, et al.,
(1998)
Trends Biochem. Sci. 23, 92-94; and Gorbalenya, et al., (1996) Perspect. Drug
Discovery Design 6, 64-86.) Cleavage activity of in-vitro translated NS2/3 is
inhibited by EDTA and activity is restored with metal ion re-addition.
(Hijikata, et
al., (1993) J. Virol. 67, 4665-4675; and Pieroni, et al., (1997) J. Virol. 71,
6373-
6380.)
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.
(Satoh, et al., J. Virol. (1995) 69, 4255-4260.) 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 X-ray crystallographic structure of NS3 bound to a peptide
containing NS4A amino acids 21-34 (amino acids 1672-1685 of the HCV
polyprotein) reveals that the NS3 N-terminal residues 2 through 9 directly
interact
with NS4A to compose one of 8 strands in an antiparallel beta-sheet extending
through the NS3 protease. (Kim, et al., (1996) Cell 87, 343-355; and Yan, et
al.,
(1998) Protein Sci. 7, 837-847, hereby incorporated by reference herein.) In
contrast,
without NS4A, the N-terminus of NS3 is poorly organized. (Love, et al., (1996)
Cell
87, 331-342.)
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SUMMARY OF THE INVENTION
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:
Z1-Ylm- X1X2X3 X4XSGX6X~ Xg X9X10_ y2n-Z2
wherein X1 is either serine, cysteine, or threonine;
X2 is either valine, leucine, or isoleucine;
X3 is either valine, leucine, isoleucine, serine, cysteine or threonine;
X4 is either valine, leucine, or isoleucine;
XS is either valine, leucine, or isoleucine;
X6 is either lysine, arginine, or histidine;
X~ is either valine, leucine, or isoleucine;
Xg is either aspartic acid, glutamic acid, valine, leucine, isoleucine,
lysine, arginine,
or histidine;
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X9 is either valine, leucine, or isoleucine;
X10 is either serine, cysteine, threonine, asparagine, glutamine, aspartic
acid, or
glutamic acid;
each Y1 is an independently selected amino acid,
each Y2 is an independently selected amino acid,
Z1 is an optionally present protecting group covalently joined to Y1,
Z2 is an optionally present protecting group covalently joined to Y2,
m is from 0 to 300 and
n is from 0 to 300.
Preferred compounds can inhibit NS2/3 autocleavage at least about
50%, at least about 75%, at least about 85%, or at least about 95%; and/or
have an
ICSO of at least about 5 ~M. Reference to "at least" with respect to ICSO
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.
Thus, 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
NS2/3. 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
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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.
Preferably, 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.
Another aspect of the present invention features a nucleic acid
comprising a nucleotide sequence encoding for a HCV inhibitor polypeptide
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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
Garner
itself is generally not active in treating or preventing a disease, but rather
facilitates
administration of the active ingredient. Examples of 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.
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"Comprising NS2/3 or a binding portion thereop' indicates that
polypeptide regions from NSZ and NS3 needed for binding to NS4A are both
present.
Preferably, at least about 70 amino acids from the NS2 carboxy terminus are
present
and at least about 150 amino acids from the NS3 amino terminus are present in
the
NS2/3 portion.
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. Preferably, 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.
Other features and advantages of the present invention are apparent
from the additional descriptions provided herein including the different
examples.
The provided examples illustrate different components and methodology useful
in
practicing the present invention. The examples do not limit the claimed
invention.
Based on the present disclosure the skilled artisan can identify and employ
other
components and methodology useful for practicing the present invention.
BRIEF DESCRIPTION OF THE DRAWINGS
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: 849-1240) at the start of a 20°C incubation (lane
1), and after 1
hour (lane 2); Ma1849-1240) 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. 1 (lane 2), SEQ. ID. NO. 2 (lane 3), SEQ. ID. NO. 3
(lane
4), SEQ. ID. NO. 11 (lane 5), SEQ. ID. NO. 4 (lane 6), SEQ. m. NO. 5 (lane 7),
SEQ.
m. NO. 6 (lane 8), DMSO control with no incubation (lane 9). Table 2, infra,
provides the sequences for the SEQ. m. NOs.
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Figure 2 illustrates titration of NS4A peptide inhibition of NS2/3.
Data shown are for peptides of SEQ. >I7. NO. 11 (circles) and SEQ. ID. NO. 12
(triangles). ICSO curves are shown after optimization of the adjustable
parameters
which produced slope coefficients (d) of 1.0 - 1.5 for all fits. ICSO values
are in Table
2.
DETAILED DESCRIPTION OF THE INVENTION
The present application identifies the NS4A binding site present on
NS2/3 as a target site for inhibiting NS2/3 protease activity. Without being
limited to
any particular theory, 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.
The compounds and methods described herein have therapeutic and
non-therapeutic applications. 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. Examples of 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
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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 occurnng NS4A polypeptide.
A variant of a polypeptide refers to a proteinaceous compound
containing one or more non-peptide groups. Examples of variants include
cyclized
peptide analogs, altered amino acid side chains, altered peptide linkages, and
the
presence of non-amino acid groups. (E.g., 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
incorporated by reference herein.)
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.
Preferably, prodrugs are used to achieve 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. Preferably, amino acids within a chiral
center
are L-amino acids.
Proteinaceuous Compounds
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.
The 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.
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Table 1
Isolate Amino Acid Se uence
HCV-BK GSVVIVGR>TLSG (SEQ. ID. NO.
20)
HCV-H''2 GCVVIVGRIVLSG (SEQ. ID. NO.
21)
6013 GSVVIVGRIVLSG (SEQ. ID. NO.
22)
HCV-13 GCVVIVGRVVLSG (SEQ. >D. NO.
23)
HCV-J63 GCVCIIGRLHVNQ (SEQ. 1D. NO.
24)
HCV-J83 GCISIIGRLHLNQ (SEQ. 1D. NO.
25)
HCV-NZL1' -CVVIVGHIEIEGK (SEQ.1D. NO.
26)
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 incorporated by
reference
herein.
ZLin, et al., (1995) J. Virol. 69, 4373-4380, hereby incorporated by reference
herein.
3Bartenshlager, et al., (1995) J. Virol. 69, 7519-7528, hereby incorporated 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. In
different embodiments the overall size of the polypeptide is 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 purpose such as producing a prodrug. The affect of additional
sequences on
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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:
zl_ylm_ X1X2X3 X4XSGX6X7 X8 X9X10_ y2n_z2
wherein X1 is either serine, cysteine, or threonine, preferably serine or
cysteine;
X2 is either valine, leucine, or isoleucine, preferably valine or isoleucine,
more
preferably valine;
X3 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;
XS is either valine, leucine, or isoleucine, preferably valine or isoleucine,
more
preferably valine;
X6 is either lysine, arginine, or histidine, preferably arginine or histidine,
more
preferably arginine;
X~ is either valine, leucine, or isoleucine, preferably isoleucine;
X8 is either aspartic acid, glutamic acid, valine, leucine, isoleucine,
lysine, arginine,
or histidine, preferably glutamic acid, valine, leucine, isoleucine, or
histidine, more
preferably valine, leucine, or isoleucine, more preferably valine;
X9 is either valine, leucine, or isoleucine, preferably leucine;
X10 is either serine, cysteine, threonine, asparagine, glutamine,
aspartic acid, or glutamic acid, preferably serine, asparagine, or glutamic
acid;
each Y1 is an independently selected amino acid,
each Y2 is an independently selected amino acid,
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Z1 is an optionally present protecting group covalently joined to Y1,
preferably, Z1 is
either an optionally substituted -C~_~o alkyl, optionally substituted -CZ_lo
alkenyl,
optionally substituted aryl, -C1_6 alkyl optionally substituted aryl, -C(O)-
(CHZ)1_~-
COOH, -C(O)- C1_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,
benzyl,
benzyloxycarbonyl or t-butyloxycarbonyl;
Z2 is an optionally present protecting group covalently joined to Y2,
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,
andOtoS.
An "optionally present protecting group covalently joined to Y1" 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
-NH2 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 a-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. Preferably, the alkyl group is 1 to 4 carbons in length.
Examples of
alkyl include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, and t-
butyl. Alkyl
substituents are selected from the group consisting of halogen (preferably -F
or-Cl)
-OH, -CN, -SH, -NHZ, -NOz, -C1_2 alkyl substituted with 1 to 6 halogens
(preferably
-F or -Cl, more preferably -F), -CF3, -OCH3, or -OCF3.
"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. Preferably, 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, -NHZ, -NO2, -C1_2 alkyl
substituted
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with 1 to 5 halogens (preferably -F or -Cl, more preferably -F), -CF3, -OCH3,
or
-OCF3.
"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. Preferably, the aryl is a 5 or 6 membered ring, more preferably
benzyl. Aryl
substituents are selected from the group consisting of -C~_4 alkyl, -Cl_4
alkoxy, halogen
(preferably -F or -Cl), -OH, -CN, -SH, -NHZ, -NO2, -C1_z alkyl substituted
with 1 to 5
halogens (preferably -F or -Cl, more preferably -F), -CF3, or -OCF3.
Proteinaceous compounds can be produced using standard techniques.
The polypeptide region of a proteinaceuous compound, and proteinaceuous
compounds that are exclusively polypeptide, 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=Ala=Alanine: codons GCA, GCC, GCG, GCU
C=Cys=Cysteine: codons UGC, UGU
D=Asp=Aspartic acid: codons GAC, GAU
E=Glu=Glutamic acid: codons GAA, GAG
F=Phe=Phenylalanine: codons UUC, UUU
G=Gly=Glycine: codons GGA, GGC, GGG, GGU
H=His=Histidine: codons CAC, CAU
I=Ile=Isoleucine: codons AUA, AUC, AUU
K=Lys=Lysine: codons AAA, AAG
L=Leu=Leucine: codons UUA, UUG, CUA, CUC, CUG, CUU
M=Met=Methionine: codon AUG
N=Asn=Asparagine: codons AAC, AAU
P=Pro=Proline: codons CCA, CCC, CCG, CCU
Q=Gln=Glutamine: codons CAA, CAG
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R=Arg=Arginine: codons AGA, AGG, CGA, CGC, CGG, CGU
S=Ser=Serine: codons AGC, AGU, UCA, UCC, UCG, UCU
T=Thr=Threonine: codons ACA, ACC, ACG, ACU
V=Val=Valine: codons GUA, GUC, GUG, GUU
W=Trp=Tryptophan: codon UGG
Y=Tyr=Tyrosine: codons UAC, UAU
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°d Edition, Cold Spring Harbor
Laboratory Press,
1989.
Preferably, 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.
Generally, 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.
Regulatory systems are available to control gene expression, such as
GENE-SWTTCHTM (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
incorporated by reference herein) and those involving the tetracycline
operator (U.S.
Patent Nos. 5,464,758 and 5,650,298, both of which are hereby incorporated by
reference herein).
The skilled artisan can readily identify expression vectors providing
suitable levels of polypeptide expression in different hosts. A variety of
mammalian
expression vectors are well known in the art including pcDNA3 (Invitrogen),
pMClneo (Stratagene), pXTI (Stratagene), pSGS (Stratagene), EBO-pSV2-neo
(ATCC 37593), pBPV-1(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),
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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 III
(Invitrogen).
Recombinant host cells may be prokaryotic or eukaryotic. Examples
of recombinant host cells include the following: bacteria such as E. coli;
fungal cells
such as yeast; mammalian cells such as human, bovine, porcine, monkey and
rodent;
and insect cells such as Drosophila and silkworm derived cell lines.
Commercially
available mammalian cell lines include L cells L-M(TK<sup>-</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-Kl
(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.
Preferably,
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.,
Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, and
Kohler,
et al., Nature 256:495-497 ( 1975).
Non-Proteinaceuous Compounds
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.
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 peptidomirnetics are well known in the art. (See, Gilon, et al.,
U.S.
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Patent 5,874,529, and Gante, Angew. Chem. Int. Ed. Engl. (1994) 33, 1699-1720,
both
of which are hereby incorporated 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. (Gene Therapy & Molecular Biology: From
Basic Mechanisms to Clinical Applications, Ed. Boulikas, Gene Therapy Press,
1998,
and Hitt, et al. (1996) Advances in Pharmacology 40:137-206, hereby
incorporated by
reference herein.) 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 & Molecular Biology: From Basic Mechanisms to Clinical
Applications, Ed. Boulikas, Gene Therapy Press, 1998 hereby incorporated by
reference herein.)
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.
COMPOUND SCREENING
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.
The effect of a compound on 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. Preferably, HCV
processing is tested for by measuring the activity or formation of NS2 or NS3.
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Targetingithe 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.
Screening in the Presence of an NS4A Agonist
Non-saturating levels of an NS4A agonist can be employed in assays
screening for HCV inhibitory compounds. Without being limited to any
particular
theory, NS4A agonists may alter NS2/3 conformation thereby increasing the
accessibility of the NS2/3 active site to HCV inhibitory compounds. However,
using
the guidance provided herein 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. Examples of 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.
Preferably, the NS4A agonist employed in the assay is present at a
level sufficient to cause a detectable inhibition of NS2/3 autocleavage. In
different
embodiments the NS4A agonist is present at a concentration no more than 2X or
1X
its Kd or IC50; or is present at a concentration about equal to its Kd or
IC50.
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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'x' Edition, Ed. Gennaro, Mack Publishing, 1990, and Modern
Pharmaceutics 2"d Edition, Eds. Banker and Rhodes, Marcel Dekker, Inc., 1990,
both
of which are hereby incorporated 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. Examples of such 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, thiocyanate, tosylate, and
undecanoate; and
base salts such as ammonium salts, alkali metal salts such as sodium and
potassium
salts, alkaline earth metal salts such as calcium and magnesium salts, salts
with
organic bases such as dicyclohexylamine salts, N-methyl-D-glucamine, and salts
with
amino acids such as arginine and lysine.
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. As immediate release tablets, these compositions
may
contain microcrystalline cellulose, dicalcium phosphate, starch, magnesium
stearate
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and lactose and/or other excipients, binders, extenders, disintegrants,
diluents and
lubricants.
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, absorption 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. When administered by injection, 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.
When rectally administered in the form of suppositories, these
compositions may be prepared by mixing the drug with a suitable non-irntating
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.
Different techniques and formulations can be used to facilitate
introduction of a peptide into a cell, including nucleic acid delivery,
prodrug
formulations, and liposomes. Examples of such techniques and formulations are
described above and in references such as Gene Therapy & Molecular Biology:
From
Basic Mechanisms to Clinical Applications, Ed. Boulikas, Gene Therapy Press,
1998.
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.
Optimal precision in achieving concentrations of drug within the range
that yields efficacy without toxicity requires a regimen based on the kinetics
of the
drug's availability to target sites. This involves a consideration of the
distribution,
equilibrium, and elimination of a drug. The daily dose for a patient is
expected to be
between 0.01 and 1,000 mg per adult patient per day.
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EXAMPLES
Examples are provided below to further illustrate different features and
advantages of the present invention. The examples also illustrate useful
methodology
for practicing the invention. These examples do not limit the claimed
invention.
Example 1: Materials and Methods
DNA constructions. Two DNA constructs were made for the synthesis
of NS2/3 J strain RNA, and its subsequent translation to proteins which lack
the
membrane binding region of NS2 but contain HCV residues 849-1240; "849-1240J"
and "Ma1849-1240)". Codons 849 to 1240 were amplified by PCR from pT7
(Santolini, et al., (1995) J. Virol. 69, 7461-7471). For 849-1240J, the HCV
sequence
was cloned into pET3c (Novagen), while for Ma1849-1240J, the DNA was inserted
into pETMaIcH (Pryor, et al., (1997) Protein Expr. Purif. 10, 309-319) to
produce an
open reading frame encoding the fusion protein, "Ma1849-1240", which includes
E.
coli maltose binding protein-His6 tag-HCV residues 849-1240 (Love, et al.,
(1996)
Cell 87, 331-342). DNA for pCTTE 810-1615BK is described by Pieroni, et al.,
(1997) J. Virol. 71, 6373-80. Upon transcription and translation, pCITE 810-
1615BK
produces HCV residues 810-1615 of the BK strain ("810-1615BK")
Site-directed mutagenesis was performed with the Stratagene Quick
Change method, to generate non-processing mutants His952A1a and Cys993A1a in
the
expression constructs described above.
Peptides. Peptides were obtained by custom synthesis from Midwest
Biotech (Fishers, IN) and were greater than 95% pure as judged from reverse
phase
HPLC. Effective molecular weights were obtained by quantitative amino acid
analysis. All peptides were dissolved and diluted in DMSO, so that the final
concentration of DMSO in every reaction was 5°Io.
In-vitro transcription and translation. Circular DNA plasmids were
linearized with BLP1 (Bpu 1102) and purified with a Qiagen QiaEX II kit before
transcription. RNA was transcribed with T7 RNA polymerise (Ambion Megascript
kit), phenol/CHC13 extracted and ethanol precipitated. Translations were with
Promega or Ambion in-vitro rabbit reticulocyte lysate translation kits at
30°C, for 30-
40 minutes using 35S-methionine as a label (NEN, Amersham). Translation was
then
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inhibited by the addition of cycloheximide (250 pM final concentration) and
samples
immediately frozen on dry ice.
NS2/3 autocleavage reactions. Translated NS2/3 was 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.
At the
completion of an experiment, frozen samples were placed in boiling water for 5
minutes, and radiolabeled proteins were separated by SDS-PAGE. ( 14%).
Prestained
Novex molecular weight standards were used in estimation of molecular weights
of
the products. For peptide inhibition measurements, incubations were initiated
by the
addition of diluted lysate to a DMSO solution of peptide in a tube held at
20°C.
The distribution of 35S-labeled proteins on dried gels was determined
with a phosphorimager (Molecular Dynamics). Product bands were quantified and
expressed as a proportion of total signal in the gel lane so that variations
in gel lane
loading were normalized. The product NS2 from 810-1615 BK was used to generate
data shown for screening of peptides and ICSO 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 ICSO values were determined by first
expressing the product level found as a fraction of the no-inhibitor control
product
level, then fitting the equation
30
b
Fractional Activity = a +
(1+xlc)
to the data, where 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
ICSO and d is a
slope coefficient.
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Example 2: NS2/3 Processing Reactions
Autocleavage reactions using NS2/3 protein translated in-vitro were
used to investigate the inhibitory potential of peptides likely to affect the
reaction.
Peptides representing the cleaved sequence had no effect upon reaction rates
and the
reaction rate was insensitive to dilution. Both results are consistent with
suggestions
that the NS2/3 cleavage is an intramolecular reaction.
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,
Ma1849-1240J, to nearly 100%. In all constructs, the mutations His952A1a or
Cys993A1a prevented the appearance of products, as previously reported
(Hijikata, et
al., (1993) J. Virol. 67, 4665-4675; and Grakoui, et al., (1993) Proc. Natl.
Acad. Sci.
USA 90, 10583-7).
Both Ma1849-1240) and 810-1615BK were used in subsequent
characterization of the NS2/3 reaction and inhibition. The translation of
Ma1849-
1240) gave the expected precursor molecular weight of 80 kDa, but also a
smaller
protein of 67 kDa (Fig. 1A), possibly due to internal initiation, thus
complicating the
use of this version of NS2/3 for quantification of processing rates and
inhibitor
potencies. In contrast, 810-1615BK was produced as a single 80 kDa band that
cleaved itself to the expected molecular weight products of 60 kDa (NS3) and
20 kDa
(NS2) (Fig. 1B). In addition, 810-1615BK did not begin cleavage until addition
to
Triton X-100, as has been reported (Pieroni, et al., (1997) J. Virol. 71, 6373-
6380),
thereby allowing reactions to be initiated at will without background cleavage
products generated during the translation phase of the experiment.
Dilution of the NS2/3 precursor 10-fold into water completely
prevented the processing reaction (data not shown). Dilution into a 10,000
molecular
weight filtrate of rabbit reticulocyte lysate supported the reaction at a rate
slightly
higher than observed in undiluted lysate. Greater dilution of precursor (up to
40-fold)
did not further change the rate of processing. The necessity of low molecular
weight
cellular components) for NS2/3 reactions was previously noted. (See, Pieroni,
et al.,
(1997) J. Virol. 71, 6373-6380, hereby incorporated by reference herein.)
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For all subsequent measurements, a 10-fold dilution of in-vitro
synthesized NS2/3 into lysate filtrate was used. The accumulation of products
for
810-1615BK occurred at a rate of 0.04 min 1.
Example 3: Peptide Inhibition of NS2/3 Processing
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.
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Table 2
Inhibition of NS2/3 by peptides.
SEQ. SEQUENCE INHIBITION ICSOa
ID.
NO. OM)
NS 2/3
Cleava
a Site-Derived
Pe tidesb
1 EQGWRLL~APITAYS 15
2 GRGLRLL~APITAYS 12
3 EQGWRLL 14
4 APITAYS -9
GRGLRLL 2
6 GWRLL~APITA 20
7 APITA 6
8 GWRLL 16
NS4A-Derived
Pe tidesc
9 KGSVVIVGRIILSGRK 61 5.7
VRLGSISVIGIVRGKK -17 137.0
11 Ac-GGSVVIVGRIIL,SGRK 66 3.4
12 GGSVVIVGRIIL.SGRG 66
13 KKGSVVIVGR1ZLSGRPAIVPRR-NHz 95
14 KKGSVVIVGRIILSGRPAIVPDRELLY 85
QEFDE
Ac-KGSVVIV-NHZ 8
16 Ac-AITLSGR -12
17 Ac-RIIL,SGRK -21
Unrelated
Pe tidesb
18 GVVNAS.Abu.RLATRR 14
19 HTYLQASEKFKM 11
aICSO values were determined as described in Example 1, and are an average of
two
determinations.
bPercent 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
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concentration corresponds to a minimum of 500 ~.M for the group of cleavage
site
peptides.
cPercent 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. The NS2/3 cleavage site-derived peptides of SEQ. ID.
NOs.
1 and 2 correspond to HCV amino acids 1020-1033, J strain and BK strain,
respectively. Other cleavage site-derived peptides are smaller segments of
SEQ. >D.
NOs. 1 or 2. The arrow indicates the cleavage point, as determined in the
NS2/3
protein. (Grakoui, et al., (1993) Proc. Natl. Acad. Sci. USA 90, 10583-10587,
and
Komoda, et al., (1994) Gene 145, 221-226.) 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. ID. NO. 9, but in a random
order.
Similar results were obtained with NS2/3 Ma1849-1240J.
Peptides containing the cleavage site sequence of NS2/3, RLL*API
(SEQ. >D. NO. 27) (where * denotes the scissile bond), were tested for effect
upon
NS2/3 processing in reactions of 30 minutes. No significant effect was
observed with
a variety of substrate or product-like peptides at a concentration of 500 p,M,
as shown
in Figure 1 and Table 2.
NS4A peptides were examined for their effect upon NS2/3
autocleavage. Significant inhibition was observed, as shown for a peptide of
SEQ.
>D. NO. 9 in Figure 1B. 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.
The inhibition by NS4A peptides could be titrated and typical results
are shown in Figure 1. Potencies of 3.4 ~.M and 5.7 ~,M were obtained for
peptides of
SEQ.1D. NOs. 9 and 1 l, respectively. Peptides that represented only a portion
of the
region known to bind to NS3, such as peptides of SEQ. )D. NOs. 15 and 16, did
not
inhibit. Similar inhibition patterns were observed with both 810-1615BK and
Ma1849-1615J. A peptide with the same amino acid composition as SEQ. >D. NO. 9
but with a randomized sequence (of SEQ. >D. NO. 10) was not inhibitory.
Peptides
unrelated to NS4A or the NS2/3 cleavage site were also not inhibitory.
-25-
CA 02383411 2002-02-27
WO 01/16379 PCT/US00/23444
Other embodiments are within the following claims. While several
embodiments have been shown and described, various modifications may be made
without departing from the spirit and scope of the present invention.
-26-
CA 02383411 2002-02-27
WO 01/16379 PCT/US00/23444
SEQUENCE LISTING
<110> Merck & Co., Inc.
<120> HEPATITIS C VIRUS REPLICATION INHIBITORS
<130> 20511 PCT
<150> 60/151,395
<151> 1999-08-30
<160> 27
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> NS2/3 Cleavage Site-Derived Peptide
<400> 1
Glu Gln Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser
1 5 10
<210> 2
<211> 14
<212> PRT
<213> Artificial Sequence
<220>
<223> NS2/3 Cleavage Site-Derived Peptide
<400> 2
Gly Arg Gly Leu Arg Leu Leu Ala Pro Ile Thr Ala Tyr Ser
1 5 10
<210> 3
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> NS2/3 Cleavage Site-Derived Peptide
<400> 3
Glu Gln Gly Trp Arg Leu Leu
1 5
<210> 4
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> NS2/3 Cleavage Site-Derived Peptide
<400> 4
-1-
CA 02383411 2002-02-27
WO 01/16379 PCT/US00/23444
Ala Pro Ile Thr Ala Tyr Ser
1 5
<210> 5
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> NS2/3 Cleavage Site-Derived Peptide
<400> 5
Gly Arg Gly Leu Arg Leu Leu
1 5
<210> 6
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> NS2/3 Cleavage Site-Derived Peptide
<400> 6
Gly Trp Arg Leu Leu Ala Pro Ile Thr Ala
1 5 10
<210> 7
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> NS2/3 Cleavage Site-Derived Peptide
<400> 7
Ala Pro Ile Thr Ala
1 5
<210> 8
<211> 5
<212> PRT
<213> Artificial Sequence
<220>
<223> NS2/3 Cleavage Site-Derived Peptide
<400> 8
Gly Trp Arg Leu Leu
1 5
<210> 9
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> NS4A-Derived Peptide
<400> 9
Lys Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Lys
1 5 10 15
-2-
CA 02383411 2002-02-27
WO 01/16379 PCT/US00/23444
<210> 10
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> NS4A-Derived Peptide
<400> 10
Val Arg Leu Gly Ser Ile Ser Val Ile Gly Ile Val Arg Gly Lys Lys
1 5 10 15
<210> 11
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<221> ACETYLATION
<222> (1)...(1)
<223> NS4A-Derived Peptide
<400> 11
Gly Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Lys
1 5 10 15
<210> 12
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> NS4A-Derived Peptide
<400> 12
Gly Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg Gly
1 5 10 15
<210> 13
<211> 23
<212> PRT
<213> Artificial Sequence
<220>
<223> NS4A-Derived Peptide
<221> AMIDATION
<222> (23)...(23)
<400> 13
Lys Lys Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg
1 5 10 15
Pro Ala Ile Val Pro Arg Arg
<210> 14
<211> 32
<212> PRT
<213> Artificial Sequence
-3-
CA 02383411 2002-02-27
WO 01/16379 PCT/US00/23444
<220>
<223> NS4A-Derived Peptide
<400> 14
Lys Lys Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly Arg
1 5 10 15
Pro Ala Ile Val Pro Asp Arg Glu Leu Leu Tyr Gln Glu Phe Asp Glu
20 25 30
<210> 15
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> NS4A-Derived Peptide
<221> ACETYLATION
<222> (1)...(1)
<221> AMIDATION
<222> (7)...(7)
<400> 15
Lys Gly Ser Val VaI Ile Val
1 5
<210> 16
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> NS4A-Derived Peptide
<221> ACETYLATION
<222> (1)...(1)
<400> 16
Ala Ile Ile Leu Ser Gly Arg
1 5
<210> 17
<211> 8
<212> PRT
<213> Artificial Sequence
<220>
<223> NS4A-Derived Peptide
<221> ACETYLATION
<222> (1)...(1)
<400> 17
Arg Ile Ile Leu Ser Gly Arg Lys
1 5
<210> 18
<211> 13
<212> PRT
<213> Artificial Sequence
-4-
CA 02383411 2002-02-27
WO 01/16379 PCT/US00/23444
<220>
<221> MOD_RES
<222> (7) ..(7)
<223> Xaa = Abu
<223> Unrelated Peptide
<400> 18
Gly Val Val Asn Ala Ser Xaa Arg Leu Ala Thr Arg Arg
1 5 10
<210> 19
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> Unrelated Peptide
<400> 19
His Thr Tyr Leu Gln Ala Ser Glu Lys Phe Lys Met
1 5 10
<210> 20
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Amino Acid Sequence Present in HCV-BK
<400> 20
Gly Ser Val Val Ile Val Gly Arg Ile Ile Leu Ser Gly
1 5 10
<210> 21
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Amino Acid Sequence Present in HCV-H
<400> 21
Gly Cys Val Val Ile Val Gly Arg Ile Val Leu Ser Gly
1 5 10
<210> 22
<211> 13
<212> PRT
<213> Artificial Sequence
<220>
<223> Amino Acid Sequence Present in G01
<400> 22
Gly Ser Val Val Ile Val Gly Arg Ile Val Leu Ser Gly
1 5 10
<210> 23
<211> 13
<212> PRT
-5-
CA 02383411 2002-02-27
WO 01/16379 PCT/US00/23444
<213> Artificial Sequence
<220>
<223>Amino Sequence Present HCV-1
Acid in
<400>23
Gly Ile Val Gly Arg Val Ser
Cys Val Leu Gly
Val
Val
1 5 10
<210>24
<211>13
<212>PRT
<213>ArtificialSequence
<220>
<223>Amino Sequence Present HCV-J6
Acid in
<400>24
Gly Ile Ile Gly Arg His Asn
Cys Leu Val Gln
Val
Cys
1 5 10
<210>25
<211>13
<212>PRT
<213>ArtificialSequence
<220>
<223>Amino Sequence Present HCV-J8
Acid in
<400>25
Gly Ile Ile Gly Arg His Asn
Cys Leu Leu Gln
Ile
Ser
1 ~ 5 10
<210>26
<211>13
<212>PRT
<213>ArtificialSequence
<220>
<223>Amino Sequence Present
Acid in HCV-NZL1
<400>26
Cys Val Gly His Ile Ile Gly
Val Glu Glu Lys
Val
Ile
1 5 10
<210>27
<211>6
<212>PRT
<213>ArtificialSequence
<220>
<223>Cleavage ite Sequence of 3
S NS2/
<400>27
Arg Pro Ile
Leu
Leu
Ala
1 5
-6-
