U.S. patent application number 13/842193 was filed with the patent office on 2013-07-25 for anti-heparan sulfate peptides that block herpes simplex virus infection in vivo.
This patent application is currently assigned to The Board of Trustees of the University of Illinois. The applicant listed for this patent is The Board of Trustees of the University of Illinois. Invention is credited to Deepak Shukla, Vaibhav Tiwari.
Application Number | 20130189784 13/842193 |
Document ID | / |
Family ID | 48797542 |
Filed Date | 2013-07-25 |
United States Patent
Application |
20130189784 |
Kind Code |
A1 |
Shukla; Deepak ; et
al. |
July 25, 2013 |
ANTI-HEPARAN SULFATE PEPTIDES THAT BLOCK HERPES SIMPLEX VIRUS
INFECTION IN VIVO
Abstract
Provided are anti-heparan sulfate peptides and methods that
employ those peptides for the prevention or treatment of viral
infections, including herpesviral infections such as
.alpha.-herpesviral, .beta.-herpesviral, and .gamma.-herpesviral
infections, which are exemplified by HSV-1. CMV, and HHV-8 viral
infections, respectively. Peptides may comprise at least 10 amino
acids of the amino acid sequences XRXRXKXXRXRX (SEQ ID NO: 2),
XRXRXXKXRXRX (SEQ ID NO: 8), XXRRRRXRRRXK (SEQ ID NO: 4), and/or
KXRRRXRRRRXX (SEQ ID NO: 10), wherein X represents any amino acid.
In some embodiments, peptides comprise at least 10 amino acids of
the sequence LRSRTKIIRIRH (SEQ ID NO: 1), HRIRIIKTRSRL (SEQ ID NO:
7), MPRRRRIRRRQK (SEQ ID NO: 3), and/or KQRRRIRRRRM (SEQ ID NO: 9).
Peptides may be coupled to one or more therapeutic compound(s) to
generate peptide-therapeutic compound conjugates, wherein the
therapeutic compound may be one or more of a nucleoside analog, an
oligosaccharide, and a small molecule.
Inventors: |
Shukla; Deepak; (Orland
Park, IL) ; Tiwari; Vaibhav; (Downers Grove,
IL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Board of Trustees of the University of Illinois; |
Urbana |
IL |
US |
|
|
Assignee: |
The Board of Trustees of the
University of Illinois
Urbana
IL
|
Family ID: |
48797542 |
Appl. No.: |
13/842193 |
Filed: |
March 15, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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PCT/US11/52002 |
Sep 16, 2011 |
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13842193 |
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61383520 |
Sep 16, 2010 |
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61651643 |
May 25, 2012 |
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Current U.S.
Class: |
435/375 ;
530/327; 530/328 |
Current CPC
Class: |
A61K 47/646 20170801;
C07K 7/06 20130101; C07K 7/08 20130101 |
Class at
Publication: |
435/375 ;
530/328; 530/327 |
International
Class: |
C07K 7/08 20060101
C07K007/08; C07K 7/06 20060101 C07K007/06 |
Goverment Interests
GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under R01
Grant Nos. AI057860 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A peptide comprising at least 10 consecutive amino acids of the
sequence XRXRXKXXRXRX (SEQ ID NO: 2) or XRXRXXKXRXRX (SEQ ID NO:
8), wherein X is any amino acid, R is arginine, and K is
lysine.
2. The peptide of claim 1 wherein said peptide comprises at least
12 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO:
2) or XRXRXXKXRXRX (SEQ ID NO: 8).
3. The peptide of claim 2 wherein said peptide is 12 amino acids in
length.
4. The peptide of claim 1 wherein each X is independently selected
from the group consisting of leucine, serine, threonine,
isoleucine, and histidine.
5. The peptide of claim 1 wherein said peptide comprises at least
10 consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO:
1) or HRIRIIKTRSRL (SEQ ID NO: 7).
6. The peptide of claim 5 wherein said peptide comprises at least
12 consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO:
1) or HRIRIIKTRSRL (SEQ ID NO: 7).
7. The peptide of claim 1 wherein said peptide comprises at least
one D-amino acid.
8. The peptide of claim 6 wherein said peptide is 12 amino acids in
length.
9. The peptide of claim 1 wherein said peptide comprises at least
10 consecutive amino acids of the sequence RSRTKIIRIR (SEQ ID NO:
5) or RIRIIKTRSR (SEQ ID NO: 11).
10. The peptide of claim 7 wherein all the amino acids in said
peptide are D-amino acids.
11. A peptide comprising at least 10 consecutive amino acids of the
sequence XXRRRRXRRRXK (SEQ ID NO: 4) or KXRRRXRRRRXX (SEQ ID NO:
10) wherein X is any amino acid, R is arginine, and K is
lysine.
12. The peptide of claim 11 wherein said peptide comprises at least
12 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO:
4) or KXRRRXRRRRXX (SEQ ID NO: 10).
13. The peptide of claim 12 wherein said peptide is 12 amino acids
in length.
14. The peptide of claim 11 wherein each X is independently
selected from the group consisting of methionine, proline,
isoleucine, and glutamine.
15. The peptide of claim 11 wherein said peptide comprises at least
10 consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO:
3) or KQRRRIRRRRPM (SEQ ID NO: 9).
16. The peptide of claim 15 wherein said peptide comprises at least
12 consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO:
3) or KQRRRIRRRRPM (SEQ ID NO: 9).
17. The peptide of claim 11 wherein said peptide comprises at least
one D-amino acid.
18. The peptide of claim 11 wherein said peptide is 12 amino acids
in length.
19. The peptide of claim 11 wherein said peptide comprises at least
10 consecutive amino acids of the sequence RRRRIRRRQK (SEQ ID NO:
6) or KQRRRIRRRR (SEQ ID NO: 12).
20. The peptide of claim 17 wherein all the amino acids in said
peptide are D-amino acids.
21. A method for blocking the binding of a virus to a target cell,
said method comprising the step of contacting said target cell with
a peptide comprising at least 10 consecutive amino acids of a
sequence selected from the group consisting of XRXRXKXXRXRX (SEQ ID
NO: 2), XRXRXXKXRXRX (SEQ ID NO: 8), XXRRRRXRRRXK (SEQ ID NO: 4),
and KXRRRXRRRRXX (SEQ ID NO: 10), wherein X is any amino acid, R is
arginine, and K is lysine.
22. A peptide-therapeutic compound conjugate, comprising: (a) at
least 10 consecutive amino acids of a sequence selected from the
group consisting of XRXRXKXXRXRX (SEQ ID NO: 2), XRXRXXKXRXRX (SEQ
ID NO: 8), XXRRRRXRRRXK (SEQ ID NO: 4), and KXRRRXRRRRXX (SEQ ID
NO: 10), wherein X is any amino acid, R is arginine, and K is
lysine; and (b) a therapeutic compound; wherein said peptide is
coupled to said therapeutic compound to generate said conjugate.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation-in-part of
International Application No. PCT/US2011/052002, filed Sep. 16,
2011 and titled "Anti-Heparan Sulfate Peptides That Block Herpes
Simplex Virus Infection In Vivo," which claims the benefit of U.S.
Provisional Patent Application No. 61/383,520, filed Sep. 16, 2010,
and the present application also claims the benefit of U.S.
Provisional Patent Application No. 61/651,643, filed May 25, 2012,
the entire disclosures of which are hereby incorporated by
reference in their entireties.
SEQUENCE LISTING
[0003] The present application includes a Sequence Listing in
electronic format as a text file entitled
"Sequence_Listing_DA015CIP.txt" which was created on Mar. 14, 2013,
and which has a size of 4,601 bytes. The contents of txt file
"Sequence_Listing_DA015CIP.txt" are incorporated by reference
herein.
BACKGROUND OF THE DISCLOSURE
[0004] 1. Technical Field
[0005] The present disclosure is directed, generally, to the
inhibition of viral infection, including herpes simplex virus (HSV)
cellular infection, and to the treatment of diseases associated
with HSV and other viral infections. More specifically, the present
disclosure provides peptides that can block viral infection of a
cell both in vitro and in vivo.
[0006] 2. Description of the Related Art
[0007] Heparan sulfate (HS) and its modified form, 3-0 sulfated
heparan sulfate (3-OS HS) provide cell surface attachment sites for
many human and non-human pathogenic viruses, including herpes
simplex virus type-1 and -2 (HSV-1 and HSV-2, respectively). HSV
infections cause a variety of medical disorders affecting the face
and mouth, the eye, the central nervous system, and other areas of
the body. Such infections may be especially severe in
immunocompromised subjects.
[0008] Existing antiviral drugs can reduce the severity of HSV
outbreaks, but cannot cure the subject of the infection. Therefore,
there is a need for prophylactic agents that can inhibit entry by
HSV and other pathogenic viruses into cells. There is also a need
for therapeutic agents that can reduce or minimize the spread of
HSV and other pathogenic viruses from infected cells to uninfected
cells within a subject.
SUMMARY OF THE DISCLOSURE
[0009] The present disclosure achieves these and other related
needs by providing peptides, including anti-HS peptides and
anti-3-OS HS peptides, that significantly inhibit viral infection
and/or receptor-mediated cell-to-cell fusion. Exemplified herein
are peptides designated G1 and G2, which represent two classes of
cationic peptides specifically isolated against HS (e.g., Group 1
peptides) and 3-OS HS (e.g., Group 2 peptides), respectively, and
which exhibit strong herpesvirus entry-inhibiting activities. The
Group 1 peptides and Group 2 peptides disclosed herein inhibit
HSV-1 spread in corneal keratitis thereby demonstrating both the in
vivo significance of HS/3-OS HS in HSV-1 pathogenesis as well as
the efficacy of the G1 and G2 peptides in the treatment of diseases
associated with viral infection.
[0010] Also exemplified herein are retro-inverso peptides,
designated riG1 and riG2, which represent retro-inverso ("ri")
forms of peptides designated G1 and G2, respectively. In some
embodiments, a "retro-inverso" form of a peptide has amino acids
that are assembled in reverse order ("retro"), and with an inverted
("inverso") chirality (L or D), relative to the peptide. Some
retro-inverso peptides may have one or more D-amino acids. In other
retro-inverso peptides, all of the amino acids may be D-amino
acids. As further described herein, retro-inverso Group 1 peptides
and retro-inverso Group 2 peptides may also inhibit viral infection
and/or receptor-mediated cell-to-cell fusion, and may be used in
the prevention and/or treatment of one or more viral infections. In
some embodiments, retro-inverso peptides may have one or more
characteristics that enhance their utility for such uses, such as
relatively low immunogenicity and/or enhanced resistance to
proteolytic degradation, relative to the corresponding G1/G2
peptide.
[0011] In various embodiments, peptides described herein may be
enriched in basic amino acid residues and classified into two major
groups:
[0012] (1) Group 1, which includes a class of peptides with
alternating charges having the sequence XRXRXKXXRXRX (SEQ ID NO:
2), as represented herein by the G1 peptide having the sequence
LRSRTKIIRIRH (SEQ ID NO: 1), and retro-inverso forms of Group 1
peptides, which have a reverse amino acid order XRXRXXKXRXRX (SEQ
ID NO: 8) and at least one D-amino acid, as represented herein by
the riG1 peptide, which has the sequence HRIRIIKTRSRL (SEQ ID NO:
7) and at least one D-amino acid;
[0013] (2) Group 2, which includes a class of peptides with
repetitive charges having the sequence XXRRRRXRRRXK (SEQ ID NO: 4),
as represented herein by the G2 peptide having the sequence
MPRRRRIRRRQK (SEQ ID NO: 3); and retro-inverso forms of Group 2
peptides, which have a reverse amino acid order KXRRRXRRRRXX (SEQ
ID NO: 10) and at least one D-amino acid, as represented herein by
the riG2 peptide, which has the sequence KQRRRIRRRRPM (SEQ ID NO:
9) and at least one D-amino acid.
[0014] Embodiments of the present disclosure provide peptides that
comprise at least 10 or at least 12 consecutive amino acids of the
sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R
is arginine, and K is lysine. Within certain aspects, these
peptides may be 10 or 12 amino acids in length. Within other
aspects, each X may be independently selected from the group
consisting of leucine (L), serine (S), threonine (T), isoleucine
(I), and histidine (H). Other embodiments of the present disclosure
provide peptides that comprise at least 10 or at least 12
consecutive amino acids of the sequence XRXRXXKXRXRX (SEQ ID NO: 8)
wherein X is any amino acid, R is arginine, K is lysine, and
wherein at least one amino acid is a D-amino acid. Within certain
aspects, these peptides may be 10 or 12 amino acids in length.
Within other aspects, each X may be independently selected from the
group consisting of leucine (L), serine (S), threonine (T),
isoleucine (I), and histidine (H). Within still further aspects,
these peptides may be composed entirely of D-amino acids.
[0015] Some embodiments of the present disclosure provide peptides
that comprise at least 10 or at least 12 consecutive amino acids of
the peptide G1, which has the amino acid sequence LRSRTKIIRIRH (SEQ
ID NO: 1), wherein L is leucine, R is arginine, S is serine, T is
threonine, K is lysine, I is isoleucine, and H is histidine. Within
certain aspects, these peptides may be 10 or 12 amino acids in
length. An exemplary 10 amino acid peptide based upon G1 is the
peptide RSRTKIIRIR (SEQ ID NO: 5).
[0016] Other embodiments of the present disclosure provide peptides
that comprise at least 10 or at least 12 consecutive amino acids of
the peptide riG1, which has the amino acid sequence HRIRIIKTRSRL
(SEQ ID NO: 7) wherein L is leucine, R is arginine, S is serine, T
is threonine, K is lysine, I is isoleucine, H is histidine, and
wherein at least one amino acid is a D-amino acid. Within certain
aspects, these peptides may be 10 or 12 amino acids in length. An
exemplary 10 amino acid peptide based upon riG1 is the peptide
RIRIIKTRSR (SEQ ID NO: 11), wherein at least one amino acid is a
D-amino acid. In some embodiments, these peptides may be composed
entirely of D-amino acids.
[0017] Further embodiments of the present disclosure provide
peptides that comprise at least 10 or at least 12 consecutive amino
acids of the sequence XXRRRRXRRRRXK (SEQ ID NO: 4) wherein X is any
amino acid, R is arginine, and K is lysine. Within certain aspects,
these peptides may be 10 or 12 amino acids in length. Within other
aspects, X may be independently selected from the group consisting
of methionine (M), proline (P), isoleucine (I), and glutamine (Q).
Embodiments of the present disclosure also provide peptides that
comprise at least 10 or at least 12 consecutive amino acids of the
sequence KXRRRXRRRRXX (SEQ ID NO: 10) wherein X is any amino acid,
R is arginine, K is lysine and wherein at least one amino acid is a
D-amino acid. Within certain aspects, these peptides may be 10 or
12 amino acids in length. Within other aspects, X may be
independently selected from the group consisting of methionine (M),
proline (P), isoleucine (I), and glutamine (Q). Within still
further aspects, these peptides may be composed entirely of D-amino
acids.
[0018] Other embodiments of the present disclosure provide peptides
that comprise at least 10 or at least 12 consecutive amino acids of
the peptide G2, which has the amino acid sequence MPRRRRIRRRQK (SEQ
ID NO: 3) wherein M is methionine, P is proline, R is arginine, I
is isoleucine, Q is glutamine, and K is lysine. Within certain
aspects, these peptides may be 10 or 12 amino acids in length. An
exemplary 10 amino acid peptide based upon G2 is the peptide
RRRRIRRRQK (SEQ ID NO: 6). Still other embodiments of the present
disclosure provide peptides that comprise at least 10 or at least
12 consecutive amino acids of the peptide riG2, which has the amino
acid sequence KQRRRIRRRRPM (SEQ ID NO: 9) wherein M is methionine,
P is proline, R is arginine, I is isoleucine, Q is glutamine, K is
lysine and wherein at least one amino acid is a D-amino acid.
Within certain aspects, these peptides may be 10 or 12 amino acids
in length. An exemplary 10 amino acid peptide based upon riG2 is
the peptide KQRRRIRRRR (SEQ ID NO: 12), wherein at least one amino
acid is a D-amino acid. Within still further aspects, these
peptides may be composed entirely of D-amino acids.
[0019] The peptides disclosed herein can block the binding of a
virus to heparan sulfate or 3-O sulfated heparan sulfate thereby
preventing the viral infection of a target cell, such as a corneal
cell. Viruses the binding of which can be blocked by these peptides
include herpesviruses, such as a herpesvirus selected from the
group consisting of an .alpha.-herpesvirus, a .beta.-herpesvirus,
and a .gamma.-herpesvirus. Within certain aspects, the
.alpha.-herpesvirus is HSV-1. Within other aspects, the
.beta.-herpesvirus is cytomegalovirus (CMV). Within still further
aspects, the .gamma.-herpesvirus is human herpesvirus-8
(HHV-8).
[0020] In some embodiments, the peptides disclosed herein may be
combined into compositions comprising two or more peptides, wherein
each of the peptides independently comprises at least 10 amino
acids of a sequence selected from the group consisting of
XRXRXKXXRXRX (SEQ ID NO: 2), XRXRXXKXRXRX (SEQ ID NO: 8),
XXRRRRXRRRXK (SEQ ID NO: 4), and KXRRRXRRRRXX (SEQ ID NO: 10),
wherein X is any amino acid, R is arginine, and K is lysine. In
some embodiments, each X is independently selected from the group
consisting of leucine, serine, threonine, isoleucine, methionine,
proline, glutamine, and histidine. In other embodiments, at least
one of said peptides comprises at least 10 consecutive amino acids
of a sequence selected from the group consisting of LRSRTKIIRIRH
(SEQ ID NO: 1), HRIRIIKTRSRL (SEQ ID NO: 7), MPRRRRIRRRQK (SEQ ID
NO: 3), and KQRRRIRRRRPM (SEQ ID NO: 9). In some embodiments, one
or more of the peptides may comprise at least 12 consecutive amino
acids of the selected sequence. In other embodiments, one or more
of the peptides is 10-12 amino acids in length. In some aspects of
these compositions, one or more of the amino acids is a D-amino
acid. In other aspects of these compositions, all of the amino
acids of at least one of the peptides may be D-amino acids.
[0021] In other embodiments, peptides disclosed herein may be
combined into compositions comprising two or more peptides wherein
each of the peptides independently comprises at least 10 amino
acids of a sequence selected from the group consisting of
XRXRXKXXRXRX (SEQ ID NO: 2) and XXRRRRXRRRXK (SEQ ID NO: 4) wherein
X is any amino acid, R is arginine, and K is lysine. In some
embodiments, each X is independently selected from the group
consisting of leucine, serine, threonine, isoleucine, methionine,
proline, glutamine, and histidine. Within certain aspects of these
compositions, at least one of the peptides comprises at least 10
amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1), such as
the peptide RSRTKIIRIR (SEQ ID NO: 5). Within other aspects of
these compositions, at least one of the peptides comprises at least
10 amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3), such as
the peptide RRRRIRRRQK (SEQ ID NO: 6). In still further aspects of
these compositions, one or more of the peptides includes at least
one D-amino acid.
[0022] In other embodiments, peptides disclosed herein may be
combined into compositions comprising two or more peptides wherein
each of the peptides independently comprises at least 10 amino
acids of a sequence selected from the group consisting of
XRXRXXKXRXRX (SEQ ID NO: 8) and KXRRRXRRRRXX (SEQ ID NO: 10),
wherein X is any amino acid, R is arginine, K is lysine, and at
least one amino acid is a D-amino acid. In some embodiments, each X
is independently selected from the group consisting of leucine,
serine, threonine, isoleucine, methionine, proline, glutamine, and
histidine. Within certain aspects of these compositions, each of
the peptides independently comprises at least 10 amino acids of a
sequence selected from the group consisting of HRIRIIKTRSRL (SEQ ID
NO: 7), such as the peptide RIRIIKTRSR (SEQ ID NO: 11), wherein at
least one amino acid is a D-amino acid. Within other aspects of
these compositions, at least one of the peptides comprises at least
10 amino acids of the sequence KQRRRIRRRRPM (SEQ ID NO: 9), such as
the peptide KQRRRIRRRR (SEQ ID NO: 12), wherein at least one amino
acid is a D-amino acid. Within still further aspects, one or more
of these peptides is composed entirely of D-amino acids.
[0023] In other embodiments, peptides disclosed herein may be
combined into compositions comprising two or more peptides wherein
one of the peptides comprises at least 10 amino acids of a sequence
selected from the group consisting of XRXRXKXXRXRX (SEQ ID NO: 2)
and XXRRRRXRRRXK (SEQ ID NO: 4), and another one of the peptides
comprises at least 10 amino acids of a sequence selected from the
group consisting of XRXRXXKXRXRX (SEQ ID NO: 8) and KXRRRXRRRRXX
(SEQ ID NO: 10), wherein X is any amino acid, R is arginine, and K
is lysine. In some embodiments, each X is independently selected
from the group consisting of leucine, serine, threonine,
isoleucine, methionine, proline, glutamine, and histidine. In
certain aspects of such compositions, at least one of the peptides
comprises at least 10 amino acids of the sequence LRSRTKIIRIRH (SEQ
ID NO: 1), such as the peptide RSRTKIIRIR (SEQ ID NO: 5), or at
least 10 amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3),
such as the peptide RRRRIRRRQK (SEQ ID NO: 6), and another one of
the peptides comprises at least 10 amino acids of a sequence
selected from the group consisting of HRIRIIKTRSRL (SEQ ID NO: 7),
such as the peptide RIRIIKTRSR (SEQ ID NO: 11), or at least 10
amino acids of the sequence KQRRRIRRRRPM (SEQ ID NO: 9), such as
the peptide KQRRRIRRRR (SEQ ID NO: 12), wherein at least one amino
acid is a D-amino acid. Within still further aspects, one or more
of these peptides is composed entirely of D-amino acids.
[0024] Other embodiments provide methods for blocking the binding
of a virus to a target cell. Embodiments of such methods may
include contacting the target cell with a peptide such as a G1,
riG1, G2, or riG2 peptide. In some embodiments, a method for
blocking the binding of a virus to a target cell may comprise
contacting the target cell with a peptide comprising at least 10 or
at least 12 consecutive amino acids of a sequence selected from the
group consisting of XRXRXKXXRXRX (SEQ ID NO: 2), XRXRXXKXRXRX (SEQ
ID NO: 8), XXRRRRXRRRXK (SEQ ID NO: 4), and KXRRRXRRRRXX (SEQ ID
NO: 10), wherein X is any amino acid, R is arginine, and K is
lysine. In some embodiments, each X is independently selected from
the group consisting of leucine, serine, threonine, isoleucine,
methionine, proline, glutamine, and histidine. Optionally, at least
one amino acid may be a D-amino acid. In other embodiments, a
method for blocking the binding of a virus to a target cell may
include contacting the target cell with a peptide comprising at
least 10 or at least 12 consecutive amino acids of a sequence
selected from the group consisting of LRSRTKIIRIRH (SEQ ID NO: 1),
HRIRIIKTRSRL (SEQ ID NO: 7), MPRRRRIRRRQK (SEQ ID NO: 3), and
KQRRRIRRRRPM (SEQ ID NO: 9). In other embodiments, a method for
blocking the binding of a virus to a target cell may include
contacting the target cell with a peptide comprising a sequence
selected from the group consisting of RSRTKIIRIR (SEQ ID NO: 5),
RRRRIRRRQK (SEQ ID NO: 6), RIRIIKTRSR (SEQ ID NO: 11), and
KQRRRIRRRR (SEQ ID NO: 12). In some embodiments, the peptide
comprises at least consecutive amino acids of the selected
sequence. In other embodiments, the peptide comprises at least 12
consecutive amino acids of the selected sequence. In other
embodiments, the peptide is 10-12 amino acids in length.
[0025] In other embodiments, a method for blocking the binding of a
virus to a target cell may include contacting the target cell with
a peptide comprising at least 10 or at least 12 consecutive amino
acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any
amino acid, R is arginine, and K is lysine. In some embodiments,
each X is independently selected from the group consisting of
leucine, serine, threonine, isoleucine, methionine, proline,
glutamine, and histidine. Within certain aspects of these methods,
the peptide comprises at least 10 or at least 12 consecutive amino
acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1). For example, in
some embodiments the peptide may comprise the sequence RSRTKIIRIR
(SEQ ID NO: 5). Optionally the peptide may comprise one or more
D-amino acids.
[0026] In some embodiments, a method for blocking the binding of a
virus to a target cell may include contacting the target cell with
a peptide comprising at least 10 or at least 12 consecutive amino
acids of the sequence XRXRXXKXRXRX (SEQ ID NO: 8) wherein X is any
amino acid, R is arginine, K is lysine, and wherein at least one
amino acid is a D-amino acid. In some embodiments, each X is
independently selected from the group consisting of leucine,
serine, threonine, isoleucine, methionine, proline, glutamine, and
histidine. Within certain aspects of these methods, the peptide
comprises at least 10 or at least 12 consecutive amino acids of the
sequence HRIRIIKTRSRL (SEQ ID NO: 7), wherein at least one amino
acid is a D-amino acid. For example, in some embodiments the
peptide may comprise the sequence RIRIIKTRSR (SEQ ID NO: 11),
wherein at least one amino acid is a D-amino acid. Within still
further aspects, these peptides may be composed entirely of D-amino
acids.
[0027] In further embodiments, a method for blocking the binding of
a virus to a target cell may include contacting the target cell
with a peptide comprising at least 10 or at least 12 consecutive
amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4) wherein X
is any amino acid, R is arginine, and K is lysine. In some
embodiments, each X is independently selected from the group
consisting of leucine, serine, threonine, isoleucine, methionine,
proline, glutamine, and histidine. Within certain aspects of these
methods, the peptide comprises at least 10 or at least 12
consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO:
3), the peptide may comprise the sequence RRRRIRRRQK (SEQ ID NO:
6). Optionally, the peptide may include one or more D-amino
acids.
[0028] Other embodiments of a method for blocking the binding of a
virus to a target cell may include contacting the target cell with
a peptide comprising at least 10 or at least 12 consecutive amino
acids of the sequence KXRRRXRRRRXX (SEQ ID NO: 10), wherein X is
any amino acid, R is arginine, and K is lysine and wherein at least
one amino acid is a D-amino acid. In some embodiments, each X is
independently selected from the group consisting of leucine,
serine, threonine, isoleucine, methionine, proline, glutamine, and
histidine. Within certain aspects of these methods, the peptide
comprises at least 10 or at least 12 consecutive amino acids of the
sequence KQRRRIRRRRPM (SEQ ID NO: 9), wherein at least one amino
acid is a D-amino acid. For example, in some embodiments the
peptide may comprise the sequence KQRRRIRRRR (SEQ ID NO: 12),
wherein at least one amino acid is a D-amino acid. Within still
further aspects, these peptides may be composed entirely of D-amino
acids.
[0029] Methods, peptides, compositions, and conjugates described
herein may be effective to block or inhibit target cell binding by
one more viruses. Such viruses may include pathogenic viruses with
target cell entry mechanisms/strategies that include binding to HS
on target cell surfaces. Viruses for which target cell entry can be
blocked or inhibited by peptides and methods described herein may
include one or more herpesviruses. In some embodiments, such
peptides and methods may be effective to block or inhibit target
cell entry by one or more herpesviruses, wherein each herpesvirus
is selected from the group consisting of an .alpha.-herpesvirus, a
.beta.-herpesvirus, and a .gamma.-herpesvirus. Within certain
aspects, the .alpha.-herpesvirus is HSV-1. Within other aspects,
the .beta.-herpesvirus is cytomegalovirus (CMV). Within yet other
aspects, the .gamma.-herpesvirus is human herpesvirus-8
(HHV-8).
[0030] Embodiments of the present disclosure provide methods for
the treatment of a patient who is susceptible to a viral infection.
In some embodiments, methods for the treatment of a patient who is
susceptible to a viral infection may include administering to the
patient a peptide comprising at least 10 consecutive amino acids of
a sequence selected from the group consisting of a sequence
selected from the group consisting of XRXRXKXXRXRX (SEQ ID NO: 2),
XRXRXXKXRXRX (SEQ ID NO: 8), XXRRRRXRRRXK (SEQ ID NO: 4), and
KXRRRXRRRRXX (SEQ ID NO: 10), wherein X is any amino acid, R is
arginine, and K is lysine. In some embodiments, each X is
independently selected from the group consisting of leucine,
serine, threonine, isoleucine, methionine, proline, glutamine, and
histidine. Optionally, at least one amino acid may be a D-amino
acid.
[0031] In other embodiments, methods for the treatment of a patient
who is susceptible to a viral infection may include administering
to the patient a peptide comprising at least 10 or at least 12
consecutive amino acids of a sequence selected from the group
consisting of LRSRTKIIRIRH (SEQ ID NO: 1), HRIRIIKTRSRL (SEQ ID NO:
7), MPRRRRIRRRQK (SEQ ID NO: 3), and KQRRRIRRRRPM (SEQ ID NO: 9).
In other embodiments, methods for the treatment of a patient who is
susceptible to a viral infection may include administering to the
patient a peptide comprising a sequence selected from the group
consisting of RSRTKIIRIR (SEQ ID NO: 5), RRRRIRRRQK (SEQ ID NO: 6),
RIRIIKTRSR (SEQ ID NO: 11), and KQRRRIRRRR (SEQ ID NO: 12). In some
embodiments, the peptide comprises at least 10 consecutive amino
acids of the selected sequence. In other embodiments, the peptide
comprises at least 12 consecutive amino acids of the selected
sequence. In other embodiments, the peptide is 10-12 amino acids in
length.
[0032] In other embodiments, methods for the treatment of a patient
who is susceptible to a viral infection may include administering
to the patient a peptide comprising at least 10 or at least 12
consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2)
wherein X is any amino acid, R is arginine, and K is lysine. In
some embodiments, each X is independently selected from the group
consisting of leucine, serine, threonine, isoleucine, methionine,
proline, glutamine, and histidine. Within certain aspects of these
methods, the peptide comprises at least 10 or at least 12
consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO:
1). For example, in some embodiments the peptide may comprise the
sequence RSRTKIIRIR (SEQ ID NO: 5). Optionally, the peptide may
comprise one or more D-amino acids.
[0033] Still further embodiments of the present disclosure provide
methods for the treatment of a patient who is susceptible to a
viral infection wherein the methods comprise the step of
administering to the patient a peptide comprising at least 10
consecutive amino acids of the sequence XRXRXXKXRXRX (SEQ ID NO: 8)
wherein X is any amino acid, R is arginine, K is lysine, and
wherein at least one amino acid is a D-amino acid. In some
embodiments, each X is independently selected from the group
consisting of leucine, serine, threonine, isoleucine, methionine,
proline, glutamine, and histidine. In certain aspects of these
methods, the peptide comprises at least 10 or at least 12
consecutive amino acids of the sequence HRIRIIKTRSRL (SEQ ID NO:
7), wherein at least one amino acid is a D-amino acid. For example,
in some embodiments the peptide may include the sequence RIRIIKTRSR
(SEQ ID NO: 11), wherein at least one amino acid is a D-amino acid.
Within still further aspects, these peptides may be composed
entirely of D-amino acids. Related embodiments of the present
disclosure provide methods for the treatment of a patient who is
susceptible to a viral infection wherein the methods comprise
administering to the patient a peptide comprising at least
consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4)
wherein X is any amino acid, R arginine, and K is lysine. In some
embodiments, each X is independently selected from the group
consisting of leucine, serine, threonine, isoleucine, methionine,
proline, glutamine, and histidine. Within certain aspects of these
methods, the peptide comprises at least 10 or at least 12
consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO:
3). For example, in some embodiments, the peptide may comprise the
sequence RRRRIRRRQK (SEQ ID NO: 6). Optionally, the peptide may
include one or more D-amino acids.
[0034] Other embodiments of methods for the treatment of a patient
who is susceptible to a viral infection may include administering
to the patient a peptide comprising at least 10 or at least 12
consecutive amino acids of the sequence KXRRRXRRRRXX (SEQ ID NO:
10), wherein X is any amino acid, R is arginine, and K is lysine
and wherein at least one amino acid is a D-amino acid. In some
embodiments, each X is independently selected from the group
consisting of leucine, serine, threonine, isoleucine, methionine,
proline, glutamine, and histidine. Within certain aspects of these
methods, the peptide comprises at least 10 or at least 12
consecutive amino acids of the sequence KQRRRIRRRRPM (SEQ ID NO:
9), wherein at least one amino acid is a D-amino acid. For example,
in some embodiments the peptide may comprise the sequence
KQRRRIRRRR (SEQ ID NO: 12), wherein at least one amino acid is a
D-amino acid. Within still further aspects, these peptides may be
composed entirely of D-amino acids.
[0035] Yet further embodiments of the present disclosure provide HS
and 3-OS HS binding peptide-therapeutic compound conjugates that
comprise an HS or a 3-OS binding peptide, as summarized above and
as described in greater detail below, that is coupled to a
therapeutic compound, such as an antiviral compound selected from a
nucleoside analog, an oligosaccharide, and a small molecule.
[0036] Within certain aspects of these embodiments, nucleoside
analogs that may be used in these HS and 3-OS HS binding
peptide-therapeutic compound conjugates include guanosine analogs
such as acyclovir (Formula I) and valacyclovir.
##STR00001##
[0037] Within other aspects of these embodiments, oligosaccharides
that may be used in these HS and 3-OS HS binding
peptide-therapeutic compound conjugates include oligosaccharides
such as tetrasaccharides, hexasaccharides, octasaccharides, and
decasaccharides that are capable of binding to one or more of HSV-1
glycoproteins gB, gC, and gD. For example an oligosaccharide can be
an HS octasaccharide 1 having the structure of Formula II:
##STR00002##
[0038] Within still further aspects of these embodiments, small
molecules that may be used in these HS and 3-OS HS binding
peptide-therapeutic compound conjugates include
Bis-2-methyl-4-amino-quinolyl-6-carbamide (Surfen) having the
structure of Formula III:
##STR00003##
BRIEF DESCRIPTION OF THE FIGURES
[0039] FIG. 1 demonstrates that the inhibition of HSV-1 entry by
12-mer synthetic peptides is not specific to any particular gD
receptor.
[0040] FIG. 2 demonstrates that G1 and G2 peptides block HSV-1
entry into human target cells.
[0041] FIG. 3 demonstrates that HSV-1 entry blocking activity of G2
peptide is not HSV-1 strain specific.
[0042] FIG. 4 presents the results of deletion analysis and alanine
scanning mutagenesis, which reveal the significance of positively
charged residues in HSV-1 entry inhibition.
[0043] FIG. 5 demonstrates that G2 blocks cellular entry by
representative members of beta and gamma herpesvirus subfamilies
(CMV and HHV-8).
[0044] FIG. 6 demonstrates that G2 functions by preventing HSV-1
attachment to cells, which results in loss of binding and viral
replication.
[0045] FIG. 7 demonstrates the activity of G2 against HSV-1
glycoprotein induced cell-to-cell fusion and spread.
[0046] FIG. 8 demonstrates that G1 or G2 effectively block
infection by HSV-1 in a mouse model of corneal keratitis.
DETAILED DESCRIPTION OF THE DISCLOSURE
[0047] The present disclosure is based upon the unexpected
discovery that certain peptides, including certain 10-mer and
12-mer peptides, can specifically bind to HS and 3-OS HS and can
block the entry of a virus, such as a herpes simplex virus (e.g.,
HSV-1), into a target cell.
[0048] Heparan sulfate (HS) and its modified form, 3-0 sulfated
heparan sulfate (3-OS HS), when present on a cell surface, may
provide an attachment site for human and non-human pathogenic
viruses such as herpes simplex virus type-1 and -2 (HSV-1 and
HSV-2, respectively) thereby contributing to viral infections. Both
wild-type and laboratory strains of HSV bind to HS. In addition to
the attachment step, HSV-1 penetration into cells can also be
mediated by 3-OS HS, which is produced after a rare enzymatic
modification in HS catalyzed by 3-O-sulfortransferases
(3-OSTs).
[0049] HSV envelope glycoproteins B and C (gB and gC) bind HS and
mediate virus attachment to cells. A third glycoprotein, gD,
specifically recognizes 3-OS HS in a binding interaction that
facilitates fusion pore formation during viral entry. Despite the
known importance of HS and 3-OS HS during HSV-1 infection in vitro,
little is known about the significance of HS and 3-OS during an
infection in vivo.
[0050] The versatility of HS to bind multiple microbes and
participate in a variety of regulatory phenomena comes from its
negatively-charged nature and highly complex structure, which is
generated by enzymatic modifications. Virtually all cells express
HS as long un-branched chains often associated with protein cores
commonly exemplified by syndecan and glypican families of HS
proteoglycans. The parent HS chain, which contains repeating
glucosamine and hexuronic acid dimers, can be 100-150 residues long
and may contain multiple structural modifications. Most common
among these structural modifications is the addition of sulfate
groups at various positions within the chain, which leads to the
generation of specific motifs, making HS highly attractive for
microbial adherence.
[0051] The role of HS in viral infection may extend beyond its
function as a low-specificity pre-attachment site. For instance, HS
mediates HSV-1 transport on filopodia during surfing and negatively
regulates virus-induced membrane fusion. Likewise, for human
papilloma virus (HPV), HS proteoglycans play a key role in the
activation of immune response, an important aspect for both vaccine
development and HPV pathogenesis. Similarly, HS expressed on
spermatozoa plays a key role in the capture of human
immunodeficiency virus (HIV) and its transmission to dendritic,
macrophage, and T-cells. 3OS HS also plays a role in hepatitis B
virus replication, and HS-binding peptides or compounds can be used
to prevent genital HPV, HIV, and cytomegalovirus infections.
[0052] The results of viral entry and gD-binding assays and the
fluorescent microscopy data presented herein demonstrate that both
G1 and G2 are potent in blocking viral entry, in particular HSV-1
entry, into primary cultures of human corneal fibroblasts (CF) and
CHO-K1 cells transiently expressing different gD-receptors.
Moreover, the G2 peptide, which was isolated against 3-OS HS,
displays a wider ability to inhibit the entry of clinically
relevant strains of HSV-1, and some divergent members of
herpesvirus family including cytomegalovirus (CMV) and human
herpesvirus-8 (HHV-8).
[0053] The entry blocking activity of the peptides disclosed herein
is independent of gD receptor, virus strain, or cell-type. In
addition to G1 and G2 peptides, the present disclosure provides
retro-inverso forms of the G1 and G2 peptides, riG1 and riG2
respectively, in which all L-amino acids are replaced with D-amino
acids and the change in chirality counterbalanced by reversing the
primary sequence. Without being bound by theory, it is believed
that the G1 and G2 peptides function by interfering with viral
binding to a cell, and that retro-inverso forms thereof (e.g., riG1
and riG2) will also interfere with viral binding to cells.
[0054] Utility of Anti-HS and Anti-3-OS HS Peptides
[0055] As described in greater detail herein, the anti-HS and
anti-3-OS peptides of the present disclosure will find broad
utility in preventing viral infection of a target cell, both in
vivo and in vitro. Thus, for example, anti-HS and anti-3-OS
peptides will find therapeutic utility as efficacious compounds for
the treatment of a viral disease, such as a herpesvirus-mediated
disease including an .alpha.-herpesvirus-, a .beta.-herpesvirus-,
and/or a .gamma.-herpesvirus-mediated disease.
[0056] The anti-HS and anti-3-OS peptides disclosed herein will
also find utility in studies seeking to demonstrate the
significance of HS during in vivo viral infection, such as HSV-1
infection. HS has been studied as an attachment receptor, but
little has been reported on its function in vivo. The experiments
presented herein, including experiments with a mouse corneal
infection model, demonstrate the efficacy of the G1 and G2 peptides
in blocking infection in vivo and indicate that HS is an important
HSV-1 co-receptor both in vitro and in vivo.
[0057] The ability of the presently disclosed peptides to act
specifically on HS/3-OS HS is significant because HS is widely
expressed on all cells and tissues and it is known to regulate many
important biological phenomena. Thus, the presently disclosed
peptides can be used to prevent the infection of a wide range of
cells and tissues, both in vitro and in vivo, and as probes to
study HS functions in a wide variety of biological contexts.
[0058] Additionally, HS moieties are frequently up-regulated during
pathological conditions and may contribute to inflammation. Thus,
the presently disclosed peptides may also find utility in blocking
the pathological effects of HS and in regulating inflammation.
[0059] The complex enzymatic regulation of HS chain gives HS a
complex ability (and affinity) to bind many proteins to perform new
functions. Therefore, 3-OS HS binding peptides disclosed herein
will be useful as probes and/or as diagnostic tools to assess
structural alterations within HS or its turnover on cell
surfaces.
[0060] Because many unrelated viruses bind HS, the G1 and G2
peptides as well as their retro-inverso forms (riG1 and riG2
peptides) will find broad utility in those applications where it is
desired to block infection by a wide variety of viruses that
utilize HS and/or 3-OS HS binding to facilitate target cell binding
and infection.
[0061] These and other utilities are contemplated by the presently
disclosed anti-HS and anti-3-OS peptides, including the Group 1 and
Group 2 peptides G1 and G2, as well as the retro-inverso forms of
these peptides riG1 and riG2, which are described in substantial
detail herein.
[0062] Anti-Heparan Sulfate and Anti-3-O Sulfated Heparan Sulfate
Peptides
[0063] As summarized above, the present disclosure provides
peptides that were identified by the screening of a random
M13-phage display library with heparan sulfate and 3-0 sulfated
heparan sulfate and the subsequent isolation of HS- and 3-O
sulfated HS binding phages. The peptides disclosed herein, which
are exemplified by those peptides that are presented in Table 1,
are characterized by the presence of the positively charged
amino-acid residues arginine and/or lysine, the unique arrangement
of which is important for blocking virus-cell binding and/or
virus-induced membrane fusion.
TABLE-US-00001 TABLE 1 Amino acid (AA) Sequences of Phage-displayed
Peptides Isolated by Three Round Screening against HS and 3-OS HS
AA sequences of peptides against HS PVFRNIRVGDPI.sup.1
RLPRLKMRNRG.sup.3 HKRRRQLRIQRR.sup.6 QRNHILTPGTSI.sup.2
CGGLDSGSGVLA.sup.1 NHRPLLIRRRRT.sup.5 RLNNPRLLNTRP.sup.3
LMSRKTNRINM.sup.2 LFGILLCGVIYV.sup.1 RKPRTSPSITLR.sup.3
KLHMRHHRSPRI.sup.5 RSMHHINRRQRR.sup.4 DLGSLYVGGACG.sup.1
PRRNLRRRRLIP.sup.4 IRKRRLRHQPRS.sup.4 RSPSQQSIMPLH.sup.3
RVCGSIGKEVLG.sup.2 LRSRTKIIRIRH.sup.8** RPRTRLHTHRNR.sup.4
PRKRRRTQQRRI.sup.5 CGILGESGGVLI.sup.1 HILIRIRRQRTP.sup.4
MNPTRRSRMRMI.sup.3 SKRSNQPIINR.sup.2 VFRINNIRVGDY.sup.1
RLRRLIRNRRGT.sup.3 RRRTQRKRRHTI.sup.5 IIQLSRRLRSIR.sup.3 G.sup.1
peptide isolated against HS AA sequences of peptides against 3-OS
HS RINKLDVLIIPV.sup.1 MPRRRRIRRRQK.sup.11** QPRHKQIPIKML.sup.3
NNNSPMRRSRNH.sup.2 LICGRVINKINK.sup.1 QKNIRRRSRSKL.sup.5
QRKTRIPRSTLP.sup.4 KRNRRPIKLRHS.sup.5 CCGIIEVTQLKG.sup.1
RKIISLTNRRLS.sup.4 RRLSSMQNLMKN.sup.3 KPQTLSIRPQLI.sup.3
RGLSQKKRHIIQ.sup.1 QLRKRQIIRSQQ.sup.2 HIIPKRTLRRNI.sup.4
RTIPNRIKTIPM.sup.4 HRIKLVAAIDVG.sup.1 LMSLRKTNRINIM.sup.2
RSNPKKSRSLQM.sup.4 INLTSKRMSLRN.sup.3 GGCTKHIDVALK.sup.1
KRSIIQINPTQS.sup.2 TPHRRHIITPSN.sup.3 IRRHRRRLSQII.sup.6
RFQKIDLIATRQ.sup.1 RKINIQRRSTLM.sup.4 PTQLHKRPRIRL.sup.4
HRPRLKMRRPTM.sup.5 G.sup.2 peptide isolated against 3OS HS
.sup.nFrequency/number of times peptide sequences isolated; **P
< 0.001, Frequently isolated peptides.
[0064] The peptides that are described herein are enriched in basic
amino acid residues and classified into two major groups:
[0065] (1) Group 1, which includes a class of peptides having
alternating charges (XRXRXKXXRXRX; SEQ ID NO: 2) and is represented
herein by the G1 peptide, which has the amino acid sequence
LRSRTKIIRIRH (SEQ ID NO: 1); and the retro-inverso forms of the
Group 1 peptides, which includes the peptides having the sequence
XRXRXXKXRXRX (SEQ ID NO: 8), wherein at least one amino acid is a
D-amino acid, and is represented herein by the riG1 peptide, which
has the amino acid sequence HRIRIIKTRSRL (SEQ ID NO: 7), wherein at
least one amino acid is a D-amino acid;
[0066] (2) Group 2, which includes a class of peptides having
repetitive charges (XXRRRRXRRRXK; SEQ ID NO: 4) and is represented
herein by the G2 peptide, which has the amino acid sequence
MPRRRRIRRRQK (SEQ ID NO: 3); and the retro-inverso forms of the
Group 2 peptides, which includes the peptides having the sequence
KXRRRXRRRRXX (SEQ ID NO: 10), wherein at least one amino acid is a
D-amino acid, and is represented herein by the riG2 peptide, which
has the amino acid sequence KQRRRIRRRRPM (SEQ ID NO: 9), wherein at
least one amino acid is a D-amino acid.
[0067] Group 1 peptides of the present disclosure comprise at least
10 or at least 12 consecutive amino acids of the sequence
XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R is
arginine, and K is lysine. Within certain Group 1 peptides, X may
be independently selected from the group consisting of leucine,
serine, threonine, isoleucine, and histidine. The retro-inverso
Group 1 peptides of the present disclosure comprise at least 10 or
at least 12 consecutive amino acids of the sequence XRXRXXKXRXRX
(SEQ ID NO: 8) wherein X is any amino acid, R is arginine, K is
lysine, and wherein at least one amino acid is a D-amino acid.
Within certain retro-inverso Group 1 peptides, X may be
independently selected from the group consisting of leucine,
serine, threonine, isoleucine, and histidine. In some aspects,
these retro-inverso peptides may be composed entirely of D-amino
acids.
[0068] Exemplified herein are Group 1 peptides that are 10, 11, or
12 amino acids in length, such as peptides that comprise at least
10 or at least 12 consecutive amino acids of the sequence
LRSRTKIIRIRH (G1; SEQ ID NO: 1) wherein L is leucine, R is
arginine, S is serine, T is threonine, K is lysine, I is
isoleucine, and H is histidine. Also exemplified herein are
retro-inverso Group 1 peptides that are 10, 11, or 12 amino acids
in length, such as peptides that comprise at least 10 or at least
12 consecutive amino acids of the sequence HRIRIIKTRSRL (riG1; SEQ
ID NO: 7) wherein L is leucine, R is arginine, S is serine, T is
threonine, K is lysine, I is isoleucine, and H is histidine and
wherein at least one amino acid is a D-amino acid. In some aspects,
a retro-inverso peptide may be composed entirely of D-amino
acids.
[0069] Group 2 peptides of the present disclosure comprise at least
10 or at least 12 consecutive amino acids of the sequence
XXRRRRXRRRXK (SEQ ID NO: 4) wherein X is any amino acid, R is
arginine, and K is lysine. Within certain Group 2 peptides, X may
be independently selected from the group consisting of methionine,
proline, isoleucine, and glutamine. The retro-inverso Group 2
peptides of the present disclosure comprise at least 10 or at least
12 consecutive amino acids of the sequence KXRRRXRRRRXX (SEQ ID NO:
10) wherein X is any amino acid, R is arginine, and K is lysine and
wherein at least one amino acid is a D-amino acid. Within certain
retro-inverso Group 2 peptides, X may be independently selected
from the group consisting of methionine, proline, isoleucine, and
glutamine. In some aspects, these retro-inverso peptides may be
composed entirely of D-amino acids.
[0070] Exemplified herein are Group 2 peptides that are 10, 11, or
12 amino acids in length, such as peptides that comprise at least
10 or at least 12 consecutive amino acids of the sequence
MPRRRRIRRRQK (SEQ ID NO: 3) wherein M is methionine, P is proline,
R is arginine, I is isoleucine, Q is glutamine, and K is lysine.
Also exemplified herein are retro-inverso Group 2 peptides that are
10, 11, or 12 amino acids in length, such as peptides that comprise
at least 10 or at least 12 consecutive amino acids of the sequence
KQRRRIRRRRPM (riG2; SEQ ID NO: 9) wherein M is methionine, P is
proline, R is arginine, I is isoleucine, Q is glutamine, and K is
lysine and wherein at least one amino acid is a D-amino acid. In
some aspects, these retro-inverso peptides may be composed entirely
of D-amino acids.
[0071] The peptides disclosed herein can block binding of a virus
to heparan sulfate or 3-O sulfated heparan sulfate and/or can
prevent a viral infection of a target cell, such as a corneal cell.
Viruses the binding of which can be blocked by these peptides
include herpesviruses, such as .alpha.-herpesviruses,
.beta.-herpesviruses, and .gamma.-herpesviruses. Within certain
aspects, the .alpha.-herpesvirus is HSV-1. Within other aspects,
the .beta.-herpesvirus is cytomegalovirus (CMV). Within still
further aspects, the .gamma.-herpesvirus is human herpesvirus-8
(HHV-8).
[0072] While the G1 and G2 peptides represent specific examples of
Group 1 and Group 2 peptides, respectively, it will be understood
that alternative Group 1 and Group 2 peptides may be identified by
the identification of alternative functional amino acids. For
example, alternative functional amino acids within Group 1 and
Group 2 peptides can be identified through the generation of point
mutations and/or via alanine scanning mutagenesis. Similarly, while
the riG1 and riG2 peptides represent specific examples of
retro-inverso Group 1 and retro-inverso Group 2 peptides,
respectively, it will be understood that alternative retro-inverso
Group 1 and retro-inverso Group 2 peptides may be identified by the
identification of alternative functional amino acids. For example,
alternative functional amino acids within retro-inverso Group 1 and
retro-inverso Group 2 peptides can also be identified through the
generation of point mutations and/or via alanine scanning
mutagenesis.
[0073] It is disclosed herein that, while both G1 and G2 peptides
are capable of blocking HSV-1-mediated cell binding and infection,
G2 exhibits the additional capacity to block the entry of divergent
herpesviruses such as, for example, CMV and HHV-8. Without being
limited by theory, because the G2 peptide can block membrane fusion
it is believed that the G2 peptide can interfere with gD's
interaction with its receptor, 3-OS HS. Among the structural
differences between G1 and G2, it appears that G2 shows more
dependence on the positively charged residues than G1, which may
depend upon the presence of a lysine residue at the N-terminus. In
general, arginine has been found important for charge-charge
interaction with HS.
[0074] The peptides disclosed herein can be combined into
compositions comprising one, two, or more peptides wherein each of
the peptides independently comprises at least 10 amino acids of the
amino acid sequences XRXRXKXXRXRX (SEQ ID NO: 2) and/or
XXRRRRXRRRXK (SEQ ID NO: 4). Exemplified herein are compositions
comprising one or more peptides each of which includes at least 10
amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1) and/or at
least 10 amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO:
3).
[0075] The peptides disclosed herein can be combined into
compositions comprising one, two, or more peptides wherein each of
the peptides independently comprises at least 10 amino acids of the
amino acid sequences XRXRXXKXRXRX (SEQ ID NO: 8), wherein at least
one amino acid is a D-amino acid, and/or KXRRRXRRRRXX (SEQ ID NO:
10), wherein at least one amino acid is a D-amino acid. Exemplified
herein are compositions comprising one or more peptides each of
which includes at least 10 amino acids of the sequence HRIRIIKTRSRL
(SEQ ID NO: 7), wherein at least one amino acid is a D-amino acid,
and/or at least 10 amino acids of the sequence KQRRRIRRRRPM (SEQ ID
NO: 9), wherein at least one amino acid is a D-amino acid. Further
exemplified herein are compositions comprising one or more peptides
each of which includes at least 10 amino acids of the sequence
HRIRIIKTRSRL (SEQ ID NO: 7), wherein all the amino acids are
D-amino acids, and/or at least 10 amino acids of the sequence
KQRRRIRRRRPM (SEQ ID NO: 9), wherein all the amino acids are
D-amino acids.
[0076] The peptides of the present disclosure can be provided to a
patient as part of a pharmaceutical composition where it is mixed
with a pharmaceutically acceptable carrier. As used herein, the
term "pharmaceutical composition" refers to a preparation of one or
more of the peptides described herein with one or more other
chemical component(s) such as a physiologically suitable carrier or
excipient. The purpose of a pharmaceutical composition is to
facilitate administration of a compound to the patient. Techniques
for formulation and administration of compositions can be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa.
[0077] As used herein, the phrases "physiologically acceptable
carrier" and "pharmaceutically acceptable carrier" refer to a
carriers, diluents, and/or adjuvants that do not cause significant
irritation to a patient and do not abrogate the biological activity
and properties of the administered peptide. Suitable carriers may
include polyethylene glycol (PEG), a biocompatible polymer with a
wide range of solubility in both organic and aqueous media.
[0078] As used herein, the term "excipient" refers to an inert
substance added to a pharmaceutical composition to further
facilitate administration of an active peptide. Exemplary
excipients include calcium carbonate, calcium phosphate, various
sugars and types of starch, cellulose derivatives, gelatin,
vegetable oils and polyethylene glycols.
[0079] Pharmaceutical compositions can be manufactured by processes
well known in the art such as, for example, by means of
conventional mixing, dissolving, granulating, dragee-making,
levigating, emulsifying, encapsulating, entrapping or lyophilizing
processes. Pharmaceutical compositions can be formulated in
conventional manner using one or more physiologically acceptable
carriers comprising excipients and auxiliaries, which facilitate
processing of the active ingredients into preparations which, can
be used pharmaceutically. Proper formulation is dependent upon the
route of administration chosen.
[0080] For injection, the active peptides of the disclosure may be
formulated in aqueous solutions, preferably in physiologically
compatible buffers such as Hank's solution, Ringer's solution, or
physiological salt buffer. For transmucosal administration,
penetrants appropriate to the barrier to be permeated are used in
the formulation. Such penetrants are generally known in the
art.
[0081] Compositions of the present disclosure that are suitable for
oral administration may be prepared as capsules, cachets, tablets
or lozenges, each containing a predetermined amount of the peptide
of the invention, or which may be contained in liposomes or as a
suspension in an aqueous liquor or non-aqueous liquid such as a
syrup, an elixir, or an emulsion. An exemplary tablet formulation
includes corn starch, lactose, and magnesium stearate as inactive
ingredients. An exemplary syrup formulation includes citric acid,
coloring dye, flavoring agent, hydroxypropylmethylcellulose,
saccharin, sodium benzoate, sodium citrate and purified water.
[0082] For oral administration, the compounds can be formulated
readily by combining the active peptides with pharmaceutically
acceptable carriers well known in the art. Such carriers enable the
compounds of the invention to be formulated as tablets, pills,
dragees, capsules, liquids, gels, syrups, slurries, suspensions,
and the like, for oral ingestion by a patient. Pharmacological
preparations for oral use can be made using a solid excipient,
optionally grinding the resulting mixture, and processing the
mixture of granules, after adding suitable auxiliaries if desired,
to obtain tablets or dragee cores. Suitable excipients are, in
particular, fillers such as sugars, including lactose, sucrose,
mannitol, or sorbitol; cellulose preparations such as, for example,
maize starch, wheat starch, rice starch, potato starch, gelatin,
gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose,
sodium carbomethylcellulose; and/or physiologically acceptable
polymers such as polyvinylpyrrolidone (PVP). If desired,
disintegrating agents may be added, such as cross-linked polyvinyl
pyrrolidone, agar, or alginic acid or a salt thereof such as sodium
alginate.
[0083] Peptide compositions may also contain one or more
pharmaceutically acceptable carriers, which may include excipients
such as stabilizers (to promote long term storage), emulsifiers,
binding agents, thickening agents, salts, preservatives, solvents,
dispersion media, coatings, antibacterial and antifungal agents,
isotonic and absorption delaying agents, and the like. The use of
such agents for pharmaceutically active compounds is well known in
the art.
[0084] The compositions may conveniently be presented in unit
dosage form and may be prepared by any of the methods well known in
the art of pharmacy. Methods typically include the step of bringing
the active ingredients of the invention into association with a
carrier that constitutes one or more accessory ingredients.
[0085] Compositions of the present disclosure suitable for
inhalation can be delivered as aerosols or solutions. An exemplary
aerosol composition includes a peptide suspended in a mixture of
trichloromonofluoromethane and dichlorodifluoromethane plus oleic
acid. An exemplary solution composition includes a peptide
dissolved or suspended in sterile saline (optionally about 5% v/v
dimethylsulfoxide ("DMSO") for solubility), benzalkonium chloride,
and sulfuric acid (to adjust pH).
[0086] Compositions of the present disclosure that are suitable for
parenteral administration include sterile aqueous preparations of
the peptides of the present disclosure and are typically isotonic
with the blood of the patient to be treated. Aqueous preparations
may be formulated according to known methods using those suitable
dispersing or vetting agents and suspending agents. Sterile
injectable preparations may also be sterile injectable solutions or
suspensions in a non-toxic parenterally-acceptable diluent or
solvent, for example as a solution in 1,3-butane diol. Among the
acceptable vehicles and solvents that may be employed are water,
Ringer's solution, and isotonic sodium chloride solution. In
aqueous solutions, up to about 10% v/v DMSO or Trappsol can be used
to maintain solubility of some peptides. Also, sterile, fixed oils
may be conventionally employed as a solvent or suspending medium
such as, for example, synthetic mono- or di-glycerides. In
addition, fatty acids (such as oleic acid or neutral fatty acids)
can be used in the preparation of injectibles. Further, Pluronic
block copolymers can be formulated with lipids for time release
from solid form over a period of weeks or months.
[0087] Compositions suitable for topical administration may be
presented as a solution of the peptide in Trappsol or DMSO, or in a
cream, ointment, or lotion. Typically, about 0.1 to about 2.5%
active ingredient is incorporated into the base or carrier. An
example of a cream formulation base includes purified water,
petrolatum, benzyl alcohol, stearyl alcohol, propylene glycol,
isopropyl myristate, polyoxyl 40 stearate, carbomer 934, sodium
lauryl sulfate, acetate disodium, sodium hydroxide, and optionally
DMSO. An example of an ointment formulation base includes white
petrolatum and optionally mineral oil, sorbitan sesquioleate, and
DMSO. An example of a lotion formulation base includes carbomer
940, propylene glycol, polysorbate 40, propylene glycol stearate,
cholesterol and related sterols, isopropyl myristate, sorbitan
palmitate, acetyl alcohol, triethanolamine, ascorbic acid,
simethicone, and purified water.
[0088] Conjugated Anti-Heparan Sulfate and Anti-3-O Sulfated
Heparan Sulfate Peptides for Intracellular Delivery of Therapeutic
Compounds
[0089] As part of the present disclosure, it was discovered that
the heparan sulfate and 3-O sulfated heparan sulfate binding
peptides, upon binding to HS and 3-OS HS, cross the plasma membrane
and are transported into the cytoplasm and nucleus. Accordingly,
the HS and 3-OS HS binding peptides described herein may be
conjugated to one or more therapeutic compound to affect the
intracellular and/or intranuclear delivery of the therapeutic
compounds. Such conjugated HS and 3-OS HS peptides will find
utility, for example, (1) in the preferential delivery of one or
more compound(s) to an infected cell and/or (2) as a multivalent
drug therapy wherein the HS and 3-OS HS peptide can (a) block viral
infection (as described herein) and (b) cross the plasma membrane
in an HS- and/or a 3-OS-dependent manner thereby delivering the
compound(s) to affect intracellular and/or intranuclear clearance
of virus-infected cells.
[0090] Nucleoside Analogs
[0091] A wide variety of therapeutic compounds can be conjugated to
the HS and 3-OS HS binding peptides, which are described herein, to
generate the presently disclosed therapeutic compound HS and 3-OS
HS binding peptide conjugates. Exemplified herein are HS and 3-OS
HS binding peptide conjugates that employ the guanosine analog
acyclovir, or its prodrug valacyclovir, which can be metabolized
and incorporated into the viral DNA within a virally infected cell.
Acyclovir is depicted in the following Formula I:
##STR00004##
[0092] Acyclovir serves as a chain terminator for viral DNA
synthesis and an inhibitor for viral DNA polymerase. Piret and
Boivin, Antimicrob. Agents Chemother. 55(2):459-72 (2011).
Acyclovir differs from other nucleoside analogues in that it
contains a partial nucleoside structure wherein the sugar ring is
replaced with an open-chain structure. Acyclovir is converted into
acyclo-guanosine monophosphate by viral thymidine kinase. Cellular
kinases subsequently convert the monophosphate form of
acyclo-guanosine into acyclo-guanosine triphosphate, which is the
high affinity substrate viral DNA polymerase. Because acyclovir has
no 3' end, its incorporation into a nascent DNA strand results in
its chain termination activity.
[0093] Acyclovir is active against a number of herpesvirus species,
including herpes simplex virus type I (HSV-1), herpes simplex virus
type II (HSV-2), and varicella zoster virus (VZV). Acyclovir is
less active against Epstein-Barr virus (EBC) and cytomegalovirus
(CMV).
[0094] Oligosaccharides
[0095] In addition to the use of nucleoside analogs, such as the
guanosine analog acyclovir in generating HS and 3-OS HS binding
peptide therapeutic compound conjugate, the present disclosure
further contemplates conjugates that comprise one or more
oligosaccharide such as a herpesvirus HS oligosaccharide.
Exemplified herein are HS and 3-OS HS binding peptides that are
conjugated to the HS octasaccharide 1 that has been shown to bind
to the HSV-1 glycoprotein gD. Copeland et al., Biochemistry
47(21):5774-83 (2008) and Liu et al., J. Biol. Chem.
277(36):33456-67 (2002). HS octasaccharide 1 is presented herein by
Formula II.
##STR00005##
[0096] Other HS oligosaccharides, including HS octasaccharides,
that are capable of binding to HSV-1 glycoproteins gB, gC, and gD
can also be employed in the HS and 3-OS HS conjugates disclosed
herein. HS structural elements that are critical to gB, gC, and gD
binding can be identified by digesting heparan polysaccharides into
oligosaccharides with heparan lyase. The resulting oligosaccharides
can then be subjected to size exclusion chromatography to separate
into tetrasaccharides, hexasaccharides, octasaccharides, and
decasaccharides. Oligosaccharide fractions can be incubated with
gB, gC, and/or gD and their binding affinities assessed using
affinity co-electrophoresis. Oligosaccharide structures can be
determined by immunoprecipitating gB-, gC-, and/or
gD-oligosaccharide complexes, eluting bound oligosaccharides, and
separating by anion exchange chromatography as described in Liu et
al., J. Biol. Chem. 277(36):33456-67 (2002). The precise structure
of an HS oligosaccharide having high gB, gC, and/or gD affinity can
be determined through a combination of disaccharide compositional
analysis and nanoelectrospray ionization (nESI) mass
spectrophotometry. Pope et al., Glycobiology 11(6):505-13 (2001)
and Liu et al., J. Biol. Chem. 285(44):34240-9 (2010).
[0097] Structurally well defined oligosaccharides can be
synthesized by employing enzymatic synthetic methodology known in
the art such as those described by Liu et al., J. Biol. Chem.
285(44):34240-9 (2010) and Linhardt et al., Semin. Thromb. Hemost.
33(5):453-65 (2007) and as presented in the following Synthetic
Pathway I for HS oligosaccharides:
##STR00006## ##STR00007##
[0098] Starting from a disaccharide primer prepared from nitrous
acid-degraded heparosan, backbone elongation can be achieved by
altering KifA and pmHS2 treatment with UDP-GlcNTFA and UDP-GlcUA as
donor substrates, which can be followed by modification with
specific sulfotransferases. To generate different HS sequences, the
enzymatic steps can be varied. For example, the C.sub.5-epi can be
removed and 2-0 sulfotransferase R189A mutant can be used for step
c of the synthetic pathway shown above. Unlike wild type 2-OST,
2-OST R189A specifically sulfates the GlcUA, not IdoUA. Bethea et
al., Proc. Natl. Acad. Sci. U.S.A. 105(48):18724-9 (2008). As a
result, the desired octasaccharide can have GlcUA2S instead of the
IdoUA2S units. Similar enzymatic variations including HS
oligosaccharides prepared without 6-O-sulfation by skipping the 6-O
sulfotransferase (6-OST) treatment step (step d) will yield a
number of unique octasaccharides for the generation of HS and 3OS
HS binding peptide conjugates. Suitable oligosaccharides, including
octasaccharides, can be tested for their ability to inhibit
multiple steps during a viral lifecycle, such as a herpesvirus
lifecycle (e.g., HSV-1). Attachment inhibition can be determined by
a flow cytometry binding assay. O'Donnell et al., Virology
397(2):389-98 (2010). Green HSV-1 (K26GFP) can be tested for
binding to HeLa cells at 4.degree. C. to prevent penetration. HeLa
cells can be preincubated with an octasaccharide or control
followed by the addition of green virus. Unbound virions are washed
away and flow cytometry performed to quantify the presence of a
green signal on a cell. Desai and Person, J. Virol. 72(9):7563-8
(1998).
[0099] Small Molecules
[0100] The present disclosure further contemplates HS and 3OS HS
binding peptides that are conjugated to one or more small
molecule(s). Small molecule inhibitors are well known, as
exemplified by the HS binding small molecule
Bis-2-methyl-4-amino-quinolyl-6-carbamide (Surfen; see Formula
III), and can be readily identified by methodology that is known in
the art.
##STR00008##
[0101] For example, robotic screening of small molecule libraries
can be performed to identify new inhibitors of HSV-1 gB, gC, and/or
gD functions. Baculovirus-expressed gB, gC, and/or gD can be
affinity purified and screened against one or more drug-like small
molecule libraries that provide: (1) diversity screening compounds;
(2) kinase targeted compounds; and (3) LOPAC (Library of
Pharmaceutically Active Compounds). Cytotoxicity of the hit
compounds can be tested as described in Bacsa et al., J. Gen.
Virol. 92(Pt 4):733-43 (2011). Potential gB, gC, and/or gD
binders/inhibitors can be analyzed by surface SPR and/or Bioforte
OCTET for affinity determinations as described in Tong et al., Cell
Res. (in press) (2011) and Abdiche et al., Anal. Biochem.
411(1):139-51 (2011).
[0102] Coupling of Therapeutic Compounds to HS and 3-OS HS Binding
Peptides
[0103] HS and 3-OS HS binding peptides can be coupled to one or
more therapeutic compound to generate HS and 3-OS HS binding
peptide-therapeutic compound conjugates by methodology that is well
known to those of skill in the art.
HS and 3-OS HS binding peptides can be coupled to HS
oligosaccharides to generate glycopeptides having enhanced affinity
through a multivalent effect through the binding to multiple viral
surface molecules, such as herpesvirus surface molecules. Peptides
can be linked to HS oligosaccharides via hydrazide/aldehyde
chemistry as presented in the following Synthetic Pathway II:
##STR00009##
[0104] Both C- and N-terminal hydrazide functionalization of the
peptides can be employed to achieve optimal linkage. Carboxylic
acid 17 is added to functionalize the peptide's N-terminus. Upon
cleavage from the resin and global deprotection, the peptide with
N-terminal hydrazide is obtained, which can undergo chemoselective
ligation reaction with an aldehyde functionalized HS
oligosaccharide followed by reduction to generate a glycopeptides
18. Alternatively, the peptide can be functionalized at its
C-terminus with amine 19 to introduce a hydrazide moiety, which can
subsequently be coupled with HS oligosaccharide to form a
glycopeptides in a similar manner as the formation of 18. HS and/or
3OS HS binding peptide-oligosaccharide conjugates can, optionally,
employ a linker of varying length to optimize binding affinity. The
binding of a multivalent glycopeptides to a virion can be
determined by using a Bioforte OCTET system as described in Abdiche
et al., Anal. Biochem. 411(1):139-51 (2011).
[0105] HS and 3-OS HS binding peptides can be coupled to one or
more nucleoside analog by methodology that known in the art. For
example, acyclovir can be modified with an HS or a 3-OS HS binding
peptide by esterification of acyclovir with a protected peptide
followed by acid promoted deprotection as presented in the
following Synthetic Pathway III:
Synthetic Pathway III
##STR00010##
[0107] Acyclovir has been shown to be stable under peptide
deprotection conditions. Friedrichsen et al., Eur. J. Pharm. Sci.
16(1-2):1-13 (2002). The attachment of a therapeutic compound to an
HS and 3-OS HS binding peptide will significantly enhance the
cellular uptake of the therapeutic compound. Once inside a cell,
the intracellular carboxyl esterases cleave the ester linkage
thereby releasing the therapeutic compound. De Clercq and Field,
Br. J. Pharmacol. 147(1):1-11 (2006). Depending upon the precise
application contemplated, and the nature of the therapeutic
compound, a linker may be employed between the HS and 3-OS HS
binding peptide and the therapeutic compound.
[0108] Methods for Blocking Viral Binding to and Viral Infection of
a Target Cell and for Treating Virus-Mediated Disease in a
Patient
[0109] In addition to the above-described Group 1, retro-inverso
Group 1, Group 2, and retro-inverso Group 2 peptides, and
compositions thereof, the present disclosure also provides methods
for blocking the binding of a virus to a target cell and/or the
infection of a target cell by a virus. In various embodiments, such
methods include contacting a target cell, either in vitro or in
vivo, with a Group 1 peptide, a retro-inverso Group 1 peptide, a
Group 2 peptide, and/or a retro-inverso Group 2 peptide.
[0110] In some embodiments, methods for blocking the binding or
infection of a target cell by a virus may include contacting the
target cell with a peptide that comprises at least 10 or at least
12 consecutive amino acids of the sequences XRXRXKXXRXRX (SEQ ID
NO: 2) and XXRRRRXRRRXK (SEQ ID NO: 4). In other embodiments, the
target cell may be contacted with a peptide that comprises at least
10 or at least 12 consecutive amino acids of the sequence
LRSRTKIIRIRH (SEQ ID NO: 1) and/or at least 10 or at least 12
consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO:
3). Optionally, the peptide may include one or more D-amino
acids.
[0111] In other embodiments, methods for blocking the binding or
infection of a target cell by a virus may include contacting the
target cell with a peptide that comprises at least 10 or at least
12 consecutive amino acids of the sequences XRXRXXKXRXRX (SEQ ID
NO: 8), wherein at least one amino acid is a D-amino acid, and
KXRRRXRRRRXX (SEQ ID NO: 10), wherein at least one amino acid is a
D-amino acid. It is further contemplated that by these methods, a
target cell is contacted, either in vitro or in vivo, with a
retro-inverso Group 1 and/or a retro-inverso Group 2 peptide that
comprises at least 10 or at least 12 consecutive amino acids of the
sequences XRXRXXKXRXRX (SEQ ID NO: 8), wherein all the amino acids
are D-amino acids, and KXRRRXRRRRXX (SEQ ID NO: 10), wherein all
the amino acids are D-amino acids.
[0112] Within certain aspects of these methods, the target cell can
be contacted with a peptide that comprises at least 10 or at least
12 consecutive amino acids of the sequence HRIRIIKTRSRL (SEQ ID NO:
7), wherein at least one amino acid is a D-amino acid, and/or at
least 10 or at least 12 consecutive amino acids of the sequence
KQRRRIRRRRPM (SEQ ID NO: 9), wherein at least one amino acid is a
D-amino acid. Within further aspects of these methods, the target
cell can be contacted with a peptide that comprises at least 10 or
at least 12 consecutive amino acids of the sequence HRIRIIKTRSRL
(SEQ ID NO: 7), wherein all the amino acids are D-amino acids,
and/or at least 10 or at least 12 consecutive amino acids of the
sequence KQRRRIRRRRPM (SEQ ID NO: 9), wherein all the amino acids
are D-amino acids.
[0113] Also disclosed herein are methods for the treatment of a
patient who is either infected with a virus or who is susceptible
to a viral infection. By these methods, a Group 1 and/or a Group 2
peptide that comprises at least 10 or at least 12 consecutive amino
acids of the sequences XRXRXKXXRXRX (SEQ ID NO: 2) and XXRRRRXRRRXK
(SEQ ID NO: 4) is administered to a patient subsequent or prior to
exposure and/or infection with a virus, such as a herpesvirus
(e.g., an .alpha.-herpesvirus, a .beta.-herpesvirus, and/or a
.gamma.-herpesvirus). Within certain aspects, the
.alpha.-herpesvirus is HSV-1. Within other aspects, the
.beta.-herpesvirus is cytomegalovirus (CMV). Within yet other
aspects, the .gamma.-herpesvirus is human herpesvirus-8
(HHV-8).
[0114] Also disclosed herein are methods for the treatment of a
patient who is either infected with a virus or is susceptible to a
viral infection. By these methods, a retro-inverso Group 1 and/or a
retro-inverso Group 2 peptide that comprises at least 10 or at
least 12 consecutive amino acids of the sequences XRXRXXKXRXRX (SEQ
ID NO: 8), wherein at least one amino acid is a D-amino acid, and
KXRRRXRRRRXX (SEQ ID NO: 10), wherein at least one amino acid is a
D-amino acid, is administered to a patient subsequent or prior to
exposure and/or infection with a virus, such as a herpesvirus
(e.g., an .alpha.-herpesvirus, a .beta.-herpesvirus, and/or a
.gamma.-herpesvirus). It is further contemplated by these methods,
a retro-inverso Group 1 and/or a retro-inverso Group 2 peptide that
comprises at least 10 or at least 12 consecutive amino acids of the
sequences XRXRXXKXRXRX (SEQ ID NO: 8), wherein all the amino acids
are D-amino acids, and KXRRRXRRRRXX (SEQ ID NO: 10), wherein all
the amino acids are D-amino acids, is administered to a patient
subsequent or prior to exposure and/or infection with a virus, such
as a herpesvirus (e.g., an .alpha.-herpesvirus, a
.beta.-herpesvirus, and/or a .gamma.-herpesvirus). Within certain
aspects, the .alpha.-herpesvirus is HSV-1. Within other aspects,
the .beta.-herpesvirus is cytomegalovirus (CMV). Within yet other
aspects, the .gamma.-herpesvirus is human herpesvirus-8
(HHV-8).
[0115] Within certain aspects of these methods, a peptide that
comprises at least or at least 12 consecutive amino acids of the
sequence LRSRTKIIRIRH (SEQ ID NO: 1) and/or at least 10 or at least
12 consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO:
3) can be administered to the patient.
[0116] Within certain aspects of these methods, a peptide that
comprises at least or at least 12 consecutive amino acids of the
sequence HRIRIIKTRSRL (SEQ ID NO: 7), wherein at least one amino
acid is a D-amino acid, and/or at least 10 or at least 12
consecutive amino acids of the sequence KQRRRIRRRRPM (SEQ ID NO:
9), wherein at least one amino acid is a D-amino acid, can be
administered to the patient. Within further aspects of these
methods, a peptide that comprises at least 10 or at least 12
consecutive amino acids of the sequence HRIRIIKTRSRL (SEQ ID NO:
7), wherein all the amino acids are D-amino acids, and/or at least
10 or at least 12 consecutive amino acids of the sequence
KQRRRIRRRRPM (SEQ ID NO: 9), wherein all the amino acids are
D-amino acids, can be administered to the patient.
[0117] As shown herein, the in vivo administration of a G1 and/or
G2 a peptide(s) prevents HSV-1 spread in the cornea. These findings
highlight the in vivo significance of HS and 3OS HS during viral
infection, such as during an ocular herpes infection. Thus, G1,
riG1, G2 and riG2 represent exemplary 12-mer peptides that exhibit
a unique ability to bind to critical domains within HS and/or 3OS
HS, respectively, which domains are believed to be required for
viral entry. Thus, the peptides presented herein will find broad
application methods for the treatment of diseases associated with
HS and/or 3OS HS-mediated viral infections, such as herpesvirus
infections.
[0118] Suitable routes of in vivo administration may, for example,
include oral, rectal, transmucosal, transnasal, intestinal or
parenteral delivery, including intramuscular, subcutaneous and
intramedullary injections as well as intrathecal, direct
intraventricular, intravenous, inrtaperitoneal, intranasal, or
intraocular injections. Alternately, peptide compositions may be
administered in a local rather than a systemic manner such as, for
example, via injection of the preparation directly into a specific
region of a patient's body.
[0119] More specifically, a therapeutically effective amount means
an amount of active ingredients effective to prevent, alleviate or
ameliorate symptoms of disease or prolong the survival of the
subject being treated. Determination of a therapeutically effective
amount is well within the capability of those skilled in the
art.
[0120] For any peptide composition used in the methods of the
present disclosure, the therapeutically effective amount and
toxicity can be estimated initially from in vitro assays and cell
culture assays. A suitable dose can be determined in animal models
and such information can be used to more accurately determine
useful doses in humans.
[0121] Toxicity and therapeutic efficacy of the peptides described
herein can be determined by standard pharmaceutical procedures in
vitro, in cell cultures, and/or in experimental animal models. The
data obtained from these in vitro and cell culture assays and
animal studies can be used in formulating a range of dosage for use
in a human patient. The dosage may vary depending upon the dosage
form employed and the route of administration utilized. The exact
formulation, route of administration, and dosage can be chosen by
the individual physician in view of the patient's condition. See,
e.g., Fingl et al., "The Pharmacological Basis of Therapeutics",
Ch. 1 p. 1 (1975).
[0122] Depending on the severity and responsiveness of the viral
infection to be treated, dosing can be of a single or a plurality
of administrations, with course of treatment lasting from several
days to several weeks or until cure is effected or diminution of
the viral infection is achieved. The amount of a peptide
composition to be administered will depend upon the patient being
treated, the severity of the affliction, the manner of
administration, and the judgment of the prescribing physician.
[0123] Doses of the pharmaceutical compositions will vary depending
upon the patient and upon the particular route of administration
used. Dosages can range from 0.1 to 100,000 .mu.g/kg a day, more
typically from 1 to 10,000 .mu.g/kg or from 1 to 100 .mu.g/kg of
body weight or from 1 to 10 .mu.g/kg. Doses are typically
administered from once a day to every 4-6 hours depending on the
severity of the condition. For acute conditions, it is preferred to
administer the peptide every 4-6 hours. For maintenance or
therapeutic use, it may be preferred to administer only once or
twice a day. Preferably, from about 0.18 to about 16 mg of peptide
are administered per day, depending upon the route of
administration and the severity of the condition. Desired time
intervals for delivery of multiple doses of a particular
composition can be determined by one of ordinary skill in the art
employing no more than routine experimentation.
[0124] All patents, patent application publications, and patent
applications, whether U.S. or foreign, and all non-patent
publications referred to in this specification are expressly
incorporated herein by reference in their entirety.
EXAMPLES
Example 1
Experimental Procedures
[0125] Selection of Phages Against HS and 3-OS HS by Library
Panning
[0126] A phage display library (PhD.TM.-12) expressing 12-mer
peptides fused to a minor coat protein (pIII) of a non-lytic
bacteriophage (M13) was purchased from New England Biolabs
(Cambridge, Mass.). A purified form of HS isolated from bovine
kidney was purchased from Sigma. Soluble 3OS HS modified by 3-OST-3
was prepared as previously described. Tiwari et al., J. Gen. Virol.
88:1075-1079 (2007).
[0127] Screening of the phage display library was accomplished by
an affinity selection (or bio-panning) process during which phage
populations were selected for their ability to bind HS and 3OS HS
(modified by 3-OST-3). Both targets at a concentration of 10
.mu.g/ml were used for overnight coating of wells of 96 well plates
(Nalge Nunc International, Naperville, Ill.) in a humidifier
chamber at 4.degree. C. The following day, the plates were blocked
for 1 hr at room temperature with 5 mg/ml bovine serum albumin
(BSA) in 0.1M NaHCO.sub.3 (pH 8.6) buffer. The plates were then
washed six times with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl,
0.1% [vol/vol] Tween-20). The phage library was added to the plate
at a concentration of 2.times.10.sup.11 in 100 .mu.l in TBST. The
plate was gently rocked for 1 hr at room temperature. Unbound
phages were removed by washing plates 10 times with 1 ml of TBST.
Bound phages were eluted by adding 100 .mu.l of Tris-HCl at pH 3.0.
The eluate containing bound phages was removed and the phages were
amplified in Escherichia coli ER2738 bacteria and partially
purified by polyethylene glycol (PEG) precipitation. The binding,
elution, and amplification steps were repeated using HS and 3-OST-3
modified HS as targets. Three rounds of selection were carried out
to select for binders of progressively higher specificity. Low
concentrations of detergent (Tween-20) in the early rounds resulted
in high eluate titers, and the stringency was gradually increased
with each successive round by raising Tween-concentration stepwise
to a maximum of 0.5%. This allowed selection of high affinity
binding phages. For final selection, the eluted phages were plaque
purified and titered on soft-agar plates.
[0128] Nucleotide Sequencing and Analysis
[0129] Automated nucleotide sequencing was performed to determine
the sequences of the peptides encoded by the phages (Research
Resource Center (RRC), University of Illinois at Chicago). Phage
DNA was purified according to the manufacture's protocol using
QIAGEN mini-prep kit (Valencia, Calif.). DNA sequencing was
initiated using ABI prism BigDye Terminator Kit (Applied Biosystem,
Foster City, Calif.) and the -96 gIII sequencing primer (New
England Biolabs, Cambridge, Mass.). Sequencing was performed on a
Hitachi 3100 gene analyzer (Applied Biosystems, Foster City,
Calif.) and the 36 nucleotide long DNA regions encoding the 12-mer
peptides were identified and used for peptide synthesis. The
synthetic peptides were resuspended at a concentration of 10 mM in
phosphate buffer saline (PBS) at pH 7.4, and stored at -80.degree.
C. until use. The purity of the peptides was >95% as verified by
high-performance liquid chromatography. The correct mass of the
peptides was confirmed by mass spectrometry.
[0130] Cell Culture and Viruses
[0131] The presently described examples employed a variety of cell
types, including wild-type Chinese hamster ovarian (CHO-K1), mutant
CHO-745, and CHO-I.beta.8 cells. In addition, primary cultures of
human corneal fibroblasts (CF), retinal pigment epithelial (RPE),
human conjunctival (HCjE), Vero, and HeLa cells were also used.
[0132] CHO cell lines were grown in Ham's F-12 medium (Gibco/BRL,
Carlsbad, Calif., USA) supplemented with 10% fetal bovine serum
(FBS), penicillin, and streptomycin (P/S) (Gibco/BRL). CF, HeLa,
and RPE cells were grown in Dulbecco's Modified Eagle Medium (DMEM)
supplemented with 10% FBS and P/S (Tiwari et al., J. Virol.
80:8970-8980 (2006) and Tiwari et al., FEBS J. 275:5272-5285
(2008)), and Vero cells were grown in DMEM with 5% FBS and P/S.
Cultured HCjE cells were grown as described in Akhtar et al.,
Invest. Ophthalmol. Vis. Sci. 49:4026-4035 (2008).
[0133] HSV-1 strains, including the .beta.-galactasidase expressing
recombinant HSV-1 (KOS) gL86 virus strain, the HSV-2 G strain, and
the HfM, F, MP and KOS strains were provided by P.G. Spear at
Northwestern University. Oh et al., Biochem Biophys Res Commun 391,
176-81 (2010). Green fluorescent protein (GFP) expressing HSV-1
(K26GFP), GFP expressing HHV-8 were provided by Drs. Prashant Desai
at Johns Hopkins University and J. Viera at University of
Washington. Desai and Person, J. Virol. 72:7563-7568 (1998). The
.beta.-galactasidase-expressing recombinant cytomegalovirus (CMV)
was obtained from ATCC.
[0134] HSV-1 Entry Assay
[0135] HSV-1 entry was assayed as described in Shukla et al., Cell
99:13-22 (1999). CHO-K1 cells were grown in 6-well plates to
subconfluence and then transfected with 2.5 g of expression
plasmids for gD receptors nectin-1 (pBG38), HVEM (pBec10), 3-OST-3
isoform (pDS43), or pcDNA3.1 (empty vector) using LipofectAMINE
(Gibco/BRL). At 16 h post-transfection, the cells were replated
into 96-well dishes for pre-incubation with peptides at different
concentrations for 60 min at room temperature. In parallel, natural
target cells (HeLa, CF, and RPE) were also pre-treated with the
peptides for the same duration. In all cases, unbound peptides were
removed after washing 3.times. with PBS. Thereafter, cells were
infected for 6 h with a recombinant virus, HSV-1 (KOS) gL86, at
multiple plaque forming units (PFUs) and .beta.-galactosidase
assays were performed using either a soluble substrate
o-nitrophenyl-.beta.-D-galactopyranoside (ONPG at 3.0 mg/ml;
ImmunoPure, Pierce) or X-gal (Sigma). For the soluble substrate,
the enzymatic activity was measured at 410 nm using a micro-plate
reader (Spectra Max 190, Molecular Devices, Sunnyvale, Calif.). For
the X-gal assay, cells were fixed (2% formaldehyde and 0.2%
glutaraldehyde) and permeabilized (2 mM MgCl.sub.2, 0.01%
deoxycholate, and 0.02% nonidet NP-40 (Sigma)). 1 ml of
.beta.-galactosidase reagent (1.0 mg/ml X-gal in ferricyanide
buffer) was added to each well and incubated at 37.degree. C. for
90 min before the cells were examined using bright field microscopy
under the 20.times. objectives of the inverted microscope (Zeiss
Axiovert 100 M).
[0136] Fluorescent Microscopy of Viral Entry
[0137] Cultured monolayers of HeLa and CF (approximately 10.sup.6
cells/well) were grown overnight in DMEM media containing 10% FBS
on chamber slides (Lab-Tek). One pool of each cell-type was
pre-treated with G1, G2, or a control peptide for 60 min. Cells
were then infected with HSV-1 K26GFP (Desai and Person J. Virol.
72:7563-7568 (1998)) at 50 PFU in serum-free media OptiMEM
(Invitrogen), which was followed by fixation of cells at 90 min
post-infection using fixative buffer (2% formaldehyde and 0.2%
glutaradehyde). The cells were then washed and permeabilized with 2
mM MgCl.sub.2, 0.01% deoxycholate, and 0.02% Nonidet NP-40 for 20
min. After rinsing, 10 nM rhodamine-conjugated phalloidin
(Invitrogen) was added for F-actin staining at room temperature for
45 min. The cells were washed and the images of labeled cells were
acquired using a confocal microscope (Leica, Solms, Germany) and
analyzed with MetaMorph software (Molecular Devices, Sunnyvale,
Calif.).
[0138] CMV Entry Assay
[0139] Natural target RPE cells were incubated with G1 and G2
peptides for 60 min at room temperature before the cells were
infected with .beta.-galactosidase expressing CMV (ATCC) for 8 h.
.beta.-galactosidase assays were performed using either a soluble
substrate o-nitrophenyl-.beta.-D-galactopyranoside (ONPG at 3.0
mg/ml; ImmunoPure, Pierce) or X-gal (Sigma).
[0140] HHV-8 Infection Assay
[0141] HCjE cells grown in chamber slides (Labteck) or in a 96 well
plate were pre-treated with G1, G2, or control peptides for 60 min
at room temperature followed by inoculation with recombinant
rHHV-8.152, expressing the green fluorescent protein (GFP). Viera
et al., J. Virol. 72:5182-5188 (2000). 48-hr post-infection,
GFP-positive cells were visualized under microscope (Zeiss
Axioverst 100M). HHV-8 infection was determined as relative
fluorescence units (RFU) using GENios Pro plate reader (TECAN) at
480-nm excitation and 520-nm emission frequencies. Five
measurements of negative control, positive control, and the test
samples were performed. Data were expressed as mean.+-.SD.
[0142] HSV-1 Glycoprotein Induced Cell-to-Cell Fusion Assay
[0143] CHO-K1 "effector" cells (grown in F-12 Ham, Invitrogen) were
co-transfected with plasmids expressing four HSV-1(KOS)
glycoproteins: pPEP98 (gB), pPEP99 (gD), pPEP100 (gH), and pPEP101
(gL), along with the plasmid pT7EMCLuc that expresses firefly
luciferase gene under transcriptional control of the T7 promoter.
Pertel et al., Virology 279:313-324 (2001). Wild-type CHO-K1 cells,
which express cell-surface HS but lack functional gD receptors,
were transiently transfected with HSV-1 entry receptors. Wild-type
CHO-K1 cultured cells expressing HSV-1 entry receptors or naturally
susceptible cells (human CF) considered as "target cells" were
co-transfected with pCAGT7 that expresses T7 RNA polymerase using
chicken actin promoter and CMV enhancer. Tiwari et al., FEBS
Letters 581:4468-4472 (2007). Untreated effector cells expressing
pT7EMCLuc and HSV-1 essential glycoproteins, and target cells
expressing gD receptors transfected with T7 RNA polymerase, were
used as positive controls. G2 or control peptide-treated target
cells were then co-cultivated (1:1 ratio) for 18 h with effector
cells for fusion. Activation of a reporter luciferase gene--as a
measure of cell fusion--was examined using reporter lysis assay
(Promega) at 24 hr post mixing.
[0144] Flow Cvtometry Analysis
[0145] Flow cytometry was performed to detect the effect of G2
peptide on GFP-HSV-1 binding to human CF. Monolayers of
approximately 5.times.10.sup.6 CF were pre-treated with G2 peptide
for 60 min followed by incubation with GFP-expressing HSV-1
(K26GFP) at 4.degree. C. A control peptide treated and untreated
cells were similarly incubated with HSV-1 GFP virions. Uninfected
CF were used as background negative control. GFP expression from
the viral capsid on cell surface was examined by a flowcytometer
(MoFlo).
[0146] Immunohistochemistry
[0147] BALB/c mice with pre-scarred corneal upper surface were
treated with PBS-based eye drops containing 0.5 mM G1, G2, or
control peptide followed by inoculation of HSV-1 (KOS). Mice were
sacrificed after 4 and 7 days for HSV-1 detection.
Immunohistochemistry was performed as described by Akhtar et al.,
Invest. Ophthalmol. Vis. Sci. 49:4026-4035 (2008). Briefly, tissue
sections were hydrated with distilled water and antigen retrieval
was performed using DAKO Target Retrieval Solution 10.times.
concentrate (DAKO, Carpinteria, Calif.). Nonspecific staining was
blocked using H.sub.2O.sub.2 solution for 10 minutes followed by a
protein block for 10 minutes. Sections were incubated with HSV-1 gD
specific antiserum (1:100 dilution) at room temperature for 1 hr
followed by a 40-minute incubation with the secondary antibody
(HRP-conjugated goat anti-rabbit IgG, 1:500; Sigma, St. Louis,
Mo.). Expression of gD was detected using the DAKO Envision.sup.+
kit. Confocal and differential interference contrast (DIC) image
acquisition was conducted with an SB2-AOBS confocal microscope
(Leica, Solms, Germany).
[0148] Statistics
[0149] The data presented herein are means.+-.SD of triplicate
measures of three or more experiments each performed independently.
Error bars represent one standard deviation (SD). Statistical
significance was calculated using Student's t-test. A p-value
<0.05 was considered statistically significant.
Example 2
Identification of HS and 3-OS HS Binding Peptides that Block HSV-1
Entry
[0150] Three rounds of screening of phages from a 12-mer peptide
phage display library resulted in the enrichment of phages that
specifically-bound to HS and/or to 3-OS HS. Peptide sequences from
individual plaques were deduced by determining the nucleotide
sequences of the portion of the phage genome that encoded them. The
predicted peptide sequences of about 200 plaques were determined
and sorted into two groups on the basis of their targets. A
frequently repeating peptide sequence from each group was
subsequently selected for further characterization. The two most
frequently isolated peptide sequences LRSRTKIIRIRH (designated G1
for HS binding group 1) and MPRRRRIRRRQK (designated G2 for 3-OS HS
binding group 2) were synthesized and examined for each peptide's
ability to inhibit HSV-1 infection of 3-OST-3 (FIG. 1A), nectin-1
(FIG. 1B), and HVEM (FIG. 1C) expressing CHO-K1 cells. Cells were
pre-incubated with G1, G2, or control peptide (Cp) at indicated
concentration in mM or mock treated (C) with 1.times. phosphate
saline buffer for 60 min at room temperature. After 60 min, a
.beta.-galactosidase-expressing recombinant virus HSV-1 (KOS) HSV-1
gL86 (25 pfu/cell) virus was used for infection. After 6 hr, the
cells were washed, permeabilized, and incubated with ONPG substrate
(3.0 mg/ml) for quantitation of .beta.-galactosidase activity
expressed from the input viral genome. The enzymatic activity was
measured at an optical density of 410 nm (OD.sub.410). Each value
shown is the mean of three or more determinations (.+-.SD).
[0151] Both of the G1 and G2 peptides were able to block HSV-1
entry into CHO-K1 cells expressing one of the three gD receptors
(i.e., 3-OS HS, nectin-1, and HVEM). Viral entry blockage occurred
in a dose-dependent manner and was independent of gD receptor used.
The concentration of each peptide that produced 50% of its maximum
potential inhibitory effect (IC.sub.50) ranged from 0.02 to 0.03
mM. A control phage bearing the sequence RVCGSIGKEVLG (designated
Cp) did not inhibit HSV-1 entry. None of the peptides exhibited
significant cytotoxicity (MTS assay, Promega) at .ltoreq.5 mM. The
highest concentration of peptides in the experiments presented
herein was 0.5 mM.
[0152] The ability of the G1 and G2 peptides to block HSV-1 entry
into natural target cells (HeLa and primary cultures of human CF)
was compared. A similar dosage response curve was generated when
HeLa (FIG. 2A) or CF (FIG. 2B) were pre-treated with G1 or G2
peptides during HSV-1 entry. The control peptide treated cells had
no effect on HSV-1 entry (FIG. 2). HeLa cells (FIG. 2A) and primary
cultures of human corneal fibroblasts (CF) (FIG. 2B) were tested.
Cells in 96-well plates were pre-treated for 60 min with indicated
mM concentrations of G1, G2, or Cp peptides. Mock-treated cells
(abbreviated as C) served as a control. Pretreated cells were
infected with a .beta. galactosidase-expressing recombinant virus
HSV-1 (KOS) HSV-1 gL86 (25 pfu/cell) for 6 hr. Viral entry was
quantitated as described above in reference to FIG. 1. Confirmation
of HSV-1 entry blocking activity of G1, G2, and control (Cp)
peptides on a per cell basis was obtained after cells were infected
as described above followed by X-gal (1.0 mg/ml) staining (Right
panels), which yields an insoluble blue product upon hydrolysis by
.beta.-galactosidase expressed from the input viral genomes. Dark
(blue) cells represent infected cells, uninfected cells do not show
any color. Microscopy was performed using a 20.times. objective of
Zeiss Axiovert 100.
[0153] Use of insoluble blue cell assay (X-gal as the substrate for
.beta.-galactosidase) further confirmed that the peptides were
effective in blocking infection of individual cells (FIGS. 2A and
2B, right panels). In virtually all cases, G2 peptide was slightly
more effective in blocking entry than G1.
Example 3
The Peptide Inhibitors are Also Active Against a Variety of HSV-1
Strains
[0154] This Example demonstrates that the inhibitory effect of the
G2 peptide is not limited by viral strain or serotype.
[0155] The anti-HSV properties of the G1 and G2 peptides were
evaluated against common strains of HSV-1 and HSV-2 (i.e., strains
F, G, Hfem, MP, KOS, and 17). Dean et al., Virology 199:67-80
(1994). 3-OST-3 expressing CHO Ig8 reporter cells were used that
express .beta.-galactosidase upon viral entry. Montgomery et al.,
Cell 87:427-436 (1996). Cells were pre-incubated with G1, G2, or
control peptide (Cp) and subsequently infected with the viruses. G2
or Cp control at 0.5 mM concentration was incubated for 60 min at
room temperature with a reporter CHO-Ig8 cells that express
.beta.-galactosidase upon HSV-1 entry. After incubation, the cells
were infected with HSV-1 (Pal, 17, Hfm, F, KOS, and MP) and HSV-2
(G) strains at 25 pfu/cell for 6 hr at 37.degree. C. Blockage of
viral entry was measured by ONPG assay as described in Example 2
and as presented in FIG. 1. These results, which are presented in
FIG. 3, demonstrated that G1 and G2 blocked entry of various HSV-1
strains by 70-80% at 0.5 mM concentration.
Example 4
Structural Aspects of G1 and G2 Peptides
G2 Shows More Dependence on Charged Residues
[0156] To better understand the inhibitory potential of G1 and G2
peptides, synthetic short variants (10-mer) were synthesized that
lacked the terminal non-positively charged amino acids. In case of
G1, N-terminus L and C-terminus H residues were removed. For G2,
the N-terminus flanking residues (M and P) were removed. Without
being bound by theory, it is believed that 12-mer peptides are too
short to adopt substantial secondary or tertiary structures and
that the primary structure of those peptides, which includes a
defined sequence or groupings of positively charged residues, plays
a critical role in mediating inhibition of HSV-1 entry. Synthetic
10-mer versions of G1 and G2 (shown above) were tested for blocking
HSV-1(KOS)gL86 entry into cultured CF. After 6 h, the viral entry
was measured by ONPG assay as described in FIG. 1.
[0157] As shown in FIG. 4A, the 10-mer version of G2 was very
similar to the 12-mer in its ability to block HSV-1 entry into CF.
In contrast, the 10-mer version of G1 peptide almost completely
lost its ability to block HSV-1 entry into target cells. Thus, it
is terminal L and H residues appear to be required for the
anti-HSV-1 activity of G1. G2, on the other hand, relies more on
its charged residues for its functional activity.
[0158] Alanine (A) scanning mutagenesis was performed to identify
specific amino acid residues responsible for each peptide's
function, stability, and conformation. O'Nuallain et al.,
Biochemistry 46:13049-13058 (2007). Twelve synthetic peptides were
made whereby each residue of G2 was sequentially replaced with an
alanine residue and corresponding changes in the G2 peptide were
evaluated for their ability to affect viral entry (FIG. 4B). The
location of alanine in the peptide is denoted by a number next to
it. Cp represents the control peptide and the oligomeric G2 is
listed as G2-O. FIG. 4B depicts the relative loss of inhibitory
potential upon substitution of a residue within G2 by an alanine.
Activity of each peptide was normalized against the wild-type G2,
which was kept at 1.00. Numbers higher than 1 show loss of activity
whereas a lower number represents gain of activity.
[0159] This Example demonstrates that the first four arginine (R)
residues and the last R and lysine (K) residue were essential. The
middle two amino acids could be substituted with only a moderate
loss of activity. The uncharged amino acids each tolerated
substitution with alanine. Under similar experimental conditions,
G2 oligomers (G2-O) were also examined and it was evident that they
blocked infection about 2-fold better than G1 (FIG. 4B). These
mutagenesis results demonstrate that the presence of positively
charged amino acid residues plays an important role in HSV-1 entry
blocking activity shown by G2.
Example 5
G2 Represents a Class of Broad Spectrum Anti-HS Peptides with
Activity Against Multiple Herpesviruses
[0160] This Example demonstrates that G2, but not G1, is effective
in blocking viral entry of herpesvirus family members in addition
to a-herpesviruses (e.g., HSV-1).
[0161] Many infectious viruses, including many herpesviruses,
utilize cell surface HS moieties during viral binding and entry.
Shukla and Spear, J. Clin. Invest. 108:503-510 (2001). As with
HSV-1 (an .alpha.-herpesvirus), .beta.-herpesvirus
(cytomegalovirus; CMV) and .gamma.-herpesvirus (human
herpesvirus-8; HHV-8) also use HS during cell entry and fusion. Liu
and Throp, Med. Res. Rev. 22:1-25 (2002) and Shukla and Spear, J.
Clin. Invest. 108:503-510 (2001).
[0162] In order to detect each peptide's effect on viral entry, Lac
Z-expressing reporter CMV and GFP-expressing HHV-8 viruses (Viera
et al., J. Virol. 72:5182-5188 (2000)) and their natural target
cells were employed for entry measurements. G2 peptide, but not G1
peptide, showed clear effects against CMV and HHV-8 (FIG. 5). A
monolayer of cultured RPE cells grown in a 96 well plate were
pre-treated with G1, G2, or control peptide (Cp) at 0.5 mM
concentration (FIG. 5A). A mock-treated population of RPE cells
served as positive control (abbreviated as C). After 60 min of
incubation at room temperature, the cells were infected with
.beta.-galactosidase expressing CMV reporter virus. After 8 h,
viral entry was measured as described for FIG. 1.
[0163] The effect of CMV entry blocking activity of G1, G2 or Cp
peptide for individual RPE cells was determined by X-gal staining,
which yields an insoluble blue product upon hydrolysis by
.beta.-galactosidase (FIG. 5B). Individual cells were examined
using a Zeiss Axiovert 100 microscope at 20.times. magnification.
Infected cells turn blue. These data demonstrate that G2 peptide
was effective in blocking CMV entry into RPE cells, whereas G1
peptide had no effect on viral entry. The effect of G1 was similar
to the control peptide (cp) or peptide untreated cells and the same
pattern was repeated when the effects of the peptides were examined
on a per cell basis by an X-gal assay.
[0164] The ability to block HHV-8 infection was examined in human
conjunctival epithelial (HCjE) cells, a natural target for HHV-8
infection. Human conjunctival (HCjE) cells were pre-incubated with
G1, G2 or control peptide (Cp) were infected with HHV-8 virions for
48 h at 37.degree. C. After incubation the cells were washed
thoroughly to remove unbound viruses. GFP-expression of HHV-8 was
quantitated by determining relative fluorescence units (RFU) using
a 96-well fluorescence reader (TECAN). Emission of fluorescence
indicates virus infection. These data, which are presented in FIG.
5C, are the means of triplicate measures and are representative of
3 independent experiments. Compared to G1 or Cp treated cells, G2
treated HcjE cells had relatively low GFP-expression. This
suggested that G2 was able to block infection. While G1 also
demonstrated a reduction in fluorescence in the representative case
shown in FIG. 5C, it was not found to be statistically significant
upon repeated experiments. This was confirmed by examination of
individual cells by fluorescence microscopy.
[0165] Viral replication in HCjE cells was visualized under
fluorescent microscope (Zeiss Axiovert 100) in cells that were
pretreated with G1, G2 or Cp, as described above (FIG. 5D).
Asterisks indicate significant difference from controls and/or
treatments (P<0.05, t test) and error bars represent SD. Cells
treated with G2 did not show fluorescence originating from GFP
virus replication. However, the fluorescence was more easily seen
with G1 or Cp treated cells. The results suggest that G2 is more
effective than G1 in blocking entry of divergent herpesviruses. G1
may have some activity but the virus can possibly overcome it
easily.
Example 6
Mechanism for HSV-1 Entry Inhibition by the Peptides
[0166] This Example demonstrates that G2 peptide prevents target
cell infection by herpesviruses by blocking viral HS binding sites
and, hence, viral attachment.
[0167] Cultured CF were pre-incubated with 0.5 mM G2 peptide or
control-peptide (cp) and then infected with a GFP-tagged
HSV-1(K26GFP) virus. Cells were fixed at 60 min post-infection and
stained for F-actin and the nucleus (DAPI). GFP-expressing
HSV-1(K26GFP) binding to CF in presence and absence of G2 peptides
was examined by fluorescence microscopy (FIG. 6A). CF were grown in
collagen coated chamber slides and incubated at room temperature
for 60 min with G2 (+) or control Cp (G2 (-)) peptide. This was
followed by the incubation of the cells in cold with GFP-expressing
HSV-1(K26GFP) for 30 min and washing of unbound virioins with PBS.
Cells were fixed, stained with phalloidin for F-actin and DAPI for
nuclei and examined by a fluorescence microscope (Leica, SP2). The
presence of the virus was shown by detecting GFP. These data
demonstrated that G2-treated cells resisted virus attachment as
compared to the control peptide treated cells.
[0168] To examine this effect on a population of 10.sup.5 cells,
GFP intensity as an indicator of virus binding, was measured after
incubation with the virus in cold and rigorous washing of the cells
afterwards (FIG. 6B). Relative virus binding to CF was estimated by
fluorescence measurements. Cultured CF were pre-incubated with G2
and control peptide (Cp) for 60-min before ice-cold incubation with
GFP-expressing HSV-1 virus for 30 min. Cells were washed 3 times
and viruses remaining on cell surfaces were assayed for GFP
fluorescent intensity using a fluorescence reader (Tecan). Clearly,
the binding was significantly higher in Cp-treated compared to G2
peptide treated cells.
[0169] GFP-expressing HSV-1(K26GFP) intensity as a surrogate for
virus binding was quantified in presence G2 or control peptide
(abbreviated as C) by flow cytometry (FIG. 6C). The cell/virus
incubation was performed as described above. G2 peptides block
HSV-1 replication into cultured human corneal fibroblasts (CF)
(FIG. 6D). Cultured CF were pre-incubated with G2 or mock-treated
(Cp) before infection with HSV-1(K26GFP) virus for 6 h. Viral
replications in CF were quantified 0-36 h post-infection by
measuring GFP fluorescent intensity using a fluorescence reader
(Tecan). The data shown are the means of triplicate measures and
are representative of three independent experiments. Asterisks
indicate significant difference from other treatments (P<0.01, t
test), error bars represent standard deviation (SD).
Example 7
G2 Peptide Acts by Inhibiting HSV-1 Binding to HS
[0170] This Example demonstrates, via flow cytometry detection, a
significant reduction of GFP reporter virus binding to cells
pretreated with G2.
[0171] Primary cultures of human CF pretreated with G2 peptide or
control peptide (C) were analyzed for HSV-1(K26GFP) binding. The
peptide untreated CF incubated with the virus served as a positive
control and uninfected CF served as a negative background control.
The results presented in FIG. 6 demonstrate that the virus failed
to bind G2 peptide treated CF (FIG. 6C). While the control peptide
exhibited a low level inhibitory activity, the activity of the G2
peptide was far more robust. This result confirms that G2 has the
ability to block virus attachment to cells.
[0172] To further confirm that blocking of viral attachment results
in a reduction of viral replication, GFP fluorescence was measured
as a function of time (K26GFP) (Desai and Person J. Virol.
72:7563-7568 (1998)) in both G2- and mock-treated cells. GFP
intensity (which reflects the degree of virus production) increased
significantly over time (FIG. 6D) in mock-treated cells but not
when the cells were treated with G2. These results demonstrate that
G2 blockage of virus binding results in a substantial reduction of
viral replication.
Example 8
[0173] Pretreatment of G2 Peptide to the Target Cell Significantly
Affects Cell-to-Cell Fusion and Viral Spread
[0174] This Example demonstrates that G2 not only blocks viral
attachment to a target cell, but it also inhibits viral penetration
by blocking membrane fusion.
[0175] Since G2 was isolated against 3-OS HS, which can mediate
viral penetration by promoting membrane fusion (Tiwari et al., J.
Gen. Virol. 85:805-809 (2004)), the ability of G2 to block HSV-1
glycoprotein-mediated membrane fusion was tested. Pertel et al.,
Virology 279:313-324 (2001). The same membrane fusion is used
during polykaryocytes formation and cell-to-cell spread. Tiwari et
al., FEBS Letters 581:4468-4472 (2007) and Tiwari et al.,
Biochemical and Biophysical Research Communications 390:382-387
(2009). 3-OST-3 expressing CHO-K1 cells and primary cultures of
human CF were pre-incubated with G2 peptide followed by co-culture
with effector CHO-K1 cells expressing HSV-1 glycoproteins. The
membrane fusion that ensues upon co-culturing the cells can be
estimated by a Luciferase based reporter assay. Pertel et al.,
Virology 279:313-324 (2001). Likewise, polykaryocyte formation can
be visualized by Giemsa staining.
[0176] FIG. 7 shows "effector" CHO-K1 cells expressing HSV-1
glycoproteins (gB, gD, gH-gL and T7 polymerase) that were
pre-incubated with G2 peptide (black bar) or 1.times.PBS (white
bar) (+) for 90 min. Control effector cells (T7 polymerase and gD,
gH-gL only) (-) were also pre-incubated with G2 for the same
duration. The effector cells were then mixed with primary cultures
of human corneal fibroblasts (CF; FIGS. 7A and 7B) or
3-OST-3-expressing CHO-K1 cells (FIGS. 7C and 7D) transfected with
Luciferase gene under T7 control. Membrane fusion as a surrogate
for viral spread was detected by monitoring luciferase activity
(FIGS. 7A and 7C). Relative luciferase units (RLUs) were determined
using a Sirius luminometer (Berthold detection systems). Error bars
represent standard deviations. *P<0.05, one way ANOVA.
Microscopic images of Gimesa (Fluka) stained polykaryocytes show
the preventative effect of G2's on cell fusion (FIGS. 7B and 7D).
Shown are 40.times. magnified photographs of cells undergoing
membrane fusion (Zeiss Axiovert 200).
[0177] These data show that prior treatment with G2 was very
effective in blocking membrane fusion (FIGS. 7A and 7C) and that
this ability translates into the loss of syncytia formation (FIGS.
7Ba and 7Da) compared to mock-treated cells (FIGS. 7Bb and
7Db).
Example 9
G1 and G2 Peptides Show Protective Effects Against HSV-1 Infection
of the Mouse Cornea
[0178] This Example demonstrates that anti-HS and anti-3OS HS
peptides exhibit efficacy as anti-HSV prophylactic agents and that
HS is an important co-receptor for an ocular HSV-1 infection in
vivo.
[0179] The abilities of G1 and G2 peptides against HSV-1 infection
was tested in a mouse cornea model. The cornea is known to express
many gD receptors including 3OS HS. Tiwari et al., J. Virol.
80:8970-8980 (2006) and Tiwari et al., FEBS Letters 581:4468-4472
(2007). The cornea is also an attractive target for HSV-1 infection
leading to the development of herpetic stromal keratitis (HSK), a
potential blinding disease common in developed countries including
United States. Liesegang, Cornea 20:1-13 (2001).
[0180] Immunohistochemistry was used to locate HSV-1 glycoprotein D
(gD) expression in the cornea pre-treated with either a control
peptide, G1 or G2 followed by HSV-1 infection. 100 .mu.l of G1, G2,
or Cp (control) peptide at 0.5 mM concentration was poured into the
mouse cornea as a prophylactic "eye drop" followed by an infection
with HSV-1 (KOS) at 10.sup.6 PFU. At 4 or 7 days post infection,
immunohistochemistry was performed using anti-HSV-1 gD polyclonal
antibody. In sections of cornea of mice euthanized at 4.sup.th and
7.sup.th day following pretreatment with Cp-peptide (control)
followed by virus inoculation, severe chronic inflammation combined
with significant staining for HSV-1 gD was demonstrated on day 4
(FIG. 8A). HSV-1 staining was gone by day 7, which is typical with
normal mice; however, damage to the corneal epithelium was still
evident (FIG. 8B). In contrast, virtually no HSV-1 protein
expression was detected in corneas treated with G1 or G2 peptide
and the epithelium remained intact at both 4 and 7 days post
infection (FIGS. 8B, 8C, 8E, and 8F).
Example 10
Synthesis and Characterization of riG1 and riG2 Peptides and
Peptide Conjugates
[0181] Retro-inverso forms of inhibitory peptides (e.g., riG1 and
riG2) may be tested and/or compared with corresponding wild-type
peptides to assess efficacy, stability, or other characteristics. A
variety of assays and testing methods may be used. Examples of
suitable assays/methods may include, but are not limited to, the
following methods/techniques, which can be used in isolation or in
any suitable combination to compare or assess inhibitory peptides
(e.g., Group 1 peptides and Group 2 peptides) and/or retro-inverso
forms of such peptides.
[0182] In some embodiments, human corneal epithelial (HCE) cell or
other suitable cell types may be used to assess
inhibitory/therapeutic activity by retro-inverso peptides. The
prophylactic potential of retro-inverso forms for inhibiting virus
entry may be assessed by adding the retro-inverso forms to human
corneal epithelial (HCE cells) before virus adsorption to the HCE
cells. The therapeutic potential of retro-inverso forms may be
assessed by adding the retro-inverso forms to the HCE cells after
virus adsorption to the HCE cells. Virus entry may be tested
before/after virus adsorption, and any reduction in cell entry and
infection may be determined.
[0183] In some embodiments, a VP16 translocation assay may be used
to assess virus entry deficiency. For example, confluent HCE cells
may be exposed to a virus, incubated at 37.degree. C. for one hour
(entry period), and subsequently processed to detect VP16 by
Western blotting.
[0184] In other embodiments, a plaque formation assay may be used
to assess the ability of retro-inverso forms to interfere with
cell-to-cell spread of a virus. For example, after HSV-1(17syn+)
adsorption (under prophylactic or therapeutic treatment condition),
HCE cells may be cultured in methylcellulose media to prevent free
virus spread. At 36-48 h later, the plaques may be counted and the
plaque sizes may be measured to assess cell-to-cell spread of the
virus.
[0185] In still other embodiments, the ability of retro-inverso
peptides to inactivate HSV-1 virions in solution may be tested by
exposing virus to a retro-inverso peptide, and subsequently
pelleting the virus to remove the peptide prior to infection.
Reporter virus entry and plaque assays may be used to determine
virus neutralization.
[0186] In various embodiments, retro-inverso peptides may be
assessed to determine whether they have enhanced stability in
comparison to the corresponding wild-type peptides. For example,
cells may be pretreated with retro-inverso peptides, and the
duration for which the pretreatment of cells induces resistance to
infection may be measured. After exposing the cells to peptide
(wild-type or ri-form), the cells may be infected with HSV-1(17)
for 12, 24, 36, 48, and 72 h. Plaques may be counted
thereafter.
[0187] These and/or other methods and techniques may be used to
assess the prophylactic and therapeutic potential of retro-inverso
forms of Group 1/2 peptides. In some embodiments, retro-inverso
forms may offer better efficacy and/or stability than G1/G2
peptides.
[0188] Peptide Conjugates
[0189] In further studies aimed at improving the antiviral activity
of the G1/G2, a C-terminal cysteine may be added as a coupling site
for peptide dimerization by cysteine oxidation with dimethyl
sulfoxide. HPLC chromatography may be performed to confirm dimer
formation. The dimer may be tested by various methods, such as the
methods described above, and compared with monomeric forms under
anaerobic conditions. Such a dimer may be more effective, and may
be at least 2-fold more effective, than the monomer. In some
embodiments, a retro-inverso peptide and/or dimer as described
herein may have efficacy in vitro against HSV-1 infection.
[0190] The efficacy of ACV, a replication blocking nucleoside
analog, is well established. However, combining it with an entry
blocking agent, such as G1, riG1, G2 and/or riG2 peptide, may
provide better delivery of ACV or other such treatments to the
nuclei, enhance efficacy, reduce non-specific cytotoxicity, and/or
prevent development of resistance against ACV. Thus, the
combination of ACV with an entry blocking agent such as G1, riG1,
G2, or riG2 peptide may enhance the efficacy of the ACV by several
fold. This ACV-peptide conjugate, or "super drug," may block virus
entry, membrane fusion and virus replication, and/or render the
virus virtually non-infectious and unable to spread from cell to
cell. In some embodiments, an ACV-peptide conjugate, or "super
drug" as described herein, may provide better therapeutic efficacy
than either of the components of the ACV-peptide conjugate alone.
In some embodiments, an ACV-peptide conjugate may include G2 or
riG2 in combination with ACV (e.g., G2/ACV or riG2/ACV). The G2 or
riG2 may block HSV-1 entry and cell-to-cell spread of the virions.
In an uninfected/infected mixed cell population, about 400% more
G2/ACV or riG2/ACV may be diverted to infected cells than to
uninfected cells because infected cells express higher amounts of
HS, to which G2 and riG2 bind. In some embodiments, G2 (riG2) may
guide G2/ACV (riG2/ACV) conjugate to the nuclei where ACV will
block viral replication
[0191] ACV is a chain terminator for viral DNA synthesis. In an
embodiment, ACV may be chemically coupled by esterification of ACV
with a protected G2 peptide or riG2 peptide followed by acid
promoted deprotection. ACV has been shown to be stable under
peptide deprotection condition. The attachment of G2 or riG2 to ACV
may significantly enhance the cellular uptake of ACV by infected
cells, which express higher HS. Once inside the cells, the
intracellular carboxyl esterases may cleave the ester linkage,
releasing the ACV.
[0192] The efficacy of an ACV-blocking agent combination (e.g.,
ACV-G1, ACV-riG1, ACV-G2, or ACV-riG2) may be assessed and/or
compared to that of other therapeutic agents (e.g., to ACV alone
and blocking agent alone) by the methods described herein. In some
embodiments, the efficacy of an ACV-blocking agent combination may
be assessed by adding the therapeutic agents to be tested (e.g.,
G2, riG2, ACV, G2:ACV (1:1 mixture), riG2:ACV (1:1 mixture),
G2-ACV, or riG2-ACV) to human corneal epithelial (HCE) cells either
before (prophylactic) or after (therapeutic) virus adsorption to
the HCE cells, and assessing/comparing loss or reduction of entry
and infection among the experimental groups. The VP16 translocation
assay may be used as described above to assess any entry deficiency
of the virus resulting from the application of the therapeutic
agents. The plaque formation assay may be used to assess the effect
of the therapeutic agents on the ability of the virus to replicate.
The ability of the virus to spread from infected cells to
uninfected cells may be determined by mixing HCE cells infected
with HSV-1 (MOI 0.1) for 2-6 h with uninfected cells and treating
the mixed cells with the therapeutic agents. At 36 h later, a
plaque assay may be performed. If peptide conjugation can uniquely
target ACV to infected cells, a significant loss of plaque
formation should be observed in the conjugate-treated cells. Such a
result would indicate improved therapeutic efficacy compared to the
other therapeutic agents/groups tested. Optionally, the efficacy of
G2/ACV and riG2/ACV conjugates may be tested in a therapeutic model
of HSV-1 infection of the murine cornea.
[0193] We have shown that G2 can block cell-to-cell fusion, a
phenomenon that is required for virus spread. Therefore, G2 and
riG2 peptides, which also block membrane fusion and entry, may
demonstrate therapeutic efficacy against an existing primary and/or
reactivated infection. Existing primary infections are slightly
different from reactivated infections because a primary infection
is caused by exposure to cell-free virions, whereas recurrent
infections originate from cell associated virus returning to cause
renewed symptoms. Therefore, G2 peptide, riG2 peptide, G2-ACV
conjugate and riG2-ACV conjugate may be assessed for therapeutic
efficacy in a primary infection model system, such as the system
described below. As G1 does not appear to block membrane fusion, G1
may have lower efficacy in this therapeutic model.
[0194] To cause an existing primary infection, the corneas of mice
may be treated topically with a saline solution containing 0.5 mM
of G2, riG2 or control peptide starting at one day after corneal
inoculation with HSV-1 McKrae (10.sup.4 pfu/eye) strain. The
peptide treatment may be continued daily for another four days.
Animals may be monitored daily for change in body weight, genital
inflammation, lesions, paralysis, and survival. Severely diseased
mice may be sacrificed when moribund. A quantitative severity scale
may be used for daily scoring of ocular lesion development, as
described further below (e.g., 0=no disease, 1=slight
erythema/swelling, 2=single or a few small lesions, 3=large or
fused vesicles and 4=ulcerated lesions). Lesions may be monitored
for up to 30 days. In order to obtain unbiased results, the
observers may be masked from the identity of the treated and the
placebo group. Groups of mice may be euthanized at 1, 2, 3, 4, 7,
and 30 days following the first day of treatment, the left eyes,
trigeminal ganglia (TG) and brain may be aseptically removed, and
in some cases, the tissues may be flash frozen.
[0195] HSV-1 replication kinetics, HSV-1 gene expression and HSV-1
DNA levels may be determined in the eye and TG at 1, 2, 3, 4, 7,
and 30 days after virus inoculation to evaluate the effectiveness
of G2 and riG2 peptides on already-established HSV-1 keratitis, HSV
spread from the eye to the TG, and establishment of latent
infection in TG. Peptide-treated cells may demonstrate
significantly less spread of the virus to the TG and hence, reduced
latency. A similar set of experiments may be repeated with one or
more other conjugates, such as G2-acylovir and riG2-ACV conjugates.
Such conjugates may have greater efficacy than G2, riG2 or ACV
alone.
[0196] Statistical Analysis:
[0197] Data can be assessed using independent samples T-test and
repeated measures analysis of variance (ANOVA) followed by
Scheffe's post hoc test. Differences between the means may be
considered statistically significant if P<0.05. The results may
be expressed as mean +/- standard deviation (SD) values.
Sequence CWU 1
1
12112PRTArtificial SequenceSynthetic construct 1Leu Arg Ser Arg Thr
Lys Ile Ile Arg Ile Arg His 1 5 10 212PRTArtificial
SequenceSynthetic construct 2Xaa Arg Xaa Arg Xaa Lys Xaa Xaa Arg
Xaa Arg Xaa 1 5 10 312PRTArtificial SequenceSynthetic construct
3Met Pro Arg Arg Arg Arg Ile Arg Arg Arg Gln Lys 1 5 10
412PRTArtificial SequenceSynthetic construct 4Xaa Xaa Arg Arg Arg
Arg Xaa Arg Arg Arg Xaa Lys 1 5 10 510PRTArtificial
SequenceSynthetic construct 5Arg Ser Arg Thr Lys Ile Ile Arg Ile
Arg 1 5 10 610PRTArtificial SequenceSynthetic construct 6Arg Arg
Arg Arg Ile Arg Arg Arg Gln Lys 1 5 10 712PRTArtificial
SequenceSynthetic construct 7His Arg Ile Arg Ile Ile Lys Thr Arg
Ser Arg Leu 1 5 10 812PRTArtificial SequenceSynthetic construct
8Xaa Arg Xaa Arg Xaa Xaa Lys Xaa Arg Xaa Arg Xaa 1 5 10
911PRTArtificial SequenceSynthetic construct 9Lys Gln Arg Arg Arg
Ile Arg Arg Arg Arg Met 1 5 10 1012PRTArtificial SequenceSynthetic
construct 10Lys Xaa Arg Arg Arg Xaa Arg Arg Arg Arg Xaa Xaa 1 5 10
1110PRTArtificial SequenceSynthetic construct 11Arg Ile Arg Ile Ile
Lys Thr Arg Ser Arg 1 5 10 1210PRTArtificial SequenceSynthetic
construct 12Lys Gln Arg Arg Arg Ile Arg Arg Arg Arg 1 5 10
* * * * *