U.S. patent application number 13/550029 was filed with the patent office on 2013-06-13 for treatment of viral infections.
This patent application is currently assigned to AI2 LIMITED. The applicant listed for this patent is Curtis Dobson. Invention is credited to Curtis Dobson.
Application Number | 20130150288 13/550029 |
Document ID | / |
Family ID | 30471239 |
Filed Date | 2013-06-13 |
United States Patent
Application |
20130150288 |
Kind Code |
A1 |
Dobson; Curtis |
June 13, 2013 |
TREATMENT OF VIRAL INFECTIONS
Abstract
The present invention concerns polypeptides derived from a
tandem repeat of apoE.sub.141-149 and their uses as medicaments.
The peptides may comprise the tandem repeat, and truncations
thereof, for which at least one Leucine (L) is replaced by an amino
acid with a side chain comprising at least 4 carbon atoms and at
least one Nitrogen atom. Such peptides are useful for preventing or
treating viral infections.
Inventors: |
Dobson; Curtis; (Manchester,
GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Dobson; Curtis |
Manchester |
|
GB |
|
|
Assignee: |
AI2 LIMITED
Manchester
GB
|
Family ID: |
30471239 |
Appl. No.: |
13/550029 |
Filed: |
July 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12702919 |
Feb 9, 2010 |
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13550029 |
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10580761 |
May 25, 2006 |
7691382 |
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PCT/GB2004/005360 |
Dec 17, 2004 |
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12702919 |
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Current U.S.
Class: |
514/3.7 ;
530/300; 530/326 |
Current CPC
Class: |
C07K 7/08 20130101; A61P
31/12 20180101; A61P 31/18 20180101; A61P 31/22 20180101; A61P
37/04 20180101; A61K 38/00 20130101; C07K 14/775 20130101 |
Class at
Publication: |
514/3.7 ;
530/300; 530/326 |
International
Class: |
C07K 7/08 20060101
C07K007/08 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 17, 2003 |
GB |
0329254.7 |
Claims
1-22. (canceled)
23. An isolated and purified antiviral polypeptide comprising
between 14 and 18 amino acids of the apoE.sub.141-149 tandem repeat
set forth in SEQ ID NO: 2, wherein said polypeptide comprises one
or more amino acid substitutions of an amino acid with a side chain
comprising at least 4 carbon atoms and at least one nitrogen atom
for leucine (L).
24. The polypeptide of claim 23, wherein the amino acid with a side
chain comprising at least 4 carbon atoms and at least one nitrogen
atom is arginine (R) or lysine (K).
25. The polypeptide of claim 23, wherein at least two substitutions
are made.
26. The polypeptide of claim 23, wherein at least one further
leucine amino acid is replaced with Phenylalanine (F) or is
deleted.
27. The polypeptide of claim 23, wherein an amino acid is added to
the N terminal, C terminal and/or between the ninth and tenth amino
acids of SEQ ID NO: 2.
28. An isolated and purified antiviral polypeptide comprising
YRKYRKRYYYRKYRKRYY (SEQ ID NO: 6).
29. An isolated and purified antiviral polypeptide comprising
LRKLRKLRKLRKLRKLRK (SEQ ID NO: 9).
30. A composition, comprising the polypeptide of claim 23.
31. A method of inhibiting viral replication, comprising
administering to a subject in need of such treatment a
therapeutically effective amount of the polypeptide of claim
23.
32. The method of claim 31, wherein the polypeptide is the
polypeptide of claim 24.
33. The method of claim 31, wherein the polypeptide is the
polypeptide of claim 25.
34. The method of claim 31, wherein the polypeptide is the
polypeptide of claim 26.
35. The method of claim 31, wherein the polypeptide is the
polypeptide of claim 27.
36. The method of claim 31, wherein the polypeptide is an isolated
and purified antiviral polypeptide comprising YRKYRKRYYYRKYRKRYY
(SEQ ID NO: 6).
37. The method of claim 31, wherein the polypeptide is an isolated
and purified antiviral polypeptide comprising LRKLRKLRKLRKLRKLRK
(SEQ ID NO: 9).
38. An isolated and purified antiviral polypeptide derived from the
apoE 141-149 tandem repeat set forth in SEQ ID NO: 2 selected from
the group consisting of: TABLE-US-00013 (SEQ ID NO: 56)
WRKCRKRCWWRKCRKRCW, (SEQ ID NO: 57) LRKLRKRLLWRKWRKRWW, (SEQ ID NO:
58) LRKLRKRLLLRKLRKRWW, (SEQ ID NO: 59) LRKLRKRLLWRKWRKRLL, (SEQ ID
NO: 60) WRKWRKRLLLRKLRKRLL, (SEQ ID NO: 61) WRKLRKRLLLRKLRKRLL,
(SEQ ID NO: 62) WRKWRKFFFRKWRKRWW, (SEQ ID NO: 63)
WRKWRKRWWFRKFRKRFF, (SEQ ID NO: 64) RRKRRKRRRRRKRRKRRR, and (SEQ ID
NO: 65) KRKKRKRKKKRKKRKRKK.
39. A method of inhibiting viral replication, comprising
administering to a subject in need of such treatment a
therapeutically effective amount of the polypeptide of claim 38.
Description
[0001] The present invention relates to polypeptides, derivatives
or analogues thereof, and to nucleic acids encoding the same with
anti-viral activity. The invention further provides the use of such
polypeptides, derivatives, analogues or nucleic acids as
medicaments, and also in methods of treatment.
[0002] Antiviral agents may target one of six stages of the viral
replication cycle, these being:
[0003] 1. Attachment of the virus to the cell;
[0004] 2. Penetration (or fusion of the viral membrane with the
cell membrane);
[0005] 3. Uncoating of the virus;
[0006] 4. Replication of the viral nucleic acids;
[0007] 5. Maturation of progeny virus particles; and
[0008] 6. Release of progeny virus into extracellular fluids.
[0009] Of these six stages, replication (stage 4 above) is the
target, which is most effectively influenced by conventional
antiviral therapies. Attachment of the virus to the cell is however
arguably a more attractive target, as the agent does not need to
pass into the host cell. However, this remains an area where few
successful therapies have been developed.
[0010] It is therefore one object of the present invention to
provide therapeutic agents that modulate viral attachment to
cells.
[0011] Lipoproteins (LPs) are globular macromolecular complexes
present in serum and other extracellular fluids, consisting of
lipid and protein, and are involved in the transport of lipid
around the body. They have been categorised according to their
density, with the main classes being high density lipoprotein
(HDL), low density lipoprotein (LDL), and very low density
lipoprotein (VLDL). Their proteins are referred to as
apolipoproteins, and a number of these have been described,
including apolipoproteins A, B, C, D, E, F, G, H, and J. In
addition, several sub-types of apolipoprote ins A, B and C have
been documented.
[0012] Various interactions have been described linking LPs with
viruses. These mostly involving binding of viruses to lipoproteins,
with this resulting in either diminished viral infectivity, or
conversely providing a `hitchhiker` method for the virus to enter
cells. Additionally, several viruses make use of cellular receptors
for LPs (e.g. the LDL receptor) as a means of entering cells,
although these receptors can also be released by cells as
endogenous antiviral agents (for example a soluble form of the VLDL
receptor is released into culture medium by HeLa cells and inhibits
human rhinovirus infection). Furthermore, direct binding between
certain apolipoproteins and viral proteins has also been reported.
For example: [0013] a. Hepatitis C virus core protein binds to
apolipoprotein AII: [0014] b. Hepatitis B virus surface antigen
binds apolipoprotein H; and [0015] c. Simian immunodeficiency virus
(SIV) gp32 protein, and human immunodeficiency virus (HIV) gp41
protein binds to apolipoprotein Al.
[0016] Work conducted in the laboratory of the inventor has shown
that the presence of latent herpes simplex virus type 1 (HSV1) in
brain and the possession of a particular allele of a specific
gene--the APOE-e4 allele of the APOE gene--increases the risk of
development of Alzheimer's disease (AD). Taken with the additional
finding that APOE-e4 carriers are more likely to suffer from cold
sores (which are lesions found after reactivation of HSV1 in the
peripheral nervous system), these results suggested that APOE-e4
carriers are more likely to suffer damage from HSV1 infections, and
suggests that there may be interactions between apolipoprotein E
and certain viruses (although such interactions need not
necessarily involve antiviral effects). One possible mode of
interaction between HSV1 and apoE relates to the independent
findings that both of these use cellular heparan sulphate
proteoglycan (HSPG) molecules as their initial site of binding to
cells, before subsequent attachment to secondary receptors, which
raises the possibility that competition may occur at these HSPG
sites between HSV1 and apoE containing LPs, which could affect
viral entry.
[0017] Apolipoprotein E has been shown to have effects on the
immune system (seemingly unrelated to its role in lipid metabolism)
including suppression of T lymphocyte proliferation. Interactions
between a number of peptides derived from residues 130-169 of apoE
with lymphocytes have been examined (Clay et al., Biochemistry, 34:
11142-11151 (1995)). The region consisting of apoE residues 141-149
are predicted to be particularly important. Similar interactions of
such peptides have been described in neuronal cell lines.
[0018] WO 94/04177 discloses that administration of particles
containing lipid and amphipathic helical peptides allows clearance
of toxins produced by microorganisms, and may increase the
effectiveness of antibacterial drugs via an effect on bacterial
membranes. However, there is no suggestion that such apoA-derived
peptide containing particles may be used as antiviral medicines. It
is also not clear whether administration of the peptides in
particles, which is a key component of the disclosed development
(whether the particles are formed before administration or
endogenously), would result in effective utilisation of any
antiviral action of either component of the particle.
[0019] An amphipathic helical peptide derived from apoA (described
by Ananatharamiah in Meth. Enz., 128: 627-647 (1986)) has been
shown to prevent fusion of viral membranes with cell membranes, and
furthermore prevent the fusion of membranes of infected cells
(Srinivas et al. J. Cellular Biochem., 45: 224-237 (1991)). The
peptide was also effective at preventing fusion for both HSV1 and
HIV (Owens et al., J. Clin. Invest., 86: 1142-1150 (1990)).
However, the peptide had no effect at all on attachment of HSV1 at
least to cells (Srinivas et al. supra).
[0020] Azuma et al. have reported that peptide derivatives of apoE
have a strong antibacterial action, comparable with that of
gentamicin (Peptides, 21: 327-330 (2000)). ApoE 133-162 was the
most effective, with apoE 134-155 having little effect.
[0021] In the light of the research described above, the inventor
conducted experiments to evaluate whether or not peptides derived
from ApoE (which are capable of forming helices) have antiviral
activity. He found that a tandem repeat of a peptide fragment of
ApoE, apoE.sub.141-149 (i.e. 2.times. LRKLRKRLL--SEQ ID No.1), did
indeed have an antiviral action. While the inventor does not wish
to bound by any hypothesis, he believes that this fragment prevents
the attachment of virus particles to cells, resulting in a decrease
in the infectivity of the virus as measured by a plaque reduction
assay technique. Example 1 illustrates how the peptide is effective
against viruses such as HSV1, HSV2 and HIV. Accordingly, this
peptide may be effective when applied to virus directly, or when
applied to virus in the presence of cells, and therefore the
peptide can be used to inactivate free virus particles long before
they reach their target cells.
[0022] In the light of the data generated for a tandem repeat of
apoE.sub.141-149 (i.e. 2.times. LRKLRKRLL--SEQ ID No.1), the
inventors decided to investigate other fragments of apolipoproteins
for antiviral activity.
[0023] According to a first aspect of the present invention, there
is provided a polypeptide, derivative or analogue thereof
comprising a tandem repeat of apoE.sub.141-149 of SEQ ID No 2 or a
truncation thereof, characterised in that at least one Leucine (L)
residue of SEQ ID No. 2 is replaced by an amino acid with a side
chain comprising at least 4 carbon atoms and at least one nitrogen
atom.
[0024] By "a tandem repeat of apoE.sub.141-149 of SEQ ID No. 2" we
mean the peptide with the amino acid sequence: LRKLRKRLLLRKLRKRLL.
The tandem repeat is referred to herein as apoE.sub.141-149dp or
apoE.sub.141-149r. This peptide is also assigned the code GIN 1 or
GIN1p (wherein p signifies N terminal protection (e.g. by an acetyl
group), and C terminal protection (e.g. by an amide group)).
[0025] By "a truncation thereof" we mean that the 18mer of SEQ ID
No. 2 is reduced in size by removal of amino acids. The reduction
of amino acids may be by removal of residues from the C or N
terminal of the peptide or may be by deletion of one or more amino
acids from within the core of the peptide (i.e. amino acids 2-17 of
SEQ ID No. 2).
[0026] By "derivative or analogue thereof" we mean that the amino
acids residues are replaced by residues (whether natural amino
acids, non-natural amino acids or amino acid mimics) with similar
side chains or peptide backbone properties. Additionally the
terminals of such peptides may be protected by N and C-terminal
protecting groups with similar properties to acetyl or amide
groups.
[0027] The inventor conducted exhaustive experiments to assess the
antiviral activity of peptides from apolipoproteins and derivatives
thereof. Peptides and derivatives from ApoE were a particular
focus. To the inventors surprise they found that most of the
peptides tested had little or no antiviral effect. The surprising
exceptions were peptides according to the first aspect of the
invention. Examples 2-7 illustrate the efficacy of the peptides
according to the invention compared to a tandem repeat of
apoE.sub.141-149 and other peptides derived from
apolipoproteins.
[0028] The inventor has identified that Tryptophan (W), Arginine
(R) or Lysine (K) may be substituted for Leucine in
apoE.sub.141-149 tandom repeats and that such peptides have
surprising antiviral activity. The inventor appreciated that these
amino acids had side chains comprising at least 4 carbons and also
containing a nitrogen atom. Accordingly it is preferred that the
amino acid used to replace the leucine is Tryptophan (W), Arginine
(R) or Lysine (K) or derivatives thereof in the peptide according
to the first aspect of the invention.
[0029] The inventor has found that peptides in which at least one L
has been substituted with a W have particular antiviral activity.
It is therefore most preferred that peptides according to the first
aspect of the invention comprise a polypeptide, derivative or
analogue thereof comprising a tandem repeat of apoE.sub.141-149 of
SEQ ID No 2 or a truncation thereof, characterised in that at least
one Leucine (L) residue of SEQ ID No. 2 is replaced by a Tryptophan
(W).
[0030] During development work the inventor noted that W
substitutions may be expected to increase the likelihood of the
peptide forming an alpha helix and wondered if this may explain the
antiviral efficacy of compounds according to the first aspect of
the invention. However, he does not believe this explains the
surprising efficacy of peptides according to the invention. This is
because a number of alternative substitutions would be expected to
increase alpha helix formation (e.g see Table 1 for calculation of
likelihood of various L substituted peptides forming an alpha
helix). However the likelihood of forming a helix (table 1) does
not correlate with the antiviral activity of peptides according to
the present invention (see Example 5).
TABLE-US-00001 TABLE 1 Predicted proportion of molecules of various
peptides forming alpha-helix in aqueous 0.15M NaCl buffer at
37.degree. C. (%) (using AGADIR secondary structure prediction
software available from http://www.embl-heidelberg.de/
Services/serrano/agadir/agadir-start.html) Amino Acid Substitution
Sequence of peptide % helix E, Glu ERKERKREEERKERKREE 6.24 A, Ala
ARKARKRAAARKARKRAA 1.85 D, Asp DRKDRKRDDDRKDRKRDD 1.59 W, Trp
WRKWRKRWWWRKWRKRWW 1.47 M, Met MRKMRKRMMMRKMRKRMM 1.01 Y, Tyr
YRKYRKRYYYRKYRKRYY 0.8 F, Phe FRKFRKRFFFRKFRKRFF 0.79 I, Ile
IRKIRKRIIIRKIRKRII 0.6 Q, Gln QRKQRKRQQQRKQRKRQQ 0.55 No swap
0.51
[0031] The inventors have also noted: [0032] 1. The increase from
the W substitution is very small (0.51% of GIN 1p molecules will
form a helix, which increases marginally to 1.47% of the W
substituted peptide); and [0033] 2. A number of other substitutions
would be predicted to increase the proportion of molecules forming
an alpha helix at any one time. For instance, substituting L for E
or A increases the likelihood of forming an alpha-helix beyond that
of a W substitution (to 6.24% and 1.87% respectively). However,
both of these substitutions in fact abolished antiviral activity
(e.g. see peptide GIN39 in Example 3 or Example 5).
[0034] Therefore there is no correlation between likelihood of
forming an alpha helix, and the strength of antiviral activity for
`L-substituted` peptides according to the invention.
[0035] The efficacy of peptides according to the invention is all
the more surprising because substitution of L (Leucine) with amino
acids according to the first aspect of the invention will make the
peptide less amphipathic. (Table 2 illustrates the accepted order
of hydrophobicity of amino acids). A skilled person may actually
suspect that making a peptide more amphipathic would confer
antiviral character. Therefore, unexpectedly, substitutions
according to the invention of SEQ ID No. 2 result in a significant
increase in their antiviral activity.
TABLE-US-00002 TABLE 2 Hydrophobicity of Amino Acids Phe > Leu =
Ile > Tyr = Trp > Val > Met > Pro > Cys > Ala
> Gly > Thr > Ser > Lys > Gln > Asn > His >
Glu > Asp > Arg
[0036] As discussed in more detail below, SEQ ID No 2 may be
manipulated according to the first aspect of the invention with a
number of different substitutions and deletions to make peptides
with antiviral activity. However, it is preferred that the
polypeptide according to the first aspect of the invention has at
least two W, R or K substitutions, and more preferably three or
more W, R or K substitutions.
[0037] In addition to one or more L substitutions with W, R or K,
it is preferred that at least one further amino acid (preferably at
least one further leucine residue) is replaced with Asparagine (N),
Tyrosine (Y), Cysteine (C), Methionine (M), Phenylalanine (F),
Isoleucine (I), Glutamine (Q) or Histidine (H). It is particularly
preferred that such a further substitution is Y or C.
[0038] The substituted polypeptide may comprise 18 amino acids (or
derivatives thereof) and thereby correspond to the full length of
SEQ ID No. 2. However the inventors have surprisingly found that
truncated peptides based on SEQ ID No.2 also have efficacy as
antiviral agents. Accordingly preferred peptides or derivatives
thereof may have less than 18 amino acids. For instance some
peptides according to the first aspect of the invention may be 17,
16, 15, 14, 13, 12, 11, 10 or less amino acids in length.
[0039] Peptides, and derivatives thereof, according to the present
invention preferably have an efficacy for inhibiting viral growth
such that their IC50 value is 30 .mu.M or less. It is preferred
that the IC50 value is 20 .mu.M or less and more preferred that the
IC50 value is 10 .mu.M or less.
[0040] Preferred peptides have similar IC.sub.50 values between
viral species. For instance preferred peptides have similar
IC.sub.50 values for inhibiting HSV 1, HSV2 and HIV growth.
[0041] It will be appreciated that modified amino acids may be
substituted into the tandem repeat of apoE.sub.141-149 with a
number of amino acid variants that may be known to those skilled in
the art Such peptides will still have antiviral activity provided
that the modification does no significantly alter its chemical
characteristics. For instance, hydrogens on the side chain amines
of R or K may be replaced with methylene groups
(--NH.sub.2.fwdarw.--NH(Me) or --N(Me).sub.2).
[0042] Preferred peptides according to the first aspect of the
invention have the amino acids sequence:
(a) WRKWRKRWWWRKWRKRWW (SEQ ID No. 3). This peptide corresponds to
the full length tandem repeat with all Leucines substituted for
Tryptophan residues. This peptide is designated GIN 7 when referred
to herein. (b) WRKWRKRWRKWRKR (SEQ ID No. 4). This peptide
corresponds to the full length tandem repeat with all Leucines
substituted for Tryptophan residues and truncated by the excision
of amino acids 9, 10, 17 and 18. This peptide is designated GIN 32
when referred to herein. (c) WRKWRKRWWLRKLRKRLL (SEQ ID No. 5).
This peptide corresponds to the full length tandem repeat with a
subset of Leucines substituted for tryptophan residues. This
peptide is designated GIN 34 when referred to herein. (d)
WRKWRKRWWRKWRKRWW (SEQ ID No. 52). This peptide corresponds to SEQ
ID No. 3 with the W residue at position 9 deleted. This peptide is
designated MU 58 when referred to herein. (e) WRKWRKRWRKWRKRW (SEQ
ID No. 53). This peptide corresponds to SEQ ID No. 3 with the W
residues at position 9, 10 and 18 deleted. This peptide is
designated MU 59 when referred to herein. (f) WRKWRKRWWFRKWRKRWW
(SEQ ID No. 54). This peptide corresponds to SEQ ID No. 3 with the
W residue at position 10 substituted with an F. This peptide is
designated MU 60 when referred to herein. (g) WRKWRKRWFFRKWRKRFF
(SEQ ID No. 55). This peptide corresponds to SEQ ID No. 3 with the
W residues at positions 9, 10, 17 and 18 substituted with F
residues. This peptide is designated MU 61 when referred to herein.
(h) WRKCRKCRCWWRKCRCW (SEQ ID No. 56). This peptide corresponds to
SEQ ID No. 3 with the W residues at positions 4, 8, 13 and 17
substituted with C residues. This peptide is designated MU 68 when
referred to herein. (i) LRKLRKRLLWRKWRKRWW (SEQ ID No. 57). This
peptide corresponds to SEQ ID No. 2 with the L residues at
positions 10, 13, 17 and 18 substituted with W residues. This
peptide is designated MU 111 when referred to herein. (j)
LRKLRKRLLLRKLRKRWW (SEQ ID No. 58). This peptide corresponds to SEQ
ID No. 2 with the L residues at positions 17 and 18 substituted
with W residues. This peptide is designated MU 112 when referred to
herein. (k) LRKLRKRLLWRKWRKRLL (SEQ ID No. 59). This peptide
corresponds to SEQ ID No. 2 with the L residues at positions 10 and
13 substituted with W residues. This peptide is designated MU 113
when referred to herein. (l) WRKWRKRLLLRKLRKRLL (SEQ ID No. 60).
This peptide corresponds to SEQ ID No. 2 with the L residues at
positions 1 and 4 substituted with W residues. This peptide is
designated MU 114 when referred to herein. (m) WRKLRKRLLLRKLRKRLL
(SEQ ID No. 61). This peptide corresponds to SEQ ID No. 2 with the
L residue at position 1 substituted with W residues. This peptide
is designated MU 115 when referred to herein. (n) WRKWRKFFFRKWRKRWW
(SEQ ID No. 62). This peptide corresponds to SEQ ID No. 3 with the
W residues at positions 8, 9 and 10 substituted with F residues and
the R residue at position 7 deleted. This peptide is designated MU
116 when referred to herein. (o) WRKWRKRWWFRKFRKRFF (SEQ ID No.
63). This peptide corresponds to SEQ ID No. 3 with the W residues
at positions 10, 13, 17 and 18 substituted with F residues. This
peptide is designated MU 117 when referred to herein. (p)
RRKRRKRRRRRKRRKRRR (SEQ ID No. 64). This peptide corresponds to the
full length tandem repeat with all Leucines substituted for
Arginine (R) residues. This peptide is designated MU 16 when
referred to herein. (q) KRKKRKRKKKRKKRKRKK (SEQ ID No. 65). This
peptide corresponds to the full length tandem repeat with all
Leucines substituted for Lysine (K) residues. This peptide is
designated MU 18 when referred to herein.
[0043] The inventor has also appreciated that peptides may be
employed according to the invention that comprise more than just a
simple dimer tandem repeat of ApoE.sub.141-149 or a truncation
thereof. For instance, peptides comprising a trimer or greater
number of repeats may be employed as antiviral agents.
[0044] In a further embodiment of the invention, antiviral peptides
may be synthesised that comprise a peptide as defined above to
which further amino acids have been added. For instance, one, two,
three or more amino acids may be added to the C or N terminals of a
peptide derived from SEQ ID No. 2. Alternatively the peptide may
comprise a tandem repeat of a peptide that is larger than the nine
amino acids of SEQ ID No. 1. Such peptides may have amino acids
added to the N terminal, C terminal and/or between the 9.sup.th and
10.sup.th amino acids of SEQ ID No. 2. It is most preferred that
the amino acid is added to C terminal and also between the 9.sup.th
and 10th amino acids of SEQ ID No. 2. It will be appreciated that
such peptides may then be modified as described above for peptides
derived from SEQ ID No. 2. By way of example WRKWRKRWWRWRKWRKRWWR
(SEQ ID No. 66) represents another preferred peptide according to
the present invention. This peptide corresponds to the full length
tandem repeat of ApoE.sub.141-150 (i.e. a tandom repeat of
LRKLRKRLLR--SEQ ID No. 67) with all Leucines substituted for
Tryptophan residues. This peptide is designated MU 83 when referred
to herein.
[0045] During the development of derivatives of tandem repeats of
apoE.sub.141-149 according to the first aspect of the invention, it
was appreciated that truncations of SEQ ID No.2, and variants
thereof, also had surprising antiviral activity. These include:
LRKLRKRLLLRKLRK (SEQ ID No. 7). This peptide corresponds to a
truncated form of the full length tandem repeat with residues 16,
17 and 18 deleted. This peptide has the advantage that the peptide
is shorter than GIN 1 and is therefore cheaper to manufacture. This
peptide is designated GIN 4 when referred to herein. LRKLRKRLRKLRKR
(SEQ ID No. 8). This peptide corresponds to the full length tandem
repeat truncated by the excision of amino acids 9, 10, 17 and 18.
This peptide is designated GIN 8 when referred to herein.
LRKLRKLRKLRKLRKLRK (SEQ ID No. 9). This peptide corresponds to a
variation of the full length tandem repeat comprising a repeat of
the LRK motif. This peptide is designated GIN 9 when referred to
herein. Furthermore YRKYRKRYYYRKYRKRYY (SEQ ID No. 6) was found to
be effect as an antiviral agent. This peptide corresponds to the
full length tandem repeat of apoE.sub.141-149 with all Leucines
substituted for Tyrosine residues. This peptide is designated GIN
41 when referred to herein.
[0046] According to a second aspect of the invention there is
provided a polypeptide, derivative or analogue thereof according to
the first aspect of the invention or a peptide of SEQ ID No. 6, 7,
8 or 9 for use as a medicament.
[0047] According to a third aspect of the invention there is
provided the use of a polypeptide, derivative or analogue thereof
according to the first aspect of the invention or a peptide of SEQ
ID No. 6, 7, 8 or 9 for the manufacture of a medicament for
treating viral infections.
[0048] It will be appreciated that the therapeutic effects of
polypeptides, derivatives or analogues according to the first
aspect of the invention may also be mediated "indirectly" by agents
that increase the activity of such polypeptides, derivatives or
analogues. The present invention provides the first medical use of
such agents.
[0049] Thus, according to a fourth aspect of the invention, there
is provided an agent capable of increasing the biological activity
of a polypeptide, derivative or analogue according to the first
aspect of the invention for use as a medicament.
[0050] Agents capable of increasing the biological activity of
polypeptides, derivatives or analogues according to the invention
may achieve their effect by a number of means. For instance, such
agents may increase the expression of such polypeptides,
derivatives or analogues. Alternatively (or in addition) such
agents may increase the half-life of polypeptides, derivatives or
analogues according to the invention in a biological system, for
example by decreasing turnover of the polypeptides, derivatives or
analogues.
[0051] Due to their increased biological activity polypeptides,
derivatives or analogues according to the first three aspects of
the invention are of utility as antiviral agents.
[0052] Polypeptides, derivatives or analogues according to the
first, second and third aspects of the invention may be used in the
treatment of a number of viral infections. The virus may be any
virus, and particularly an enveloped virus. Preferred viruses are
poxviruses, iridoviruses, togaviruses, or toroviruses. A more
preferred virus is a filovirus, arenavirus, bunyavirus, or a
rhabdovirus. An even more preferred virus is a paramyxovirus or an
orthomyxovirus. It is envisaged that virus may preferably include a
hepadnavirus, coronavirus, flavivirus, or a retrovirus. Preferably,
the virus includes a herpesvirus or a lentivirus. In preferred
embodiments, the virus may be Human Immunodeficiency Virus (HIV),
Human herpes simplex virus type 2 (HSV2), or Human herpes simplex
virus type 1 (HSV1).
[0053] Polypeptides, derivatives or analogues according to the
first, second and third aspects of the invention may be used to
treat viral infections as a monotherapy (i.e. use of the compound
alone) or in combination with other compounds or treatments used in
antiviral therapy (e.g. acyclovir, gangcylovir, ribavirin,
interferon, anti-HIV medicaments including nucleoside, nucleotide
or non-nucleoside inhibitors of reverse transcriptase, protease
inhibitors and fusion inhibitors.)
[0054] The polypeptides, derivatives or analogues may be used as a
prophylactic (to prevent the development of a viral infection) or
may be used to treat existing infections.
[0055] Derivatives of polypeptides according to the invention may
include derivatives that increase or decrease the polypeptide's
half-life in vivo. Examples of derivatives capable of increasing
the half-life of polypeptides according to the invention include
peptoid derivatives of the polypeptides, D-amino acid derivatives
of the polypeptides, and peptide-peptoid hybrids.
[0056] Polypeptides according to the invention may be subject to
degradation by a number of means (such as protease activity in
biological systems). Such degradation may limit the bioavailability
of the polypeptides and hence the ability of the polypeptides to
achieve their biological function. There are wide ranges of
well-established techniques by which peptide derivatives that have
enhanced stability in biological contexts can be designed and
produced. Such peptide derivatives may have improved
bioavailability as a result of increased resistance to
protease-mediated degradation. Preferably a peptide derivative or
analogue suitable for use according to the invention is more
protease-resistant than the peptide from which it is derived.
Protease-resistance of a peptide derivative and the peptide from
which it is derived may be evaluated by means of well-known protein
degradation assays. The relative values of protease resistance for
the peptide derivative and peptide may then be compared.
[0057] Peptoid derivatives of the peptides of the invention may be
readily designed from knowledge of the structure of the peptide
according to the first aspect of the invention. Commercially
available software may be used to develop peptoid derivatives
according to well-established protocols.
[0058] Retropeptoids, (in which all amino acids are replaced by
peptoid residues in reversed order) are also able to mimic
antiviral peptides derived from apolipoproteins. A retropeptoid is
expected to bind in the opposite direction in the ligand-binding
groove, as compared to a peptide or peptoid-peptide hybrid
containing one peptoid residue. As a result, the side chains of the
peptoid residues are able point in the same direction as the side
chains in the original peptide.
[0059] A further embodiment of a modified form of polypeptides
according to the invention comprises D-amino acid forms of the
polypeptides. The preparation of peptides using D-amino acids
rather than L-amino acids greatly decreases any unwanted breakdown
of such an agent by normal metabolic processes, decreasing the
amounts of agent which need to be administered, along with the
frequency of its administration.
[0060] The polypeptides, analogues, or derivatives of the invention
represent products that may advantageously be expressed by
biological cells.
[0061] Thus, the present invention also provides, in a fifth
aspect, a nucleic acid sequence encoding a polypeptide, derivative
or analogue according to the first aspect of the invention.
[0062] The nucleic acids encoding apoE.sub.141-149 has the DNA
sequence cttcgtaaacttcgtaaacgtcttctt (SEQ ID. No. 10) whereas GIN 1
has the sequence
cttcgtaaacttcgtaaacgtcttcttcttcgtaaacttcgtaaacgtcttctt (SEQ ID. No.
11).
[0063] Preferred nucleic acids according to the fifth aspect of the
invention encode the peptides identified herein as GIN 4, 7, 8, 9,
32, 34 and 41 have the following respective sequences:
TABLE-US-00003 (SEQ ID No. 16) cttcgtaaac ttcgtaaact tcgtaaactt
cgtaaacttc gtaaacttcg taaa; (SEQ ID No. 12) tggcgtaaat ggcgtaaacg
ttggtggtgg cgtaaatggc gtaaacgttg gtgg; (SEQ ID No. 17) cttcgtaaac
ttcgtaaacg tcttcgtaaa cttcgtaaac gt; (SEQ ID No. 18) cttcgtaaac
ttcgtaaact tcgtaaactt cgtaaacttc gtaaacttcg taaa; (SEQ ID No. 13)
tggcgtaaat ggcgtaaacg ttggcgtaaa tggcgtaaac gt; (SEQ ID No. 14)
tggcgtaaat ggcgtaaacg ttggtggctt cgtaaacttc gtaaacgtct tctt,; and
(SEQ ID No. 15) tatcgtaaat atcgtaaacg ttattattat cgtaaatatc
gtaaacgtta ttat.
[0064] A skilled person will appreciate that the nucleic acid
sequence of other preferred peptides according to the present
invention may be readily generated.
[0065] It will be appreciated that, due to redundancy in the
genetic code, a nucleic acid sequence in accordance with the fifth
aspect of the invention may vary from the naturally occurring ApoE
gene providing a codon encodes a polypeptide, derivative or
analogue thereof in accordance with the first aspect of the
invention.
[0066] It will be appreciated that polypeptides, derivatives and
analogues according to the invention represent favourable agents to
be administered by techniques involving cellular expression of
nucleic acid sequences encoding such molecules. Such methods of
cellular expression are particularly suitable for medical use in
which the therapeutic effects of the polypeptides, derivatives and
analogues are required over a prolonged period.
[0067] Thus according to a sixth aspect of the present invention
there is provided a nucleic acid sequence according to the fifth
aspect of the invention for use as a medicament.
[0068] The nucleic acid may preferably be an isolated or purified
nucleic acid sequence. The nucleic acid sequence may preferably be
a DNA sequence.
[0069] The nucleic acid sequence may further comprise elements
capable of controlling and/or enhancing its expression. The nucleic
acid molecule may be contained within a suitable vector to form a
recombinant vector. The vector may for example be a plasmid, cosmid
or phage. Such recombinant vectors are highly useful in the
delivery systems of the invention for transforming cells with the
nucleic acid molecule.
[0070] Recombinant vectors may also include other functional
elements. For instance, recombinant vectors can be designed such
that the vector will autonomously replicate in the cell. In this
case elements that induce nucleic acid replication may be required
in the recombinant vector. Alternatively, the recombinant vector
may be designed such that the vector and recombinant nucleic acid
molecule integrates into the genome of a cell. In this case nucleic
acid sequences, which favour targeted integration (e.g. by
homologous recombination) are desirable. Recombinant vectors may
also have DNA coding for genes that may be used as selectable
markers in the cloning process.
[0071] The recombinant vector may also further comprise a promoter
or regulator to control expression of the gene as required.
[0072] The nucleic acid molecule may (but not necessarily) be one,
which becomes incorporated in the DNA of cells of the subject being
treated. Undifferentiated cells may be stably transformed leading
to the production of genetically modified daughter cells (in which
case regulation of expression in the subject may be required e.g.
with specific transcription factors or gene activators).
Alternatively, the delivery system may be designed to favour
unstable or transient transformation of differentiated cells in the
subject being treated. When this is the case, regulation of
expression may be less important because expression of the DNA
molecule will stop when the transformed cells die or stop
expressing the protein (ideally when the required therapeutic
effect has been achieved).
[0073] The delivery system may provide the nucleic acid molecule to
the subject without it being incorporated in a vector. For
instance, the nucleic acid molecule may be incorporated within a
liposome or virus particle. Alternatively a "naked" nucleic acid
molecule may be inserted into a subject's cells by a suitable means
e.g. direct endocytotic uptake.
[0074] The nucleic acid molecule may be transferred to the cells of
a subject to be treated by transfection, infection, microinjection,
cell fusion, protoplast fusion or ballistic bombardment. For
example, transfer may be by ballistic transfection with coated gold
particles, liposomes containing the nucleic acid molecule, viral
vectors (e.g. adenovirus) and means of providing direct nucleic
acid uptake (e.g. endocytosis) by application of the nucleic acid
molecule directly.
[0075] It will be appreciated that the polypeptides, agents,
nucleic acids or derivatives according to the present invention may
be used in a monotherapy (i.e. use of polypeptides, agents, nucleic
acids or derivatives according to the invention alone to prevent
and/or treat a viral infection). Alternatively, polypeptides,
agents, nucleic acids or derivatives according to the invention may
be used as an adjunct, or in combination with, known therapies.
[0076] Polypeptides, agents, nucleic acids or derivatives according
to the invention may be combined in compositions having a number of
different forms depending, in particular on the manner in which the
composition is to be used. Thus, for example, the composition may
be in the form of a powder, tablet, capsule, liquid, ointment,
cream, gel, hydrogel, aerosol, spray, micelle, transdermal patch,
liposome or any other suitable form that may be administered to a
person or animal. It will be appreciated that the vehicle of the
composition of the invention should be one which is well tolerated
by the subject to whom it is given, and is preferably adapted to
enable delivery of the polypeptides, agents, nucleic acids or
derivatives to the target tissue.
[0077] Compositions comprising polypeptides, agents, nucleic acids
or derivatives according to the invention may be used in a number
of ways. For instance, oral administration may be required in which
case the compound may be contained within a composition that may,
for example, be ingested orally in the form of a tablet, capsule or
liquid. Alternatively the composition may be administered by
injection into the blood stream. Injections may be intravenous
(bolus or infusion) or subcutaneous (bolus or infusion). The
compounds may be administered by inhalation (e.g.
intranasally).
[0078] Compositions may be formulated for topical use. For
instance, ointments may be applied to the skin, areas in and around
the mouth or genitals to treat specific viral infections. Topical
application to the skin is particularly useful for treating viral
infections of the skin or as a means of transdermal delivery to
other tissues. Intravaginal administration is effective for
treating sexually transmitted diseases (including AIDS).
[0079] Polypeptides, agents, nucleic acids or derivatives may also
be incorporated within a slow or delayed release device. Such
devices may, for example, be inserted on or under the skin, and the
compound may be released over weeks or even months. Such devices
may be particularly advantageous when long term treatment with a
polypeptide, agent, nucleic acid or derivative according to the
invention is required and which would normally require frequent
administration (e.g. at least daily injection).
[0080] It will be appreciated that the amount of a polypeptide,
agent, nucleic acid or derivative that is required is determined by
its biological activity and bioavailability which in turn depends
on the mode of administration, the physicochemical properties of
the polypeptide, agent, nucleic acid or derivative employed and
whether the polypeptide, agent, nucleic acid or derivative is being
used as a monotherapy or in a combined therapy. The frequency of
administration will also be influenced by the above-mentioned
factors and particularly the half-life of the polypeptide, agent,
nucleic acid or derivative within the subject being treated.
[0081] Optimal dosages to be administered may be determined by
those skilled in the art, and will vary with the particular
polypeptide, agent, nucleic acid or derivative in use, the strength
of the preparation, the mode of administration, and the advancement
of the disease condition. Additional factors depending on the
particular subject being treated will result in a need to adjust
dosages, including subject age, weight, gender, diet, and time of
administration.
[0082] It will be appreciated that a skilled person will be able to
calculate required doses, and optimal concentrations of the
peptides at a target tissue, based upon the pharmacokinetics of the
peptides and in particular the IC.sub.50 values given in the
Examples.
[0083] Generally, a daily dose of between 0.01 .mu.g/kg of body
weight and 0.5 g/kg of body weight of polypeptides, agents, nucleic
acids or derivatives according to the invention may be used for the
prevention and/or treatment of a viral infection, depending upon
which specific polypeptide, agent, nucleic acid or derivative is
used. More preferably, the daily dose is between 0.01 mg/kg of body
weight and 200 mg/kg of body weight, and most preferably, between
approximately 1 mg/kg and 100 mg/kg.
[0084] Known procedures, such as those conventionally employed by
the pharmaceutical industry (e.g. in vivo experimentation, clinical
trials, etc.), may be used to establish specific formulations of
polypeptides, agents, nucleic acids or derivatives according to the
invention and precise therapeutic regimes (such as daily doses of
the polypeptides, agents, nucleic acids or derivatives and the
frequency of administration).
[0085] Daily doses may be given as a single administration (e.g. a
single daily injection). Alternatively, the polypeptide, agent,
nucleic acid or derivative used may require administration twice or
more times during a day. As an example, polypeptides, agents,
nucleic acids or derivatives according to the invention may be
administered as two (or more depending upon the severity of the
condition) daily doses of between 25 mg and 7000 mg (i.e. assuming
a body weight of 70 kg). A patient receiving treatment may take a
first dose upon waking and then a second dose in the evening (if on
a two dose regime) or at 3 or 4 hourly intervals thereafter.
Alternatively, a slow release device may be used to provide optimal
doses to a patient without the need to administer repeated
doses.
[0086] This invention provides a pharmaceutical composition
comprising a therapeutically effective amount of a polypeptide,
agent, nucleic acid or derivative according to the invention and
optionally a pharmaceutically acceptable vehicle. In one
embodiment, the amount of the polypeptide, agent, nucleic acid or
derivative is an amount from about 0.01 mg to about 800 mg. In
another embodiment, the amount of the polypeptide, agent, nucleic
acid or derivative is an amount from about 0.01 mg to about 500 mg.
In another embodiment, the amount of the polypeptide, agent,
nucleic acid or derivative is an amount from about 0.01 mg to about
250 mg. In another embodiment, the amount of the polypeptide,
agent, nucleic acid or derivative is an amount from about 0.1 mg to
about 60 mg. In another embodiment, the amount of the polypeptide,
agent, nucleic acid or derivative is an amount from about 0.1 mg to
about 20 mg.
[0087] This invention provides a process for making a
pharmaceutical composition comprising combining a therapeutically
effective amount of a polypeptide, agent, nucleic acid or
derivative according to the invention and a pharmaceutically
acceptable vehicle. A "therapeutically effective amount" is any
amount of a polypeptide, agent, nucleic acid or derivative
according to the first aspect of the invention which, when
administered to a subject provides prevention and/or treatment of a
viral infection. A "subject" is a vertebrate, mammal, domestic
animal or human being.
[0088] A "pharmaceutically acceptable vehicle" as referred to
herein is any physiological vehicle known to those of ordinary
skill in the art useful in formulating pharmaceutical
compositions.
[0089] In a preferred embodiment, the pharmaceutical vehicle is a
liquid and the pharmaceutical composition is in the form of a
solution. In another embodiment, the pharmaceutically acceptable
vehicle is a solid and the composition is in the form of a powder
or tablet. In a further embodiment, the pharmaceutical vehicle is a
gel and the composition is in the form of a cream or the like.
[0090] A solid vehicle can include one or more substances which may
also act as flavouring agents, lubricants, solubilisers, suspending
agents, fillers, glidants, compression aids, binders or
tablet-disintegrating agents; it can also be an encapsulating
material. In powders, the vehicle is a finely divided solid that is
in admixture with the finely divided active polypeptide, agent,
nucleic acid or derivative. In tablets, the active polypeptide,
agent, nucleic acid or derivative is mixed with a vehicle having
the necessary compression properties in suitable proportions and
compacted in the shape and size desired. The powders and tablets
preferably contain up to 99% of the active polypeptide, agent,
nucleic acid or derivative. Suitable solid vehicles include, for
example, calcium phosphate, magnesium stearate, talc, sugars,
lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidine,
low melting waxes and ion exchange resins.
[0091] Liquid vehicles are used in preparing solutions,
suspensions, emulsions, syrups, elixirs and pressurized
compositions. The active polypeptide, agent, nucleic acid or
derivative can be dissolved or suspended in a pharmaceutically
acceptable liquid vehicle such as water, an organic solvent, a
mixture of both or pharmaceutically acceptable oils or fats. The
liquid vehicle can contain other suitable pharmaceutical additives
such as solubilisers, emulsifiers, buffers, preservatives,
sweeteners, flavouring agents, suspending agents, thickening
agents, colours, viscosity regulators, stabilizers or
osmo-regulators. Suitable examples of liquid vehicles for oral and
parenteral administration include water (partially containing
additives as above, e.g. cellulose derivatives, preferably sodium
carboxymethyl cellulose solution), alcohols (including monohydric
alcohols and polyhydric alcohols, e.g. glycols) and their
derivatives, and oils (e.g. fractionated coconut oil and arachis
oil). For parenteral administration, the vehicle can also be an
oily ester such as ethyl oleate and isopropyl myristate. Sterile
liquid vehicles are useful in sterile liquid form compositions for
parenteral administration. The liquid vehicle for pressurized
compositions can be halogenated hydrocarbon or other
pharmaceutically acceptable propellent.
[0092] Liquid pharmaceutical compositions which are sterile
solutions or suspensions can be utilized by for example,
intramuscular, intrathecal, epidural, intraperitoneal, intravenous
and particularly subcutaneous, intracerebral or
intracerebroventricular injection. The polypeptide, agent, nucleic
acid or derivative may be prepared as a sterile solid composition
that may be dissolved or suspended at the time of administration
using sterile water, saline, or other appropriate sterile
injectable medium. Vehicles are intended to include necessary and
inert binders, suspending agents, lubricants, flavourants,
sweeteners, preservatives, dyes, and coatings.
[0093] Polypeptides, agents, nucleic acids or derivatives according
to the invention can be administered orally in the form of a
sterile solution or suspension containing other solutes or
suspending agents (for example, enough saline or glucose to make
the solution isotonic), bile salts, acacia, gelatin, sorbitan
monoleate, polysorbate 80 (oleate esters of sorbitol and its
anhydrides copolymerized with ethylene oxide) and the like.
[0094] Polypeptides, agents, nucleic acids or derivatives according
to the invention can also be administered orally either in liquid
or solid composition form. Compositions suitable for oral
administration include solid forms, such as pills, capsules,
granules, tablets, and powders, and liquid forms, such as
solutions, syrups, elixirs, and suspensions. Forms useful for
parenteral administration include sterile solutions, emulsions, and
suspensions.
[0095] The invention will be further described, by way of example
only, with reference to the following Examples and figures in
which:--
[0096] FIG. 1 shows the effect of apoE.sub.141-149dp and
apoE.sub.263-286 on HSV1 infectivity. (points are derived from the
average of up to four values) as described in Example 1;
[0097] FIG. 2 shows the effect of apoE.sub.141-149dp or
apoE.sub.263-286 on HSV2 infectivity (points are derived from the
average of up to four values) as described in Example 1;
[0098] FIG. 3 illustrates inhibition of HIV-1 p24 production, as
measured by ELISA, by apoE.sub.141-149r, and apoE.sub.263-286 in
acutely infected U937 cells (values are the average of three
experiments) as described in Example 1 (ApoE.sub.141-149dp was
significantly active against HIV (ANOVA, p<0.001), whereas the
activity of apoE.sub.263-286 against HIV did not reach significance
(ANOVA; 0.06<p<0.62));
[0099] FIG. 4 illustrates the effect of 4 peptides (GIN 1, 1p, 2
and 3) on HSV 1 infectivity as described in Example 2;
[0100] FIG. 5 illustrates the effect of 4 peptides (GIN 4-7) on
HSV1 infectivity as described in Example 2;
[0101] FIG. 6 illustrates the effect of 4 peptides (GIN 8-11) on
HSV1 infectivity as described in Example 2;
[0102] FIG. 7 compares and illustrates the effect of peptides GIN
7, GIN 32, GIN 34, and GIN 1p on HSV1 infectivity as described in
Example 2;
[0103] FIG. 8 illustrates the anti-HIV action of peptide GIN7
against HIV isolate SF162, grown in NP-2 glioma cells
overexpressing CCR5 co-receptors as described in Example 4;
[0104] FIG. 9 shows typical mass spectrometry data for GIN7 and
illustrates that the peptide was >95% purity;
[0105] FIG. 10 shows typical HPLC data for GIN7 and illustrates
that the peptide was >95% purity.
EXAMPLE 1
[0106] Experiments were conducted with ApoE.sub.141-149 to
establish whether or not the peptide had any efficacy as an
antiviral agent.
[0107] 1.1 HSV1
[0108] FIG. 1 and table 1 show typical results for the test for
anti-HSV1 activity. The assay involved treating confluent Vero
cells in 24-well plates with medium containing virus and varying
amounts of peptide for one hour, followed by removal of this
inoculum, and addition of viscous `overlay` medium, containing 0.2%
high viscosity carboxymethylcellulose. The overlay medium only
allows infection of those cells immediately adjacent to an infected
cell. After 2 days incubation and then fixation and staining, small
patches of infected cells (or `plaques`) are visible, which are
counted. Each of these corresponds to the infection of a single
cell during the one hour inoculation. ApoE.sub.141-149dp produced a
40% reduction in plaque number at a concentration of around 20
.mu.M. Note the peptide was only present in the experimental system
for 1 hour.
TABLE-US-00004 TABLE 1 HSV1 plaque formation in Vero cells after
inoculation with virus containing either apoE.sub.141-149dp or
apoE.sub.263-286. Values for untreated wells are underlined.
ApoE.sub.141-149dp ApoE.sub.263-286 [.mu.M] 1 2 3 4 Mean .+-. sd 1
2 3 4 Mean .+-. sd 0 96 102 123 107 .+-. 14.2 5 129 106 103 100 110
.+-. 13.2 113 119 122 126 120 .+-. 5.5 10 73 87 76 89 81 .+-. 7.9
116 124 102 114 .+-. 11.1 20 68 67 63 63 65 .+-. 2.6 148 112 133
114 127 .+-. 17.0 30 72 71 56 66 .+-. 9.0 134 109 114 125 121 .+-.
11.2 40 64 65 53 68 63 .+-. 6.6 120 113 125 144 126 .+-. 11.2
[0109] 1.2 HSV2
[0110] FIG. 2 and table 2 show typical results for the test for
anti-HSV2 activity. The assay was carried out as for the anti-HSV 1
assay, except Hep-2 cells were used rather than Vero cells.
ApoE.sub.141-149dp produced a 50% reduction in plaque number at a
concentration of around 20 .mu.M. Again note that the peptide was
only present in the experimental system for 1 hour.
TABLE-US-00005 TABLE 2 HSV2 plaque formation in HEp-2 cells after
inoculation with virus containing either apoE.sub.141-149dp or
apoE.sub.263-286. Values for untreated wells are underlined.
ApoE.sub.141-149dp ApoE.sub.263-286 [.mu.M] 1 2 3 4 Mean .+-. sd 1
2 3 4 Mean .+-. sd 0 156 137 162 152 152 .+-. 10.7 5 160 134 140
130 141 .+-. 13.3 135 160 161 152 152 .+-. 12.0 10 125 113 131 132
125 .+-. 8.7 157 121 151 134 141 .+-. 16.1 20 82 72 73 81 77 .+-.
5.2 118 150 182 134 146 .+-. 27.3 30 76 77 71 72 74 .+-. 2.9 118
117 103 159 124 .+-. 24.2 40 51 59 69 49 57 .+-. 9.1 132 144 125
124 131 .+-. 24.2
[0111] 1.3. HIV
[0112] FIG. 3 and table 3 show typical results for the test for
anti-HIV activity. The assay was carried out by incubating HIV
infected U937 cells in the presence of various levels of peptide
for 7 days, followed by assay for levels of the HIV protein p24 in
the cells using an Enzyme Linked Immunoabsorbant Assay (ELISA)
technique. ApoE.sub.141-149dp produced a 95% reduction in
infectivity at 20 .mu.M. ApoE.sub.263-286 produced a 20% reduction
in infectivity at 20 .mu.M, which did not reach statistical
significance. The effect on HIV appears at lower peptide
concentrations, though this may be due to peptide being in contact
with cells for 7 days, as opposed to just 1 hour in plaque
reduction assays with herpes viruses.
TABLE-US-00006 TABLE 3 Inhibition of HIV-1 p24 production, as
measured by ELISA, by apoE.sub.141-149dp, and apoE.sub.263-286 in
acutely infected U937 cells. % Decrease in HIV p24 Production
ApoE.sub.141-149dp ApoE.sub.263-286 [.mu.M] Exp. 1 Exp. 2 Exp. 3
Mean .+-. sd Exp. 1 Exp. 2 Exp. 3 Mean .+-. sd 0 0 0 0 0 0 0 0 0 10
91.66 70.31 89.85 83.94 .+-. 11.84 31.75 8.50 29.38 23.21 .+-.
12.79 20 96.87 95.08 93.10 95.02 .+-. 1.89 7.69 29.71 30.91 22.77
.+-. 13.07 30 95.94 88.63 87.77 90.78 .+-. 4.49 37.94 27.83 41.78
35.85 .+-. 7.21 40 96.80 95.47 95.33 95.87 .+-. 0.81 23.50 30.08
48.04 38.87 .+-. 12.70 50 95.73 93.25 95.38 94.79 .+-. 1.34 33.36
41.45 45.66 40.16 .+-. 6.25
[0113] The results presented in 1.1-1.3 illustrate that
ApoE.sub.141-149dp was more efficacious than ApoE.sub.263-286.
[0114] In the light of these results, the inventors proceeded to
test other peptides generated from apolipoproteins to investigate
whether or not such peptides had antiviral activity (see Example
2).
EXAMPLE 2
[0115] Given the knowledge gained by the inventors following the
work reported in Example 1, experiments were conducted to evaluate
the antiviral effects of a large number of peptides derived from
apolipoproteins. Surprisingly, the inventors found that only a
minority of the peptides tested had antiviral effects (see 2.2).
Such peptides represent peptides according to the invention.
2.1 Materials and Methods
2.1.1 Cell Culture.
[0116] African Green Monkey Kidney (Vero) cells were maintained in
Eagle's minimum essential medium with Earle's salt (EMEM) and
supplemented with 10% foetal calf serum (heat-inactivated), 4 mM
L-glutamine, and 1% (v/v) nonessential amino acids, plus penicillin
and streptomycin (100 IU/mg and 100 mg/ml, respectively)
(maintenance medium referred to as 10% EMEM). The cells were
incubated at 37.degree. C. in a humidified atmosphere of air with
5% CO.sub.2.
[0117] On harvesting, monolayers were washed in phosphate-buffered
saline (PBS), and dislodged by incubating with trypsin in PBS for
30 min, before inactivating trypsin by addition of an equal volume
of 10% EMEM and centrifuging at 500 g (5 min, 4.degree. C.). Cell
pellets were resuspended in 10% EMEM, prior to cell counting and
seeding of 24-well plates. For antiviral assays, medium containing
only 0.5% FCS was used (referred to as 0.5% EMEM).
2.1.2 Virus
[0118] Three separate passages of HSV1 virus were prepared by
infecting Vero cells, and preparing semi-pure suspensions of virus
from tissue culture supernatant and cell lysates, before freezing
aliquots of virus at -85.degree. C. Viral infectivity was assessed
by carrying out plaque assays on serial dilutions of thawed
aliquots (expressed in pfu/ml).
2.1.3 Peptides
[0119] Peptides were obtained in lyophilised form from a commercial
supplier (AltaBioscience, University of Birmingham or Advanced
Biomedical), and were produced at 5 micromole scale. N-terminals
were protected by addition of an acetyl group, and the C-terminals
were protected by addition of an amide group.
[0120] Molecular weight of peptides was confirmed by laser
desorption mass spectrometry using a Finnigan LASERMAT 2000
MALDI-time of flight mass analyzer or a Scientific Analysis Group
MALDI-TOF mass spectrometer. HPLC purification of peptides was
performed using a Vydac analytical C-4 reverse phase column, using
0.1% TFA and 0.1% TFA/80% acetonitrile as solvents, or for some
peptides an ACE C18 Reverse Phase column, using 0.05% IFA and 60%
acetonitrile as solvents. Typical mass spectrometry data and high
performance liquid chromatography (HPLC) traces (purity>95%) for
peptide GIN 7 (SEQ ID No. 3) are shown in FIGS. 9 and 10.
[0121] Small quantities of peptide were weighed in sterile
Eppendorf tubes, before addition of sufficient 0.5% EMEM to produce
a 1.5 mM stock solution, which was frozen at -20.degree. C. in
aliquots.
2.1.4 Plaque Reduction Assays.
[0122] Vero cells were seeded at 125,000 cells per well in 10%
EMEM, and were incubated overnight resulting in confluent
monolayers. Peptides were diluted in 0.5% EMEM to give 2.times.
final desired concentration, and 100 .mu.l aliquots were arranged
on 96-well plates in arrangement to be used for 24-well plate;
control wells containing normal 0.5% EMEM were also prepared. Virus
stocks (p3) were thawed, and diluted in 0.5% EMEM such that there
were around 100 pfu in 100 .mu.l. Each 24-well plate was inoculated
separately. Firstly 100 .mu.l of virus stock was added to the
peptide or control medium arranged on a 96-well plate. This was
incubated at 37.degree. C. for ten minutes before inoculation.
Medium was removed from four wells of a 24-well plate containing
confluent Vero, and the 200 .mu.l inoculum added to the appropriate
well. Once all wells were treated, the 24-well plate was incubated
for a further 60-80 minutes. Finally the peptide-containing
inoculum was removed, and 1 ml of 1% EMEM containing 1%
carboxymethylcellulose was added to each well. Plates were
incubated for a further 22 hours or in some experiments 40 hours,
before removal of overlay, and addition of 10% formaldehyde in PBS.
After a further one hour incubation, fixative was removed,
monolayers washed several times with tap water, and stained with
carbol fuchsin solubilised in water. After 30 minutes stain was
removed, and plates washed several times with tap water, before
being air dried. Plaques were counted using an Olympus IX70
Inverting Microscope, and antiviral effect expressed as a
percentage of the control value for each peptide concentration. The
1050 was calculated from plots of inhibitory effect against peptide
concentration.
2.1.5 Toxicity Testing.
[0123] Vero cells were seeded in 96-well plates at 30,000 cells per
well in 10% EMEM, and were incubated overnight resulting in
confluent monolayers. GIN peptides were diluted in 0.5% EMEM to
give final desired concentration, and 100 .mu.l aliquots were
arranged on separate non-cell containing 96-well plates, prior to
taking Vero 96-well plates, removing 10% EMEM, and adding 0.5% EMEM
containing peptides. After incubating for 48 hours, 25 .mu.l of 1.5
mg/ml MTT solution (in 0.5% EMEM) was added per well, and plates
returned to incubator for one hour. Finally, medium was removed
from wells, and blue formazan crystals solubilised by addition of
100 .mu.l of dimethylsulphoxide (DMSO). Absorbance of resulting
solutions was then measured at 570 nm, and toxic effect expressed
as a percentage of the control value for each peptide
concentration. Where possible, the EC50 was calculated from plots
of toxic effect against peptide concentration. Fortunately, no
evidence of toxicity was found for the cell line tested, using
peptide at 40 .mu.M exposed to cells for 2 days.
2.2 Results
[0124] Table 4 summarises data obtained for 16 peptides identified
as GIN1, GIN 1p and GIN 2-15.
TABLE-US-00007 TABLE 4 SEQ Apparent ID IC50 Peptide No. (.mu.M)
Sequence Size GIN 1 2 16.5 LRKLRKRLLLRKLRKRLL 18 GIN 1p 2 10
LRKLRKRLLLRKLRKRLL 18 GIN 2 24 >40 LRKRLLLRKLRKRLL 15 GIN 3 31
No RLLLRKLRKRLL 12 Activity GIN 4 7 29.5 LRKLRKRLLLRKLRK 15 GIN 5
25 >40 LRKLRKRLLLRK 12 GIN 6 26 >40 ERKERKREEERKERKREE 18 GIN
7 3 <5 WRKWRKRWWWRKWRKRWW 18 GIN 8 8 13 LRKLRKRLRKLRKR 14 GIN 9
9 15.5 LRKLRKLRKLRKLRKLRK 18 GIN 10 22 39 RLLRLLRLLRLLRLLRLL 18 GIN
11 20 36.5 QSTEELRVRLASHLRKLRKRLL 22 GIN 12 27 >40 LRKLRKRLLR
DADDLQKRLA 20 GIN 13 28 >40 RDADDLQKR RDADDLQKR 20 GIN 14 29
>40 GERLRARMEGERLRARME 18 GIN 15 30 >40 RLRARMEEMRLRARMEEM
18
[0125] FIG. 4 illustrates that the ApoE.sub.141-149dp (labelled as
GIN 1) had good efficacy for reducing HSV1 infectivity. A related
peptide GIN 1p (GIN 1 with N and C terminal protection) had similar
efficacy.
[0126] As illustrated in Table 4 the inventors tested a number of
other related peptides (identified as GIN 2, GIN 3, GIN 4, GIN 5,
GIN 6, GIN 10, GIN 11, GIN 12, GIN 13, GIN 14 and GIN 15) and it
was found that they had no, or poor, efficacy for reducing viral
infectivity.
[0127] In addition, the inventor found to his surprise, that a
subset of the tested peptides (which are peptides according to the
present invention) were effective as antiviral agents. FIG. 5
illustrates that the peptide designated GIN 7 had efficacy for
reducing HSV-1 infectivity.
[0128] FIG. 6 illustrates that the peptides designated GIN 8 and
GIN 9 also had efficacy for reducing HSV-1 infectivity.
[0129] Table 5 and FIG. 4a illustrate that a number of peptides
related or similar to the ApoE.sub.141-149dp peptide (identified as
peptides GIN 17-31 in Table 4a) had no, or poor, efficacy for
reducing viral infectivity. The inventors had rationally designed
these molecules in the expectation that they may have anti-HSV1
activity and, based on the data presented in Table 4, a skilled
person may have expected such peptides to have similar efficacy to
those claimed according to the invention. The fact that these
peptides had little effect makes the usefulness of the claimed
peptides all the more surprising.
TABLE-US-00008 TABLE 5 SEQ Apparent ID IC50 Peptide No. (.mu.M)
Sequence Size GIN 17 33 NA RALVDTLKFVTQAEGAK 17 GIN 18 34 NA
PYLDDFQKKWQEEMELYRQKVE 22 GIN 19 35 NA PLGEEMRDRARAHVDALRTHLA 22
GIN 20 36 NA PYSDELRQRLAARLEALKENGG 22 GIN 21 37 NA
ARLAEYHAKATEHLSTLSEKAK 22 GIN 22 19 36 DWLKAFYDKVAEKLKEAF 18 GIN 23
38 NA PVLDEFREKLNEELEALKQKMK 22 GIN 24 39 NA VTDYGKDLMEKVKSPELQ 18
GIN 25 40 NA VTDYGKDLMEKVKEWLNS 18 GIN 26 41 NA NFHAMFQPFLEMIHEAQQ
28 GIN 27 42 NA CKNKEKKCCKNKEKKC 18 GIN 28 43 NA LRKEKKRLLLRKEKKRLL
18 GIN 29 21 38.5 HMLDVMQDHFSRASSIIDEL 20 GIN 30 44 NA
LQVAERLTRKYNELLKSYQ 19 GIN 31 45 NA KFMETVAEKALQEYRK 16
EXAMPLE 3
[0130] A further set of experiments was conducted on an expanded
number of peptides to further evaluate the effect of peptides
according to the invention against HSV-1. Table 6 confirms that the
peptides designated GIN 1p and GIN 7 had anti-HSV-1 properties,
whereas the peptides designated GIN 32, 34 and 41 also had
efficacy. The efficacy of these peptides is surprising given that
the majority of peptides tested had little or no activity.
[0131] FIG. 7 compares and illustrates the effect of peptides GIN
7, GIN 32, GIN 34, and GIN 1p on HSV1 infectivity.
TABLE-US-00009 TABLE 6 Nucleic acid IC.sub.50 Peptide Code SEQ ID
No. SEQ ID No. Sequence (.mu.M) 7 SEQ ID No. 3 SEQ ID No. 12
WRKWRKRWWWRKWRKRWW 3.5 34 SEQ ID No. 5 SEQ ID No. 14
WRKWRKRWWLRKLRKRLL 6 32 SEQ ID No. 4 SEQ ID No. 13 WRKWRKRWRKWRKR
10 41 SEQ ID No. 6 SEQ ID No. 15 YRKYRKRYYYRKYRKRYY 16 1p SEQ ID
No. 2 SEQ ID No. 11 LRKLRKRLLLRKLRKRLL 17 activity low: 4 SEQ ID
No. 7 SEQ ID No. 16 LRKLRKRLLLRKLRK 29.5 22 SEQ ID No. 19 NA
DWLKAFYDKVAEKLKEAF 36 11 SEQ ID No. 20 NA QSTEELRVRLASHLRKLRKRLL
36.5 29 SEQ ID No. 21 NA HMLDVMQDHFSRASSIIDEL 38.5 10 SEQ ID No. 22
NA RLLRLLRLLRLLRLLRLL 39 44 SEQ ID No. 23 NA LRQLRQRLLLRQLRQRLL 40
2 SEQ ID No. 24 NA LRKRLLLRKLRKRLL >40 5 SEQ ID No. 25 NA
LRKLRKRLLLRK >40 6 SEQ ID No. 26 NA ERKERKREEERKERKREE >40 12
SEQ ID No. 27 NA LRKLRKRLLR DADDLQKRLA >40 13 SEQ ID No. 28 NA
RDADDLQKR RDADDLQKR >40 14 SEQ ID No. 29 NA GERLRARMEGERLRARME
>40 15 SEQ ID No. 30 NA RLRARMEEMRLRARMEEM >40 No activity:
apoE 141-149 SEQ ID No. 1 SEQ ID No. 10 LRKLRKRLL NA 3 SEQ ID No.
31 NA RLLLRKLRKRLL NA 6 SEQ ID No. 32 NA ERKERKREEERKERKREE NA 17
SEQ ID No. 33 NA RALVDTLKFVTQAEGAK NA 18 SEQ ID No. 34 NA
PYLDDFQKKWQEEMELYRQKVE NA 19 SEQ ID No. 35 NA
PLGEEMRDRARAHVDALRTHLA NA 20 SEQ ID No. 36 NA
PYSDELRQRLAARLEALKENGG NA 21 SEQ ID No. 37 NA
ARLAEYHAKATEHLSTLSEKAK NA 23 SEQ ID No. 38 NA
PVLDEFREKLNEELEALKQKMK NA 24 SEQ ID No. 39 NA VTDYGKDLMEKVKSPELQ NA
25 SEQ ID No. 40 NA VTDYGKDLMEKVKEWLNS NA 26 SEQ ID No. 41 NA
NFHAMFQPFLEMIHEAQQ NA 27 SEQ ID No. 42 NA CKNKEKKCCKNKEKKC NA 28
SEQ ID No. 43 NA LRKEKKRLLLRKEKKRLL NA 30 SEQ ID No. 44 NA
LQVAERLTRKYNELLKSYQ NA 31 SEQ ID No. 45 NA KFMETVAEKALQEYRK NA 39
SEQ ID No. 46 NA ARKARKRAAARKARKRAA NA 40 SEQ ID No. 47 NA
MRKMRKRMMMRKMRKRMM NA 42 SEQ ID No. 48 NA LRWLRWRLLLRWLRWRLL NA 45
SEQ ID No. 49 NA LWKLWKWLLLWKLWKWLL NA 46 SEQ ID No. 50 NA
LYKLYKYLLLYKLYKYLL NA 47 SEQ ID No. 51 NA LQKLQKQLLLQKLQKQLL NA
EXAMPLE 4
[0132] Similar experiments to those described in Example 2 were
conducted to test the efficacy of the peptides according to the
invention against HIV infection.
[0133] The glioma cell line NP2 over-expressing both CD4 and the
appropriate co-receptor (CCR5 or CXCR4) were maintained in DMEM
supplemented with 10% FCS. 2.times.10.sup.4 cells were plated per
well of a 48-well plate 24 h prior to infection and grown at 37 C.
The cells were then washed, and incubated in DMEM/FCS containing
peptide concentrations ranging from 0.1 to 10 micromolar, at 37 C
for 30 minutes. 200 focus-forming units of HIV-1 stocks were then
added to each well, and the cells incubated at 37 C for a further 2
hours. The cells were then washed twice in PBS and fresh medium
replaced. After 3 day's growth the cells were fixed in cold
methanol:acetone, and stained in situ for expression of HIV-1 p24
using a monoclonal anti-p24 followed by a secondary anti-mouse
beta-galactosidase conjugate. Expression was visualised by X-Gal
staining and infectious foci enumerated by light-microscopy.
[0134] It was found that peptides according to the invention had
similar efficacy against HSV-1 and HIV.
[0135] FIG. 8 illustrates the anti-HIV action of peptide GIN 7
against HIV isolate SF162, grown in NP-2 glioma cells
overexpressing CCR5 co-receptors.
[0136] Similar data was generated for other HIV strains, and in
other host cells types. Notably GIN 1p (apoEdp) had no detectable
anti-HIV activity in the one combination of HIV strain and cell
type against which this peptide was tested, and at the
concentrations used here (up to 10 .mu.M). This would suggest the W
substituted peptides according to the present invention are more
potent against HIV than GIN 1p (apoE.sub.141-149 dp).
EXAMPLE 5
[0137] Further experiments were conducted to test the efficacy of
peptides according to the present invention against HSV 1.
5.1 Methods
[0138] The methods employed were as described in Examples 1-4
expect peptides were prepared as 400 .mu.M stocks in phosphate
buffered saline (PBS).
5.2 Results
5.2.1 Effect of Complete Substitution of Leucine
[0139] Experiments were conducted to investigate the effect of full
substitution of L residues in the apoE.sub.141-149 tandem repeat
with a single amino acid. Table 7 illustrates that peptides
according to the present invention have efficacy for inhibiting the
growth of HSV1 (i.e. W, R or K substitution). The peptides
according to the first aspect of the invention surprisingly have
more efficacy than the apoE tandom repeat (GIN 1/MU 10).
[0140] It is interesting to note that substitution with M, Y, F, I,
Q, H or N had some efficacy (comparable with the apoE tandem
repeat) and as such further substitutions according to the
invention may comprise these amino acids.
[0141] Substitutions with E, A, D, S, V, T, G or P resulted in
antiviral activity being abolished.
[0142] Although the inventors do not wish to be bound by any
hypothesis they have noted that substitution with amino acids with
small side chains tends to abolish antiviral activity whereas as
amino acids with larger side chains maintain the antiviral effects.
However substitution of L with amino acids as defined by the first
aspect of the invention confers surprising antiviral activity.
TABLE-US-00010 TABLE 7 SEQ HSV1 ID IC.sub.50 Peptide Code No.
Sequence (.mu.M) MU 1 (GIN 6) 67 ERKERKREEERKERKREE NA MU 2 (GIN
39) 68 ARKARKRAAARKARKRAA NA MU 3 69 DRKDRKRDDDRKDRKRDD NA MU 4
(GIN 7) 3 WRKWRKRWWWRKWRKRWW 3.5 MU 5 (GIN 40) 70
MRKMRKRMMMRKMRKRMM >30 MU 6 (GIN 41) 6 YRKYRKRYYYRKYRKRYY >30
MU 7 71 FRKFRKRFFFRKFRKRFF >30 MU 8 72 IRKIRKRIIIRKIRKRII >30
MU 9 73 QRKQRKRQQQRKQRKRQQ >30 MU 10 (GIN 1p) 2.
LRKLRKRLLLRKLRKRLL 30 MU 10 (GIN 1) 2. LRKLRKRLLLRKLRKRLL 16.5 (no
N/C protect) MU 11 74 NRKNRKRNNNRKNRKRNN 27.5 MU 13 75
SRKSRKRSSSRKSRKRSS NA MU 14 76 VRKVRKRVVVRKVRKRVV NA MU 15 77
TRKTRKRTTTRKTRKRTT NA MU 16 64 RRKRRKRRRRRKRRKRRR 7.5 MU 17 78
GRKGRKRGGGRKGRKRGG NA MU 18 65 KRKKRKRKKKRKKRKRKK 7.5 MU 19 79
HRKHRKRHHHRKHRKRHH NA MU 20 80 PRKPRKRPPPRKPRKRPP NA
5.2.2 Testing of Further apoE Derived Peptides.
[0143] The inventor constructed an expanded library of peptides to
evaluate what apoE peptide derivatives may have antiviral
activity.
[0144] Table 8 illustrates that a number of peptides, which were
based on SEQ ID No. 2 but fall outside the definition of peptides
according to the first aspect of the invention, had no or poor
antiviral activity (e.g. MU 24-46). It is interesting to note that
MU 43 and MU 44 correspond to tandem repeats of the murine and
bovine equivalents to apoE.sub.141-149 (SEQ ID No. 1).
[0145] The data presented in Table 8 for MU 58-117 demonstrate that
each of these peptides, which are peptides according to the present
invention have good antiviral activity which is surprisingly
superior to that of apoE.sub.141-149 dp (SEQ ID No. 2)
TABLE-US-00011 TABLE 8 SEQ HSV1 Peptide ID IC.sub.50 Code No.
Sequence (.mu.M) MU 24 81 LLRKRLKRLLLRKRLKRL 40 MU 38 82
LRRLRRRLLLRRLRRRLL >30 MU 39 83 LKKLKKKLLLKKLKKKLL 30 MU 40 84
LHHLHHHLLLHHLHHHLL NA MU 41 85 LDDLDDDLLLDDLDDDLL NA MU 42 86
LEELEEELLLEELEEELL NA MU 43 87 MRKLRKRLMMRKLRKRLM NA MU 44 88
LRKLPKRLLLRKLPKRLL >30 MU 45 89 WRKWRKRWW NA MU 46 1 LRKLRKRLL
NA MU 58 52 WRKWRKRWWRKWRKRWW 4.25 MU 59 53 WRKWRKRWRKWRKRW 9.75 MU
60 54 WRKWRKRWWFRKWRKRWW MU 61 55 WRKWRKRFFWRKWRKRFF 4.75 MU 68 56
WRKCRKRCWWRKCRKRCW 4.25 MU 83 66 WRKWRKRWWRWRKWRKRW 2.5 WR MU 111
57 LRKLRKRLLWRKWRKRWW 12.5 MU 112 58 LRKLRKRLLLRKLRKRWW >20 MU
113 59 LRKLRKRLLWRKWRKRLL 18.5 MU 114 60 WRKWRKRLLLRKLRKRLL 16 MU
115 61 WRKLRKRLLLRKLRKRLL 17.5 MU 116 62 WRKWRKFFFRKWRKRWW 3.3 MU
117 63 WRKWRKRWWFRKFRKRFF 3.3
EXAMPLE 6
[0146] Further experiments were conducted to test the efficacy of
peptides according to the present invention against HSV2.
6.1 Methods
[0147] Plaque assays were performed. The methodology was as
described in previous Examples for HSV1 plaque assays (including
usage of Vero cells) except HSV2 clinical isolates (provided by
Prof. Anthony Hart of Liverpool University) were employed
instead.
6.2 Results
[0148] A number of peptides that were found to have efficacy
against HSV 1 were also tested against HSV2. Table 9 illustrates
that peptides according to the present invention were effective
against both HSV 1 and HSV2. This illustrates that the peptides
will have broad spectrum activity against viruses.
TABLE-US-00012 TABLE 9 SEQ HSV2 Peptide ID IC.sub.50 code No.
Sequence (.mu.M) GIN 34 5 WRKWRKRWWLRKLRKRLL <3.3 GIN 32 4
WRKWRKRWRKWRKR 6.25 MU 4 3 WRKWRKRWWWRKWRKRWW <3.3 (GIN 7) MU 59
53 WRKWRKRWRKWRKRW 10 MU 83 66 WRKWRKRWWRWRKWRKRWWR <3.3 MU 111
57 LRKLRKRLLWRKWRKRWW 6.5 MU 112 58 LRKLRKRLLLRKLRKRWW 16 MU 113 59
LRKLRKRLLWRKWRKRLL 11.75 MU 114 60 WRKWRKRLLLRKLRKRLL 9
EXAMPLE 7
[0149] Further experiments were conducted to test the efficacy of
peptides according to the present invention against Human
Immunodeficiency Virus (HIV). The effect of a peptide according to
the present invention was tested against a different HIV strain to
that tested in Example 4.
7.1 Methods
[0150] Peptides (prepared as described previously) were diluted in
50 .mu.l aliquots and mixed with T-cells (C8166) at 40,000 cells
per well. Next HIV-1 111B was added at a multiplicity of infection
(MOI) of 0.01, and the mixture incubated for 5 days at 37.degree.
C. Syncytia formation was assessed visually using an inverting
microscope, and viral gp120 levels in supernatants assessed by a
gp120 ELISA using GNA for antigen capture. 96-well plates coated
with 50 .mu.l GNA (Galanthus nivalis) were washed, then treated
with 100 .mu.l RPMI (10% foetal calf serum) and left for one hour.
After further washing, 250 test sample supernatants were added to
wells, along with dilutions of infected control samples. After
lysis by 3 hr treatment with 0.5% Empigen (detergent used to lyse
virus) to all wells, and washing, 50 .mu.l of human anti-HIV sera
was added, and plates incubated overnight. After further washing,
50 .mu.l of a 1000.times. dilution of anti-human Ig peroxidase
conjugate was added, and plates incubated at 37.degree. C. for 90
min. After a final wash, 50 ul peroxidase substrate was added to
each well, and plates incubated for 10-30 min. Reaction was stopped
with 25 .mu.l 2M H.sub.2SO.sub.4, and A450 measured.
7.2 Results
[0151] Further tests were conducted to support the data presented
in Example 4 illustrating that peptides according to the present
invention were effective against HIV as well as both HSV1 and
HSV2.
[0152] GIN 32 (SEQ ID No. 4) had an IC.sub.50 of 7.5 .mu.M for
inhibiting HIV-1 growth. The efficacy of this was similar in HSV1,
HSV2 and HIV. This confirms that peptides according to the present
invention have broad antiviral effects.
Sequence CWU 1
1
5119PRTHomo sapiens 1Leu Arg Lys Leu Arg Lys Arg Leu Leu1
5218PRTHomo sapiens 2Leu Arg Lys Leu Arg Lys Arg Leu Leu Leu Arg
Lys Leu Arg Lys Arg1 5 10 15Leu Leu318PRTHomo sapiens 3Trp Arg Lys
Trp Arg Lys Arg Trp Trp Trp Arg Lys Trp Arg Lys Arg1 5 10 15Trp
Trp414PRTHomo sapiens 4Trp Arg Lys Trp Arg Lys Arg Trp Arg Lys Trp
Arg Lys Arg1 5 10518PRTHomo sapiens 5Trp Arg Lys Trp Arg Lys Arg
Trp Trp Leu Arg Lys Leu Arg Lys Arg1 5 10 15Leu Leu618PRTHomo
sapiens 6Tyr Arg Lys Tyr Arg Lys Arg Tyr Tyr Tyr Arg Lys Tyr Arg
Lys Arg1 5 10 15Tyr Tyr715PRTHomo sapiens 7Leu Arg Lys Leu Arg Lys
Arg Leu Leu Leu Arg Lys Leu Arg Lys1 5 10 15814PRTHomo sapiens 8Leu
Arg Lys Leu Arg Lys Arg Leu Arg Lys Leu Arg Lys Arg1 5 10918PRTHomo
sapiens 9Leu Arg Lys Leu Arg Lys Leu Arg Lys Leu Arg Lys Leu Arg
Lys Leu1 5 10 15Arg Lys1027DNAHomo sapiens 10cttcgtaaac ttcgtaaacg
tcttctt 271154DNAHomo sapiens 11cttcgtaaac ttcgtaaacg tcttcttctt
cgtaaacttc gtaaacgtct tctt 541254DNAHomo sapiens 12tggcgtaaat
ggcgtaaacg ttggtggtgg cgtaaatggc gtaaacgttg gtgg 541342DNAHomo
sapiens 13tggcgtaaat ggcgtaaacg ttggcgtaaa tggcgtaaac gt
421454DNAHomo sapiens 14tggcgtaaat ggcgtaaacg ttggtggctt cgtaaacttc
gtaaacgtct tctt 541554DNAHomo sapiens 15tatcgtaaat atcgtaaacg
ttattattat cgtaaatatc gtaaacgtta ttat 541645DNAHomo sapiens
16cttcgtaaac ttcgtaaacg tcttcttctt cgtaaacttc gtaaa 451742DNAHomo
sapiens 17cttcgtaaac ttcgtaaacg tcttcgtaaa cttcgtaaac gt
421854DNAHomo sapiens 18cttcgtaaac ttcgtaaact tcgtaaactt cgtaaacttc
gtaaacttcg taaa 541918PRTHomo sapiens 19Asp Trp Leu Lys Ala Phe Tyr
Asp Lys Val Ala Glu Lys Leu Lys Glu1 5 10 15Ala Phe2022PRTHomo
sapiens 20Gln Ser Thr Glu Glu Leu Arg Val Arg Leu Ala Ser His Leu
Arg Lys1 5 10 15Leu Arg Lys Arg Leu Leu 202120PRTHomo sapiens 21His
Met Leu Asp Val Met Gln Asp His Phe Ser Arg Ala Ser Ser Ile1 5 10
15Ile Asp Glu Leu 202218PRTHomo sapiens 22Arg Leu Leu Arg Leu Leu
Arg Leu Leu Arg Leu Leu Arg Leu Leu Arg1 5 10 15Leu Leu2318PRTHomo
sapiens 23Leu Arg Gln Leu Arg Gln Arg Leu Leu Leu Arg Gln Leu Arg
Gln Arg1 5 10 15Leu Leu2415PRTHomo sapiens 24Leu Arg Lys Arg Leu
Leu Leu Arg Lys Leu Arg Lys Arg Leu Leu1 5 10 152512PRTHomo sapiens
25Leu Arg Lys Leu Arg Lys Arg Leu Leu Leu Arg Lys1 5 102618PRTHomo
sapiens 26Glu Arg Lys Glu Arg Lys Arg Glu Glu Glu Arg Lys Glu Arg
Lys Arg1 5 10 15Glu Glu2720PRTHomo sapiens 27Leu Arg Lys Leu Arg
Lys Arg Leu Leu Arg Asp Ala Asp Asp Leu Gln1 5 10 15Lys Arg Leu Ala
202818PRTHomo sapiens 28Arg Asp Ala Asp Asp Leu Gln Lys Arg Arg Asp
Ala Asp Asp Leu Gln1 5 10 15Lys Arg2918PRTHomo sapiens 29Gly Glu
Arg Leu Arg Ala Arg Met Glu Gly Glu Arg Leu Arg Ala Arg1 5 10 15Met
Glu3018PRTHomo sapiens 30Arg Leu Arg Ala Arg Met Glu Glu Met Arg
Leu Arg Ala Arg Met Glu1 5 10 15Glu Met3112PRTHomo sapiens 31Arg
Leu Leu Leu Arg Lys Leu Arg Lys Arg Leu Leu1 5 103218PRTHomo
sapiens 32Glu Arg Lys Glu Arg Lys Arg Glu Glu Glu Arg Lys Glu Arg
Lys Arg1 5 10 15Glu Glu3317PRTHomo sapiens 33Arg Ala Leu Val Asp
Thr Leu Lys Phe Val Thr Gln Ala Glu Gly Ala1 5 10 15Lys3422PRTHomo
sapiens 34Pro Tyr Leu Asp Asp Phe Gln Lys Lys Trp Gln Glu Glu Met
Glu Leu1 5 10 15Tyr Arg Gln Lys Val Glu 203522PRTHomo sapiens 35Pro
Leu Gly Glu Glu Met Arg Asp Arg Ala Arg Ala His Val Asp Ala1 5 10
15Leu Arg Thr His Leu Ala 203622PRTHomo sapiens 36Pro Tyr Ser Asp
Glu Leu Arg Gln Arg Leu Ala Ala Arg Leu Glu Ala1 5 10 15Leu Lys Glu
Asn Gly Gly 203722PRTHomo sapiens 37Ala Arg Leu Ala Glu Tyr His Ala
Lys Ala Thr Glu His Leu Ser Thr1 5 10 15Leu Ser Glu Lys Ala Lys
203822PRTHomo sapiens 38Pro Val Leu Asp Glu Phe Arg Glu Lys Leu Asn
Glu Glu Leu Glu Ala1 5 10 15Leu Lys Gln Lys Met Lys 203918PRTHomo
sapiens 39Val Thr Asp Tyr Gly Lys Asp Leu Met Glu Lys Val Lys Ser
Pro Glu1 5 10 15Leu Gln4018PRTHomo sapiens 40Val Thr Asp Tyr Gly
Lys Asp Leu Met Glu Lys Val Lys Glu Trp Leu1 5 10 15Asn
Ser4118PRTHomo sapiens 41Asn Phe His Ala Met Phe Gln Pro Phe Leu
Glu Met Ile His Glu Ala1 5 10 15Gln Gln4216PRTHomo sapiens 42Cys
Lys Asn Lys Glu Lys Lys Cys Cys Lys Asn Lys Glu Lys Lys Cys1 5 10
154318PRTHomo sapiens 43Leu Arg Lys Glu Lys Lys Arg Leu Leu Leu Arg
Lys Glu Lys Lys Arg1 5 10 15Leu Leu4419PRTHomo sapiens 44Leu Gln
Val Ala Glu Arg Leu Thr Arg Lys Tyr Asn Glu Leu Leu Lys1 5 10 15Ser
Tyr Gln4516PRTHomo sapiens 45Lys Phe Met Glu Thr Val Ala Glu Lys
Ala Leu Gln Glu Tyr Arg Lys1 5 10 154618PRTHomo sapiens 46Ala Arg
Lys Ala Arg Lys Arg Ala Ala Ala Arg Lys Ala Arg Lys Arg1 5 10 15Ala
Ala4718PRTHomo sapiens 47Met Arg Lys Met Arg Lys Arg Met Met Met
Arg Lys Met Arg Lys Arg1 5 10 15Met Met4818PRTHomo sapiens 48Leu
Arg Trp Leu Arg Trp Arg Leu Leu Leu Arg Trp Leu Arg Trp Arg1 5 10
15Leu Leu4918PRTHomo sapiens 49Leu Trp Lys Leu Trp Lys Trp Leu Leu
Leu Trp Lys Leu Trp Lys Trp1 5 10 15Leu Leu5018PRTHomo sapiens
50Leu Tyr Lys Leu Tyr Lys Tyr Leu Leu Leu Tyr Lys Leu Tyr Lys Tyr1
5 10 15Leu Leu5118PRTHomo sapiens 51Leu Gln Lys Leu Gln Lys Gln Leu
Leu Leu Gln Lys Leu Gln Lys Gln1 5 10 15Leu Leu
* * * * *
References