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.
Antiviral agents may target one of six stages of the viral replication cycle, these being:
1. Attachment of the virus to the cell;
2. Penetration (or fusion of the viral membrane with the cell membrane);
3. Uncoating of the virus;
4. Replication of the viral nucleic acids;
5. Maturation of progeny virus particles; and
6. Release of progeny virus into extracellular fluids.
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.
It is therefore one object of the present invention to provide therapeutic agents that modulate viral attachment to cells.
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 apolipoproteins A, B and C have been documented.
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:
a. Hepatitis C virus core protein binds to apolipoprotein AII;
b. Hepatitis B virus surface antigen binds apolipoprotein H; and
c. Simian immunodeficiency virus (SIV) gp32 protein, and human immunodeficiency virus (HIV) gp41 protein binds to apolipoprotein A1.
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.
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.
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.
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).
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.
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, apoE141-149 (i.e. 2x 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.
In the light of the data generated for a tandem repeat of apoE141-149 (i.e. 2x LRKLRKRLL—SEQ ID No.1), the inventors decided to investigate other fragments of apolipoproteins for antiviral activity.
According to a first aspect of the present invention, there is provided a polypeptide, derivative or analogue thereof comprising a tandem repeat of apoE141-149 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.
By “a tandem repeat of apoE141-149 of SEQ ID No. 2” we mean the peptide with the amino acid sequence: LRKLRKRLLLRKLRKRLL. The tandem repeat is referred to herein as apoE141-149dp or apoE141-149r. 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)).
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).
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.
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 apoE141-149 and other peptides derived from apolipoproteins.
The inventor has identified that Tryptophan (W), Arginine (R) or Lysine (K) may be substituted for Leucine in apoE141-149 tandem 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.
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 apoE141-149 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).
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).
Predicted proportion of molecules of various
peptides forming alpha-helix in aqueous 0.15M
NaCl buffer at 37° C. (%) (using AGADIR
secondary structure prediction software available
Amino Acid Substitution
Sequence of peptide