WO1996022999A1

WO1996022999A1 - Peptides which inhibit viruses

Info

Publication number: WO1996022999A1
Application number: PCT/GB1996/000156
Authority: WO
Inventors: Brian John Boughton
Original assignee: Brian John Boughton
Priority date: 1995-01-24
Filing date: 1996-01-24
Publication date: 1996-08-01
Also published as: GB9501300D0

Abstract

Novel polypeptides are described which have anti-viral, especially anti-herpes goup anti-viral, activities. The polypeptide sequences are formed of up to 50, usually 5-20 amino acid residues and are generally made by protein synthetic techniques. The residues are primarily hydrophobic in nature and have a high propensity towards α-helical conformation.

Description

PEPTIDES WHICH INHIBIT VIRUSES The present invention relates to peptides, usually synthetic peptides, which inhibit viral infection of cells. The peptides may be used as treatment or prophylaxis for viral infections, for instance by herpes group viruses of various types, and/or to treat autoimmune or neoplastic sequelae of such infections. The peptides are relatively hydrophobic and have a high proportion of α-helical secondary structure.

The viral infectious cycle consists of a number of steps. In the first step the virus attaches to a receptor on a host cell. Subsequently the virus or at least the genetic component thereof enters the cell. Subsequently the biosynthetic machinery of the host cell is utilised to reproduce the components of the virus which subsequently assemble to form complete viral particles. Various strategies for preventing viral infection have been investigated, including immunological means and otter ways of preventing initial attachment of viral particles to host cells, for instance by inhibiting receptor-viral particle interaction. Inhibiting entry, for instance by inhibiting endocytosis is a potential means for preventing infection, for instance by affecting membrane structure, by interacting with lipids or membrane proteins. Various means for preventing operation of the biosynthetic machinery of the host cell operating have been utilised, for instance inhibiting the nucleic acid synthetic machinery or other components of the host cell machinery hijacked by the virus. Alternatively proteins, for instance enzymes encoded by the virus itself which are required for the replication may have their activity inhibited. Inhibiting assembly of the components to form complete viral particles is another strategy for interrupting the cycle.

Polypeptides may have potential anti-viral activity in inhibiting any of the steps in the viral reproductive cycle. Liuzzi et al in Nature, 372, page 695 et seg (1994) describe a peptide inhibitor of an enzyme coded by herpes simplex virus, namely ribonucleotide reductase. A polypeptide formed of six amino acid residues is shown to have in vitro and in vivo anti-viral activity.

Cell surface intergrins on host cells have been implicated as the cell surface receptors for various human viruses, for instance coxsackievirus, by Roivainen et al in Virology 203, 357-365 (1994) and human adenovirus, by Wickham et al in J. Cell Biology, 127 (1), 257-264 (1994) .

The Herpes group viruses which infect man are Herpes

Simplex Viruses 1 and 2, Varicella Zoster, Cytomegalovirus,

Epstein Barr Virus, Human Herpes Virus 6, HHV7 and HHV8.

The acute infections caused by these viruses are asymptomatic in some individuals and life threatening in others. These viruses subsequently have a life-long latency which is associated with recurrent infections, autoimmune disorders and malignant tumour formation. All current antivirals have toxic side effects, and innate or acquired viral resistance renders them ineffective against many herpes viruses. Any new antiviral agents are therefore important new developments.

In Platelets, 6, 75-82 (1995) (which was published after the priority date claimed in the present application) I and my co-workers describe sequence homologies between human virus proteins and the integrin Human Platelet Ilb/IIIa. We discovered that synthetic peptides representing viral sequences having high levels of homologies with that integrin inhibited serum auto antibodies of adults with acute autoimmune thrombocytopenic purpura (AITP) . The pattern and degree of auto antibody inhibition suggested that polyclonal GPIIb/IIIa auto antibodies are directed to different GP epitopes and are cross reactive to viral proteins. We concluded that the results indicated a role for human viruses in the aetiology of AITP via molecular mimicry of platelet GPIIb/IIIa such that chronic autoimmunity may be related to a persistent antigenic stimulus from life-long latent viral infections. My further work on the polypeptides described in the article in Platelets led to the discovery that some of these have antiviral activity, illustrated by their ability to inhibit the growth of viruses in tissue culture.

According to the present invention there is a provided a new synthetic or recombinant peptide formed from 5-50 amino acid residues having an α-helical secondary structure over a stretch of at least 5 amino acid residues and having a Kyte and Doolittle hydropathy measure for the said stretch of at least 5 amino acid residues which is hydrophobic. The presence of the alpha-helical secondary structure may be determined by use of H-nmr, C-nmr or N-nmr or by x-ray crystallographic techniques. The H-nmr tests may for instance be carried out in model membrane structures formed of appropriate surfactants, for instance deuterated sodium dodecyl sulphate (SDS) .

The propensity measure for α-helix secondary structure of a polypeptide is calculated as described by Chou and

Fasman, in Advances in Enzymology, 47, pages 45-147 (1978). The propensity measures for the sequences can be plotted on a scale between 0.5 and 1.5, in which 1.03 represents the threshold for α-helical forming structure. If the curve which represents the average of the attribute over a window of 4 residues, drops below 1.0 and if there is at least one breaking residue (that is which breaks the structure) in 4, then the structure may terminate. The relative propensities for α-helix and jS-sheet are also relevant for determining the likelihood of either of these secondary structures. The curves can be plotted using a program devised by the University of Wisconsin Genetics Computer Group called PepPlot. The Chou and Fasman propensities have been found to give a reasonable prediction of the alpha-helical structures for the polypeptides desired in the present invention. The hydrophobicity is calculated by the technique of Kyte and Doolittle in J. Mol. Biol. 157, 105-132 (1982). The curve (printed using the PepPlot software) is the average of a residue-specific hydrophobicity index over a window of 9 residues, on a scale of -3 to +3. When the curve has a value of more than 0, it indicates a hydrophobic region. Although the hydropathy measure has been calculated based on a window of 9 residues during the present work, it is possible to set the window to a number other than 9 if desired, for instance for short polypeptides.

In the invention the polypeptide should generally be formed of at least 5 amino acid residues, preferably at least 7 amino acid residues. It may be up to 50 amino acid residues long, but is generally up to 20, preferably up to 15 amino acids in length. The sequence should be long enough for an alpha helical structure to be formed, for instance at least 6 residues long, whilst it should preferably not be so long that the polypeptide is antigenic. The alpha helical structure and the hydrophilic hydropathy values are generally present along a stretch of at least 7 residues, usually over more than 50% of the length of the polypeptide.

The polypeptide may be made by synthetic or recombinant DNA techniques. For instance, where the polypeptide is derived from a viral peptide sequence, such as one of those described in my article (op . cit . ) , the viral genome sequence can be identified from the location information in that document, and that information used to produce an appropriate rDNA sequence for expression in a transformed microorganism. Alternatively, where the polypeptide is derived from a viral sequence from a known translated viral protein, the polypeptide may be produced from that isolated translated protein by any necessary protein cleavage steps. It is preferred for polypeptide synthetic methods to be used, for instance using standard solid state peptide synthesis techniques, apparatus and starting materials. Where the polypeptide is less than 20 amino acids in length peptide synthesis methods are preferred and are readily available.

The present invention is based on the discovery that polypeptides which have a propensity to α-helical structure and which are hydrophobic appear to have anti-viral properties. These properties are illustrated by the inhibition of growth of viruses in appropriate tissue cultures. Some of the polypeptides which have been found to have anti-viral, specifically against herpes group viruses, properties, are themselves viral peptide sequences. Some of the peptides with anti-viral properties are those having high homologies with integrins as described in my article mentioned above. Without wishing to be bound by theory, it is believed that the anti-viral properties of the polypeptides described herein may be due to an inhibitory effect on the entry into the host cells of the viruses. Although the mechanism of the inhibition is not known, the hydrophobicity and the propensity to α-helical structure suggest that the polypeptides may have the ability to become incorporated into the host cell membrane and, whilst incorporated, have some effect on the lipid and/or protein components of the cell membrane, for instance on receptors for viral particles. Some or all of the polypeptides may have alternative or additional activities.

The polypeptides of the present invention are generally formed substantially or entirely of relatively hydrophobic amino acid residues, that is having R groups which are non-polar. These residues are, for instance, selected from alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, methionine and proline. Other residues present in minor amounts, that is less than 50 mole %, are residues with polar R groups, although these are usually uncharged. However, the number of α-helix breaking residues should be minimised, ie the number of proline, glycine, tyrosine and asparagine should not be such as to prevent alpha-helical structures of the necesary length forming.

In my early work on the anti-viral peptides from sequences disclosed in my article (op . cit . ) , I discovered that viral sequence WWTLLVDLLWLLL and non-viral sequence ISVTGPLLRWEFCAL gave very high anti-viral sequences, AWLPSLS and CMRIRSLLCS had significant anti-viral properties and HFLWMVRLYG had some anti-viral properties whilst VLFLRLADSVPRPLD and SWLLEYSLLC had no reproducible anti-viral properties.

From the two strongly inhibitory compounds further synthetic sequences were developed which had conservative substitutions for some of the amino acid residues. Conservative substitutions for some of the amino acid residues. Conservative substitutions can be selected by a person skilled in the art from recognised tables which compare amino acid residues which, when substituted one for the other in a polypeptide or protein, cause minimal chemical and secondary structural changes. Also, other viral peptide sequences having similar hydrophobicities and propensity to alpha-helical structure were selected from the sequences investigated in my work reported in my article (op . cit . ) . From these original and further sequences the following polypeptides have been indentified as having particular utility in the invention:

1. TWLLWWITILL, which is a viral sequence from cytomegalovirus (CMV) ;

2. WWTLLVDLLWLLL, which is a viral sequence from the Epstein Barr virus (EBV) tested in my original work; 3. LLSLWLLNAWLLLA, a viral sequence from the Varicella

Zoster Virus (which is somewhat less active than the other preferred polypeptides) ; and

4. LTVIIIWKLLRT, a viral peptide sequence from the

Epstein Barr virus. Non-viral synthetic polypeptides which have been found to have antiviral activity are: WAPTLGSLFELIRCV,

ISVTGPLLRWEFCAL and FSMPCTLWVAEIWLY. The above-mentioned polypeptides all have high hydropathy values and a high propensity to alpha-helix formation as can be seen in the PepPlots Figures 1-7. the predictions for the alpha-helix-forming tendency for these polypeptides has been shown to have a good correlation with alpha helical structure as identified from analysis of some of the polypeptides by H-nmr in SDS.

Apart from the specific polypeptide sequences mentioned above, longer peptides including those sequences may also be used in the invention, as may polypeptides having one or more conservative substitutions and polypeptides including most but not the entire sequences.

The amino acid residues used are preferably naturally occurring amino acids, but other amino acids or amino acid derivatives can be used as one or more of the residues, for instance one or both terminals. Thus peptidomimetic compounds can also be used and are included in the invention.

The present invention comprises also the new polypeptides for use in a method of treatment of the human or animal, as well as pharmaceutical compositions containing the novel polypeptides. Such pharmaceutical compositions generally comprise a pharmacological carrier and may be in the form of an injectable, for instance IV or IM injectable, composition, or compositions for oral administration or for topical application, for instance to skin or mucous membranes at the site of an acute outbreak of an infection. Prophylactic compositions may also be topically applied, for instance at the site of a likely infection, to prevent the initial infection.

Pharmaceutical compositions may contain pharmaceutically acceptable salts or other derivatives of the polypeptides.

The polypeptides of the present invention may also have utility as laboratory reagents for use in the selection of viruses for further investigation.

Although the present invention is of particular value to inhibit infection by herpes viruses, it is believed that the novel polypeptides may also show anti-viral properties for other types and classes of virus.

The present invention is illustrated further in the following examples and the accompanying graphs. In the graphs, figures 1-7 represent partial PepPlots of some of the polypeptides tested in the examples, in particular showing the Chou and Fasman α-helix forming propensities and the Kyte and Doolittle hydropathy.

EXAMPLE 1

The polypeptides listed in Table 1 were synthesised in vitro using a Biotech BT 7300 synthesizer which utilises conventional FMOC chemistry at 10 synthetic cycles per 24 hours. The peptides were dissolved in dimethyl sulphoxide (DMSO) at a concentration of about 2.0 mg/ml and subsequently added to cultures of human epithelial cell mono-layer cultures (HEp2) to give a final concentration of about 10 μg/ml of polypeptide. The cultures were subsequently infected with a laboratory strain of human simplex virus type 1 (HSV1 (strain MP) ) , and cultured in 5% carbon dioxide at 37°C for 48 hours. The laboratory strain of HSV1 is understood to lack an envelope glycoprotein, which makes the plaques formed in the test more readily visible. This is not believed to affect the infectivity of the virus, however. Viral proliferation and its inhibition by synthetic peptides was assessed at 48 hours by fixing the cell culture with formal saline, staining with basic fuchsin and counting the plaques of lysed cells under an inverted microscope. The results are shown in the following table: TABLE 1

SEQUENCE NO. PEPTIDE SEQUENCE VIRUS INHIBITION %

MEAN

ViralSeσuences

1 HFLWMVRLYG 46

2 VLFLRLADSVPRPLD NSf

3 SWLLEYSLLC NSt

4 CMRIRSLLCS 25

5 AWLPSLS 73

6 WWTLLVDLLWLLL 77 Non-Viral Seσuences

7 ISVTGPLLRWEFCAL 93

fNS means not significant

In addition to the specific polypeptides mentioned above DMSO control and 42 other peptide sequences (from my Platelets article (op. cit . ) were tested and shown to give low and insignificant virus inhibition values (ie in the range 0.15%) .

The above results for viral inhibition and the PepPlot for the sequences having good anti-viral properties as compared to the sequences having poor anti-viral properties, show that those sequences having a high propensity to α-helical structure and having relatively hydrophobic residues have good anti-viral properties against HSVl.

EXAMPLE 2

Using the same techniques as in Example 1, further viral peptide sequences were synthesized, which were derived from the sequences 6 and 7 with conservative substitutions. Sequence 7* is the same as 7 but the carboxy terminus is amidated. In addition further non- viral, hydrophobic polypeptide having viral sequences with similar hydropathy and α-helical propensity as sequences 6 and 7 which had been investigated in my article in Platelets (op. cit . ) were selected for testing of their anti-viral properties. These were tested for their anti¬ viral properties against the MP strain of HSVl used in Example 1 to give the results shown in Table 2. PepPlots of these sequences are shown in figures 1-7.

TABLE 2

SEQUENCE PEPTIDE MEAN RANGE

NO. SEQUENCE INHIBITION (%) (%) (OF 3 ASSAYS)

Non-viral sequences

8 WAPTLGSLFELIRCV 94 89-98

7* ISVTGPLLRWEFCALJ 94 91-96

7 ISVTGPLLRWEFCAL 93 88-96

9 FSMPCTLWVAEIWLY 52 40-64

Viral sequences

10 TWLLWWTTILL 87 78-100

6 WWTLLDVDLLWLLL 77 42-98

11 LLSLWLLNAWLLLA 70 23-94

12 LTVIIIWKLLRT 61 30-99 1

These results show that there appears to be a correlation between the propensity to α-helical structure and hydrophobic properties of the peptide sequences (as illustrated in the PepPlots) and the anti-viral properties of the polypeptides.

EXAMPLE 3

Some of the polypeptides synthesized in Example 2 have been tested for their anti-viral properties on another strain of HSVl (the clinical isolate Troisbel) , as well as on a herpes simplex virus type 2 and on cytomegalovirus. The host cell line used for HSV2 (as well as the clinical isolate of HSVl) was the human epithelial cell line used in examples 1 and 2. The host cell line for CMV was, however, MRC5 cells, a human lung fibroblast cell line.

The peptide sequences and results are shown in Table 3.

TABLE 3

EXAMPLE PEPTIDE VIRUS IN HIBITION (%) SEQUENCE

HSVl HSV2 CMV (Troisbel)

Non-Viral sequences

7* ISVTGPLLRWEFCALJ 36 75 28

7 ISVTGPLLRWEFCAL 32 42 21 8 WAPTLGSLFELIRCV 41 42 35

Viral sequences

10 TWLLWWJTILL 37 54 33

12 LTVIIIWKLLRT 38 47 29

6 WWTLLVDLLWLLL 34 23 23

11 LLSLWLLNAWLLLA 8 18 Not tested

The above results show that, although the viral inhibitory properties of the peptide sequences are lower in the clinical isolate of HSVl, than in the laboratory strain, the polypeptides do have significant anti-viral activity. The activity is also demonstrated in other herpes viruses, for instance HSV2 and CMV. From these further results polypeptide no. 11 has less reproducible anti-viral properties than the other sequences tested.

Claims

1. A synthetic or recombinant peptide formed from 5-50 amino acid residues having a predicted secondary structure with an α-helical secondary structure over a stretch of at least 5 amino acid residues and having a Kyte and Doolittle hydropathy measure for the said stretch of at least 5 amino acid residues which is hydrophobic.

2. A polypeptide according to claim 1 which is synthetic.

3. A polypeptide according to claim 1 or claim 2 which is formed of 5-20 amino acid residues, preferably 7-15 residues.

4. A polypeptide according to any preceding claim which includes a sequence selected from the following sequences: TWLLWWITILL, WWTLLVDLLWLLL, LLSLWLLNAWLLLA, LTVIIIWKLLRT, WAPTLGSLFELIRCV, ISVTGPLLRWEFCAL and FSMPCTLWVAEIWLY.

5. A polypeptide according to any of claims 1 to 3 which includes a sequence selected from the following sequences: VLFLRLADSVPRPLD, CMRIRSLLCS, AWLPSLS, WWTLLVDLLWLLL, and ISVTGPLLRWEFCAL.

6. A polypeptide according to claim 1 which is an analogue of a polypeptide according to claim 4 or claim 5 in which up to 80% of the residues are conservatively substituted.

7. Use of a polypeptide according to any preceding claim in the manufacture of a pharmaceutical composition for use in the treatment of a viral infection in a human or animal.

8. A polypeptide according to any of claims 1 to 6 for use in a method of treatment of the human or animal body by therapy.

9. A pharmaceutical composition comprising a polypeptide according to any of claims 1 to 6 and a pharmaceutically acceptable excipient.

10. A pharmaceutical composition according to claim 9 which is suitable for i.m. or i.v. injection or for topical application to skin or mucous membrane.