Method of preventing virus:cell fusion by inhibiting the function of the fusion initiation region in rna viruses having class I membrane fusogenic envelope proteins

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DESCRIPTION

Method to prevent virus: cell fusion by inhibiting the function of the fusion initiation region in RNA viruses having class I fusogenic membrane envelope proteins

This application claims the benefit of US Provisional Application Serial Number 60 / 517,181, filed November 4, 2003.

Field of the invention

The present invention relates to peptides for use in preventing or inhibiting viral infection by a retrovirus of a cell (thereby preventing insertion of the viral genome into the cell cytoplasm, a required step for viral infection). The present invention provides compositions and uses thereof to prevent infection by a retrovirus by interfering in its fusion inhibiting region (FIR).

Introduction

All viruses must bind to, and invade, their target cells in order to replicate. For enveloped animal viruses, including RNA viruses having class I membrane fusion proteins (type I viruses), the process involves (a) binding of the virion to the target cell, (b) fusion of the envelope of the virus with the plasma membrane or an inner cell membrane, (c) destabilization of the virus envelope and cell membrane in the fused zone to create a fusion pore, (d) the transfer of the viral RNA through the pore and (e) the modification of cell function by the viral RNA.

The fusion of the viral membrane and the cell envelope, steps (b) and (c) above, is mediated by the interaction of a viral transmembrane glycoprotein (fusion protein) with surface proteins and membranes of the target cell. These interactions cause conformational changes in the fusion protein that result in the insertion of a viral fusion peptide into the membrane of the target cell. This insertion is followed by additional conformational changes within the fusion protein that bring the viral envelope and cell membranes into close proximity and results in the fusion of the two membrane bilayers.

A virus cannot spread and spread within its host if this fusion process is disturbed. Intentional disruption of this fusion process can be accomplished by targeting homologous peptides and peptidomimetics to fusion protein sequences, antibodies that recognize the fusion protein, and other factors that act against the fusion protein.

Background of the invention

Structural similarities between RNA virus class I fusion proteins.

Influenza virus hemagglutinin 2 (HA2), an orthomyxovirus, is the prototypical RNA virus class I fusion protein and contains an amino-terminal hydrophobic domain, termed a fusion peptide, that is exposed during cleavage of the precursor protein of hemagglutinin. The membrane fusion proteins of RNA viruses of several diverse families, including arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses, share several common structural features with HA2 and have been termed class I viral fusion proteins. The HIV-1 fusion protein, transmembrane glycoprotein, and other retroviral transmembrane proteins, such as orthomyxoviruses and paramyxoviruses, have been shown to have a hydrophobic fusion peptide domain exposed during cleavage of a precursor (gp160) (Gallaher , 1987; Gonzalez-Scarano et al., 1987). Based on these similarities and computer algorithms that predict protein configurations, it has been suggested (Gallaher et al., 1989) that the outer part (ectodomain, amino-terminal end) of the HIV-1 transmembrane protein and the transmembrane proteins of others retrovirus, could fully fit the HA2 structure scaffold as determined by X-ray crystallography (Wilson, Skehel, & Wiley, 1981).

Based on these observations, retroviral transmembrane proteins were predicted to contain several structural features in addition to the fusion peptide in common with the known structure of HA2, including an extended amino-terminal helix (N-helix, usually a "heptad repeat "Or" leucine zipper "), a carboxyl-terminal helix (C-helix), and an aromatic motif proximal to the transmembrane domain. The presence of at least four of these five domains defines a viral envelope protein as a class I fusion protein. This retroviral transmembrane protein pattern was subsequently confirmed by structural determinations and mutational analyzes (Chan et al., 1997; Kowalski et al., 1991; Weissenhorn et al., 1997). Common structural motifs are present not only in fusion proteins of orthomyxovirus and retrovirus, but also in those of paramyxovirus, filovirus (such as Ebola virus, VEbo) (Gallaher, 1996) and arenavirus (Gallaher, DiSimone and Buchmeier, 2001) . Gallaher's structural model of the VEbo fusion protein (GP2) has also been shaped by X-ray crystallography methods (Malashkevich et al., 1999; Weissenhorn et al., 1998).

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Figure 1 shows the five domains, previously described, of the fusion proteins of the six families of type I viruses. Fusion proteins originate from a hydrophobic fusion peptide, terminate on an anchor peptide, and incorporate an extended amino-terminal alpha helix (N-helix, usually a "heptad repeat" or "leucine zipper"), a helix alpha carboxyl-terminal (C-helix) (Carr and Kim, 1993; Suarez et al., 2000; Wilson, Skehel, and Wiley, 1981), and sometimes an aromatic motif proximal to the virion envelope. Also shown is the sixth domain, the fusion initiation region (FIR), discovered by the present inventors.

Inhibition of fusion in type I viruses

Previous attempts by the present inventors (Garry) and others to design peptides and peptidomimetics, antibodies, and other factors that inhibit fusion in type I viruses have focused on the fusion peptide, the N-helix, and the C-helix of the fusion proteins. In the case of fusion peptides, it has been found that orthomyxovirus and paramyxovirus analogues (Richardson, Scheid & Choppin, 1980) and HIV-1 fusion peptide domains (Gallaher et al., 1992; Owens et al., 1990; Silburn et al., 1998) block viral infection, presumably forming inactive heteroaggregates. Peptides corresponding to parts of the Nhelix and the C-helix have also been found to be effective in inhibiting viral infection both in vitro and in vivo. For example, a 17 amino acid peptide corresponding to the carboxy-terminal part of the N-helix of the HIV-1 fusion protein, defined as the CS3 region, blocked HIV infection (Qureshi et al., 1990). In addition, other N-helix and C-helix inhibitory peptides were developed based on the fusion protein structural model (Wild, Greenwell and Matthews, 1993; Wild et al., 1992), including the anti-HIV peptide drug- 1 of C-helix DP178 (T20 or FUZEON®). DP178 overlaps the C-helix and proximal aromatic anchor domain and inhibits virion: cell fusion of HIV-1 at very low concentrations (50% inhibition at 1.7 nM) that can be achieved in vivo after injection. In a clinical trial, 100 mg / day of DP178 caused an approximately 100-fold reduction in plasma of the HIV-1 load of infected individuals (Kilby et al., 1998). This result has greatly motivated the search for other HIV-1 inhibitory peptides based on the transmembrane protein structure (Pozniak, 2001; Sodroski, 1999). Peptide inhibitors of paramyxovirus have also been shown to inhibit viral replication (Lambert et al., 1996; Young et al., 1999). Studies by Watanabe et al. Suggest that a similar approach to N-helix and C-helix targeting of VEbo GP2 may also lead to useful inhibitors (Watanabe et al., 2000). Neutralizing antibodies directed against parts of fusion protein domains have also been shown to inhibit virion: cell fusion.

Observations on HIV-1

A great deal of study has been devoted to the inhibition of fusion in the human immunodeficiency virus HIV-1, one of the type I RNA viruses. Bolognesi et al. (5,464,933) and the present inventors (Garry, USPN 5,567,805) teach that HIV-mediated cell killing can be inhibited by introducing peptides that bind to parts of the HIV-1 virion transmembrane fusion protein parts . The DP178 binding region of Bolognesi, labeled FUZEON® in Figure 7, is primarily based on the C-helix and is outside of what is described in the present application the fusion initiation region (FIR). Bolognesi demonstrates inhibition but does not teach any method of inhibition. The present inventors (Garry) previously demonstrated inhibition in the CS3 region of HIV-1 TM, labeled CS3 in Figure 7, but did not identify any inhibition method, suggesting only that the CS3: CS3 receptor interaction is inhibited. . The unexpected discovery of the FIR by the present inventors (as currently described herein) and the fact that the CS3 sequences lie within the FIR indicates that the CS3: CS3 receptor binding described in USPN 5,567,805 is in fact the binding that occurs between the CS3 part of the FIR and parts of the cell membrane for which the CS3 part of the FIR has an affinity. Furthermore, although Melikyan, Watanabe, Bewley, and others have described the inhibition of fusion with introduced peptides, they have not explained the mechanisms through which the inhibition occurs. Correspondingly, the location of the FUZEON® peptide is remote from the FIR, strongly suggesting that other elements of the fusion process operate in the FUZEON® region. WO 88/08429 describes HIV peptides for use in immunotherapy. The synthesis of an HIV peptide and its subsequent use to produce antibodies for the treatment of HIV infection has been reported (Viveros et al., 2000). Studies on the HIV type 1 gp41 supercoiled helix have also been reported (Chan et al., 1998).

In view of the foregoing, it is clear that there is a need in the art for more effective means to identify those regions of the virus that are involved in the infection process and for compositions effective to prevent or inhibit viral infection. The invention described and disclosed herein provides an effective solution to these needs.

Summary of the invention

According to a first aspect of the invention, there is provided an isolated peptide selected from the group consisting of:

(a) a peptide having an amino acid sequence consisting of SEQ ID NO: 7,

ES 2 425 600 T3 (b) a peptide consisting of 8 to 40 contiguous amino acid residues of SEQ ID NO: 7 and not including residues 65-81 of SEQ ID NO: 21 (conserved sequence 3 of transmembrane protein human immunodeficiency virus 1; HIV-1 TM CS3), (c) a peptide analog having an amino acid sequence consisting of SEQ ID NO: 7 that includes one or more conservative amino acid substitutions and in which most residues of the analog are identical to the sequence of SEQ ID NO: 7; and (d) a peptide analog consisting of 8 to 40 contiguous amino acid residues of SEQ ID NO: 7 that includes one or more conservative amino acid substitutions and in which the majority of the analog residues are identical to the sequence of the 8 to 40 contiguous amino acid residues of SEQ ID NO: 7; wherein the analog does not include amino acid residues 65-81 of SEQ ID NO: 21, for use in inhibiting virus: cell fusion in the treatment of a retroviral infection.

The peptide can include an acetyl group, a carbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, a hydrophobic group, or a macromolecular group at the amino terminus of the peptide. In addition, such peptides can include an amido group, a hydrophobic group, or a macromolecular group at the carboxyl-terminus of the peptide. Retroviral infection can be an HIV-1 infection.

According to a second aspect of the invention, there is provided the use of a peptide according to the first aspect above for the preparation of a medicament for treating a retroviral infection in a patient. Retroviral infection can be an HIV-1 infection.

According to a third aspect of the invention, there is provided a recombinant DNA molecule that enables, or stimulates, a patient to produce the peptide according to the first aspect above to treat a retroviral infection in a patient. This aspect includes the use of a recombinant DNA molecule that enables, or stimulates, a patient to produce the peptide according to the first aspect above for the preparation of a medicament for treating a retroviral infection in a patient. Retroviral infection can be an HIV-1 infection.

According to a fourth aspect of the invention, there is provided a viral fusion inhibiting agent comprising a peptide having an amino acid sequence consisting of 8 to 50 amino acid residues for use in treating a retroviral infection, in where the peptide comprises an amino acid sequence of a peptide of the first aspect above. The viral fusion inhibiting agent may include an acetyl group, a carbobenzoxyl group, a dansyl group, a t-butyloxycarbonyl group, a hydrophobic group, or a macromolecular group at the amino terminus thereof. Furthermore, the viral fusion inhibiting agent may include an amido group, a hydrophobic group, or a macromolecular group at the carboxyl-terminus thereof. This aspect includes the use of a viral fusion inhibiting agent of this aspect for the preparation of a medicament for treating a retroviral infection in a patient.

Illustrative embodiments of the invention

The sixth domain of RNA viruses that have class I membrane fusion proteins

Arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses are the six currently identified families of RNA viruses that have class I membrane fusion envelope proteins. The present inventors (Garry) and others have previously shown that the proteins Fusion of these type I viruses incorporate five conserved domains or motifs (Carr and Kim, 1993; Gallaher et al., 1989; Suarez et al., 2000; Wilson, Skehel and Wiley, 1981). These domains comprise a fusion peptide, an N-helix, a C-helix, and an aromatic motif, which are all ectodomains, and an anchor peptide, which is an endodomain.

Using computational analysis, secondary structure modeling, surface hydrophobicity calculations, and other techniques, the present inventors have made the surprising discovery of a highly conserved sixth domain that is present in the fusion proteins of a wide variety of viruses (this sixth domain is described in this document). Viruses exhibiting this domain include, but are not necessarily limited to, the six classes of RNA viruses listed above. To emphasize the critical function of this newly identified domain, which is an ectodomain, the domain is referred to herein as the fusion initiation region (FIR) of viruses.

As used herein, the term "extended" alpha helix refers to an alpha helix that has more than four "turns of alpha helix" (specifically, more than 14 amino acids).

Other embodiments envision "factors" that the inventors have unexpectedly found to be effective in preventing or preventing viral infection and / or virus: cell fusion.

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As used herein the term "factors" includes, but is not limited to, isolated peptides or functional peptide segments (or peptide analogs thereof) from fusion initiation region (FIR) domains. Just described, peptidomimetics ("peptidomimetic" refers to any compound or substance that could serve as a substitute for a peptide that interacts with the FIR, that is, any compound that mimics the properties of a functional segment of the FIR), antibodies specific for functional FIR domains (for example, idiotypic or anti-idiotypic antibodies) and other molecular compounds that interfere with virus binding and / or fusion: cell.

As used herein the term "functional segment" or "functional fragment" of a fusion initiation region (FIR) refers to a fragment that can inhibit virus: cell fusion, inhibit viral infectivity, which can produce an antibody that can specifically recognize and bind FIR and / or interfere with FIR-mediated cell infection.

As used herein, a "peptide analog" or "modified peptide" is preferably defined as an FIR peptide modified to contain an amino group, an acetyl group, a hydrophobic group (eg, carbobenzoxyl, dansyl, or t-butyloxycarbonyl) or a macromolecular carrier group (eg, lipid conjugate, polyethylene glycol, a carbohydrate, or a protein) at the amino terminus. A further class of FIR peptide analogs contains a carboxyl group, an amido group, a hydrophobic group, or a macromolecular carrier group at the carboxyl terminus. Other peptide analogs are defined as FIR peptides in which at least one bond that joins adjacent amino acid residues is a non-peptide bond (eg, an imido, ester, hydrazine, semicarbazoid, or azo bond), a peptide in which at The least one amino acid residue is in a D-isomer configuration or a peptide in which the order of the amino acids is reversed. Additional peptide analogs are FIR peptides that commit at least one amino acid substitution in which a first amino acid residue is replaced by a different second amino acid residue (the amino acid substitution can be a conserved substitution or a non-conserved substitution). As used herein, such peptide analogs can comprise analogous amino acid sequences, with the analogous sequences containing most identical or chemically similar amino acids in the same order as the primary sequences.

As used herein, the term "fusion initiation region" (FIR) generally refers to a region of a viral fusion protein involved in the initial stage (s) of viral infection and / or fusion with a host cell.

As used herein the term "peptidomimetic" includes, but is not limited to, organic compounds or other chemicals that mimic the structure or function of the FIR peptide. Examples of peptidomimetics include, but are not limited to, organic compounds that comprise the functional side groups of an amino acid or peptide, but that lack the carbon / nitrogen backbone or peptide bonds. Peptidomimetic also refers to compounds that mimic the action of these functional side groups with other moieties.

Other molecules, such as idiotypic or anti-idiotypic antibodies or proteins selected by phage display methods, that bind to the peptides, peptide analogs or peptidomimetics described in the present application may also function as inhibitors of viral infection and / or the virus: cell fusion. Also contemplated by the present invention are plasmids, or recombinant viruses, or other molecules or compounds that allow the, or stimulate, the patient to produce an analog of the inhibitory compounds. For example, a recombinant protein, produced in an engineered bacterial, fungal, or mammalian cell can be used to produce an immunogenic FIR analog of a viral fusion protein. Similarly, an anti-idiotypic antibody response could be induced in the individual using an engineered protein comprising a sequence corresponding to the binding site of a FIR-specific antibody.

As used herein, the term "fusion peptide" preferably refers to a hydrophobic sequence at or near the amino terminus of a class I viral fusion protein (see, Gallaher et al. , 1987; 1992).

As used herein, the term "substantially purified" peptide or peptide analog preferably refers to a peptide or peptide analog that is greater than about 80% pure. More preferably, "substantially purified" refers to a peptide or peptide analog that is more than about 90% pure or more than about 95% pure. Most preferably, it refers to a peptide or peptide analog that is more than 96%, 97%, 98%, or 99% pure. Functionally, "substantially purified" means that it is free of contaminants to a degree that makes it suitable for the purposes envisaged herein. Methods for evaluating purity are well known to those of skill in the art. Suitable methods include, but are not limited to, analysis by gas chromatography (GC) coupled to mass spectrophotometry, high performance liquid chromatography (HPLC), and functional assays in cell culture systems that, among others, evaluate cytotoxicity. .

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As used herein, the term "stable analog" refers to a peptide that has a pharmacologically active half-life in biological systems. Biological half-lives of more than 60 minutes are contemplated.

As used herein, the term "peptide derivative" refers to a peptide having substituted amino acids other than those in the FIR sequence of a viral fusion protein. In them, the substitutions do not render the peptide useless for the present invention.

According to various aspects of the present embodiment of the invention, peptides, isolated nucleic acid sequences, or antibodies can be produced by any means known in the art, including but not limited to chemical synthesis, recombinant DNA methods, and combinations thereof.

As defined herein, the present invention provides compositions and uses for treating or preventing retrovirus infection. One possible mechanism by which the present invention may prevent and / or inhibit infection is by interfering with FIR-mediated virus: cell fusion.

Brief description of the figures

Figure 1 shows the domains of the fusion proteins of one member of each of these six viral families (specifically, arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses). The circles in Figure 1 show the approximate location of the FIR in each illustrated virus.

Figures 2 to 7 show the amino acid sequences of these fusion proteins (corresponding to SEQ ID NO 16-21, respectively) and a schematic representation of their ectopic structure. Specifically, the five domains described previously are shown, which are the fusion peptide, ie, the N-helix, the C-helix, the aromatic motif (if present), and the anchor peptide. The newly discovered sixth domain, the fusion initiation region or FIR, is also identified. Each FIR is indicated by a polygon in Figures 2 to 7.

The circled area behind the fusion proteins in each of Figures 2-7 represents the primary virus: cell binding protein (VCBP) of the virus. VCBP usually interacts with the part of the fusion protein that is most distal to the viral membrane and is therefore shown to be so located in the figures. Unlike the highly conserved fusion protein, the VCBP of each family of viruses is more divergent. Usually it is the VCBP that dictates the host range of the virus and determines which of the host's cell types are targeted for infection. VCBP acts in this capacity by recognizing and binding to specific cell surface proteins. Binding of VCBP to targeted cell proteins occurs prior to, and is usually a prerequisite for, virus: cell fusion.

Figure 8: Inhibition of coronavirus infectivity by fusion initiation region peptides. Between 50 and 100 PFU of mouse hepatitis virus strain A59 or SARS coronavirus (severe acute respiratory syndrome) strain Urbani with or without the indicated peptides (~ 100 pM) were preincubated in serum-free DMEM during 1 hour. Cells were then exposed to peptide treated inoculum or a vehicle control (no peptide). After 1 hr of adsorption, the inoculum was removed, the cells were washed twice with 1X phosphate buffered saline, and the cells were coated with DMEM containing 10% FBS and 0.5% agarose. Forty-eight hours after infection, infected monolayers were fixed and stained with crystal violet to determine plaque numbers.

Figure 9: Inhibition of Lassa virus infectivity by fusion initiation region peptides. Between 50 and 100 PFU of Lassa virus with or without the indicated peptides (-100 pM) were preincubated in serum-free BME for 1 h. The cells were then exposed to the peptide treated inoculum or vehicle control (no peptide). After 1 hr of adsorption, the inoculum was removed, cells were washed twice with 1X phosphate buffered saline, and cells were coated with BME containing 5% FBA, 10 mM HEPES, and 0.5% agarose. Four days after infection, a second coating containing 5% neutral red was applied, and plaques were counted 24 h later.

The six families of RNA viruses that are now known to have class I membrane fusion proteins (type I viruses) and representative members of each family are as follows:

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Representative RNA Viruses Having Type I Membrane Fusion Proteins (Type I Viruses)

Representative Virus Family Shown in Figures

<td colspan="2">Lassa virus</td><td>Yes</td>

<td></td><td>Lymphocytic choriomeningitis virus (MLV)</td><td>No</td>

<td></td><td>Junín virus</td><td>No</td>

<td></td><td>Machupo virus</td><td>No</td>

<td></td><td>Guanarito virus</td><td>No</td>

<td></td><td>Sabia virus</td><td>No</td>

<td></td><td>Severe acute respiratory syndrome virus (SARS)</td><td>Yes</td>

<td></td><td>Murine hepatitis virus (MHV)</td><td>No</td>

<td></td><td>Bovine coronavirus</td><td>No</td>

<td></td><td>Canine coronavirus</td><td>No</td>

<td>Arenavirus</td><td>Feline infectious peritonitis virus</td><td>No</td>

<td>Coronavirus</td><td>Ebola virus</td><td>Yes</td>

<td>Filovirus</td><td>Marburg virus</td><td>No</td>

<td>Orthomyxovirus</td><td>Influenza A virus</td><td>Yes</td>

<td>Paramyxovirus</td><td>Influenza B virus</td><td>No</td>

<td>Retrovirus</td><td>Influenza C virus</td><td>No</td>

<td></td><td>Measles virus</td><td>Yes</td>

<td></td><td>Mumps virus</td><td>No</td>

<td></td><td>Canine distemper virus</td><td>No</td>

<td></td><td>Newcastle disease virus</td><td>No</td>

<td></td><td>Immunodeficiency virus 1 (HIV-1)</td><td>Yes</td>

<td></td><td>Immunodeficiency virus 2 (HIV-2)</td><td>No</td>

<td></td><td>Human T-cell lymphotropic virus type 1 (HTLV-1)</td><td>No</td>

<td></td><td>Human T-cell lymphotropic virus type 2 (HTLV-2)</td><td>No</td>

<td></td><td>Human intracisternal type A particle 1 (HIAP-1)</td><td>No</td>

<td></td><td>Human intracisternal type A particle 2 (HIAP-2)</td><td>No</td>

The viruses shown in the figures are as follows: