Influenza virus inhibiting peptides

This application is a continuation of U.S. Ser. No. 10/578,013, filed on May 3, 2006, now U.S. Pat. No. 7,491,793, which is the National Stage of PCT/US2004/36578, filed on Nov. 3, 2004, which claims the benefit of U.S. Provisional Application Ser. No. 60/517,181, filed Nov. 4, 2003, each of which is incorporated herein by reference in its entirety.

The present invention relates to a method of preventing or inhibiting viral infection of a cell and/or fusion between the envelope of a virus and the membranes of a cell targeted by the virus (thereby preventing delivery of the viral genome into the cell cytoplasm, a step required for viral infection). The present invention provides methods for identifying a fusion initiation region, or FIR, of the viruses. The present invention provides for a method of identifying the FIR in these viruses. The present invention further provides for methods of preventing infection by a Type I virus by interfering with its FIR.

All viruses must bind to and invade their target cells 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 internal cellular membrane, (c) destabilisation of the viral envelope and cellular membrane at the fused area to create a fusion pore, (d) transfer of the viral RNA through the pore, and (e) modification of cellular function by the viral RNA.

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 target cell membrane. This insertion is followed by further 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 is unable to spread and propagate within its host if this fusion process is disrupted. Intentional disruption of this fusion process can be achieved by directing peptides and peptide mimics homologous to fusion protein sequences, antibodies that recognize the fusion protein, and other factors that act against the fusion protein.

Structural Similarities among RNA Virus Class I Fusion Proteins. Hemagglutinin 2 (HA2) of influenza virus, an orthomyxovirus, is the prototypic RNA virus Class I fusion protein and contains an amino terminal hydrophobic domain, referred to as the fusion peptide, that is exposed during cleavage of the hemagglutinin precursor protein. The membrane fusion proteins of RNA viruses from several diverse families, including arenaviruses, coronaviruses, filoviruses, orthomyxoviruses, paramyxoviruses, and retroviruses, share several common structural features with HA2 and have been referred to as Class I viral fusion proteins.

It has been observed that the fusion protein of HIV-1, the transmembrane glycoprotein and other retroviral transmembrane proteins, like those of orthomyxoviruses and paramyxoviruses, possess 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 external portion (ectodomain, amino terminus) of HIV-1 transmembrane protein and the transmembrane proteins of other retroviruses, all could fit the scaffold of HA2 structure as determined by x-ray crystallography (Wilson, Skehel, and Wiley, 1981).

Based on these observations, it was predicted that retroviral transmembrane proteins 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 out of these five domains defines a viral envelope protein as a Class I fusion protein. This retroviral transmembrane protein model was subsequently confirmed by structural determinations and mutational analyses (Chan et al., 1997; Kowalski et al., 1991; Weissenhorn et al., 1997). Common structural motifs are present not only in orthomyxovirus and retrovirus fusion proteins, but also in those of paramyxoviruses, filoviruses (such as Ebola virus, EboV) (Gallaher, 1996) and arenaviruses (Gallaher, DiSimone, and Buchmeier, 2001). The Gallaher structural model of the EboV fusion protein (GP2) has also been confirmed by x-ray crystallographic methods (Malashkevich et al., 1999; Weissenhom et al., 1998).

FIG. 1 shows the five, previously-described, domains of the fusion proteins of the six families of Type I viruses. The fusion proteins originate in a hydrophobic fusion peptide, terminate in an anchor peptide, and incorporate an extended amino terminal alpha-helix (N-helix, usually a “heptad repeat” or “leucine zipper”), a carboxyl terminal alpha-helix (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.

Fusion Inhibition in Type I Viruses. Previous attempts by the present inventors (Garry) and others to design peptides and peptide mimics, 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, analogs of the orthomyxoviruses and paramyxoviruses (Richardson, Scheid, and Choppin, 1980) and HIV-1 fusion peptide domains (Gallaher et al., 1992; Owens et al., 1990; Silburn et al., 1998) have been found to block viral infection, presumably by forming inactive heteroaggregates. Peptides corresponding to portions of the N-helix and 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 portion 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 C-helix anti-HIV-1 peptidic drug DP178 (T-20 or FUZEON®). DP178 overlaps the C-helix and the aromatic anchor-proximal domain and inhibits HIV-1 virion: cell fusion at very low concentrations (50% inhibition at 1.7 nM) achievable in vivo following injection. In a clinical trial, 100 mg/day of DP178 caused an approximately 100-fold reduction in plasma 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 transmembrane protein structure (Pozniak, 2001; Sodroski, 1999). Peptidic inhibitors of paramyxoviruses have also been shown to inhibit viral replication (Lambert et al., 1996; Young et al., 1999). Studies by Watanabe and coworkers suggest that a similar approach of targeting the N-helix and the C-helix of EboV GP2 may also lead to useful inhibitors (Watanabe et al., 2000). Neutralizing antibodies directed against portions of the fusion protein domains have also been shown to inhibit virion: cell fusion.

Observations in HIV-1. A great deal of study has been devoted to fusion inhibition in human immunodeficiency virus HIV-1, one of the Type I RNA viruses. Bolognesi et al. (U.S. Pat. No. 5,464,933) and the present inventors (Garry, U.S. Pat. No. 5,567,805) teach that HIV-mediated cell killing can be inhibited by introducing peptides that bind to portions of the transmembrane fusion protein of the HIV-1 virion. The Bolognesi DP178 binding region, labeled FUZEON® in FIG. 7, lies primarily on the C-helix and is outside what is described in the present application the fusion initiation region (FIR). Bolognesi demonstrates inhibition but teaches no method of inhibition. The present inventors (Garry) previously demonstrated inhibition at the CS3 region of HIV-1 ™, labeled CS3 in FIG. 7, but identified no method of inhibition, suggesting only that 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 U.S. Pat. No. 5,567,805 is in fact binding that occurs between the CS3 portion of the FIR and portions of the cell membrane for which the CS3 portion of the FIR has an affinity. In addition, although Melikyan, Watanabe, Bewley, and others have described fusion inhibition with introduced peptides, they have not explained the mechanisms through which the inhibition occurs. Correspondingly, the location of the FUZEON® peptide is distant from the FIR, which strongly suggests that other elements of the fusion process operate in the FUZEON® region.

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

Various embodiments of the instant invention provide for methods of identifying “factors” (compounds) capable of inhibiting membrane fusion between viruses and their host cells and, thereby, preventing or inhibiting infection of the host cell by the virus. Aspects of this embodiment of the invention provide for methods of identifying these inhibitory “factors” where the method comprises the steps of (a) identifying a virus having an envelope fusion protein having two, or more, extended alpha helices, a fusion peptide, and a fusion initiation region (FIR); (b) preparing a “target” wherein the target comprises the amino acid sequence of the FIR, (c) exposing the “target” to one or more test compounds, and (d) identifying those test compounds that physically interact with the “target”. For example, physical interaction can be detected using a “target” bound to a solid substrate and a fluorescently or radioactively labeled test compound in a standard binding assay. Target and test compounds having dissociation coefficients (K_d) in the micromolar range or lower (i.e. ≦about 9×10⁻⁶) are considered to be positively interacting.

Other aspects of the instant invention provide for compositions comprising an isolated peptide having the amino acid sequence of a viral fusion initiation region (FIR) or a functional segment of the FIR or having an amino acid sequence which is analogous to the sequence of a FIR or a functional segment of a FIR. As used herein, an analogous amino acid or peptide sequence is a sequence containing a majority of identical or chemically similar amino acids in the same order as a primary sequence. Such chemical similarities are well known to those skilled in the art.

Other aspects of this embodiment of the invention provide for isolated, typically substantially purified, peptides or peptide analogs that are capable of preventing or inhibiting viral infection of a host cell and/or inhibiting membrane fusion of a virus with a host cell, where the virus comprises a membrane fusion protein having two (extended) alpha helices, a fusion peptide and a FIR.

Additional embodiments of the instant invention provide for methods of treating or preventing viral infection by administering to a patient one or more of the compounds identified by the methods described herein as capable of inhibiting viral infection. In various aspects of this embodiment of the invention the compounds administered are peptides or peptide analogs comprising all or a functional segment of a viral FIR sequence. In any aspect of this embodiment of the invention the administered compound is antigenic and is administered in an amount sufficient to eliciting an immune response.