# Patent

## Title
Flavivirus fusion inhibitors

## Abstract

The present invention relates to peptides and methods of inhibiting fusion between the virion envelope of Flaviviruses and membranes of the target cell, the process that delivers the viral genome into the cell cytoplasm. The invention provides for methods which employ peptides or peptide derivatives to inhibit Flavivirus:cell fusion. The present invention is based in part on the discovery that E1 envelope glycoprotein of hepaciviruses and E2 envelope glycoprotein of pestivirus have previously undescribed structures, truncated class II fusion proteins. The present invention provides peptides and methods of treatment and prophylaxis of diseases induced by Flaviviruses.

## Background
This application is a divisional of Appl. Ser. No. 10/532,480, filed Apr. 22, 2005, now U.S. Pat. No. 7,416,733, which is a §371 U.S. national stage filing of international application PCT/US2003/035666, filed Nov. 7, 2003, which claims priority to U.S. Provisional Appl. Ser. No. 60/424,746, filed Nov. 8, 2002, each of which is incorporated by reference herein in its entirety.


1. FIELD OF THE INVENTION
The present invention relates to peptides and methods of inhibiting cell infection and/or virion:cell fusion by members of the Flaviviridae family.
2. BACKGROUND OF THE INVENTION
2.1. Entry of enveloped animal viruses requires fusion between the viral membrane and a cellular membrane, either the plasma membrane or an internal membrane. Class I fusion proteins possess a “fusion peptide” at or near the amino terminus, a pair of extended α helices and, generally, a cluster of aromatic amino acids proximal to a hydrophobic transmembrane anchoring domain (Carr and Kim, 1993; Suarez et al., 2000; Wilson, Skehel, and Wiley, 1981). Several otherwise disparate viruses, including orthomyxoviruses, paramyxoviruses, retroviruses, arenaviruses, and filoviruses encode class I fusion proteins varying in length and sequence, but highly similar in overall structure (Gallaher, 1996; Gallaher et al., 1989). X-ray crystallography of the E glycoprotein (E-protein) of tick-borne encephalitis virus (TBEV), a member of the genus flavivirus of the Flaviviridae family, revealed a structure for this fusion protein distinct from other fusion proteins (Rey et al., 1995). E-protein possesses an internal fusion peptide stabilized by dicysteine linkages and three domains (I-III) comprised mostly of antiparallel β sheets. In the slightly curved rod-like configuration of the E-protein present in the virion, the fusion peptide is located at the tip of domain II, the furthest point distal from the C-terminal transmembrane anchor. Examination by Lescar and coworkers (2001) of E1, the fusion protein of the Togavirus Semliki Forest virus (SFV), revealed a remarkable fit to the scaffold of TBEV E. Recently, the E-glycoprotein of dengue virus, a medically important flavivirus, was also shown to have a class II structure (Kuhn et al., 2002).
2.2. The Flaviviridae family consists of three genera, flaviviruses, hepaciviruses and pestiviruses. In the United States alone, 4 million people are infected with a member of the hepacivirus genus, hepatitis C virus (HCV). This is four times the number infected by HIV. Each year in the US, 30-50,000 new HCV infections occur, and about 15-20,000 people die. These numbers are expected to increase dramatically. The infection is spread primarily through needle sharing among drug users, although there is some risk from accidental needle sticks, blood products before 1992, chronic blood dialysis, and frequent sexual contact. Current treatments for HCV using ribavirin and interferon cost $8,000 to $20,000 per year, and help about half of patient only partly. End stage HCV disease is the most frequent indication for liver transplants and this costs $250,000 to $300,000. Better drugs to treat HCV infection and an effective vaccine to prevent HCV infection are urgently needed. Members of the flavivirus genus, dengue virus, Japanese encephalitis virus, yellow fever virus, and West Nile virus, cause important human diseases world-wide. Pestiviruses, such as bovine viral diarrhea virus and border disease virus, cause significant veterinary illnesses.
3. SUMMARY OF THE INVENTION
Based on sequence similarities, it is likely that the E glycoproteins of other members of the flavivirus genus within the family Flaviviridae, including West Nile virus, are also class II fusion proteins. Analyses presented herein indicate that glycoproteins of viruses from members of the other two genera of the Flaviviridae family, hepaciviruses and pestiviruses, have previously undescribed structures. The envelope glycoprotein E1 of hepatitis C virus, a hepacivirus, and the envelope glycoprotein E2 of pestiviruses have novel structures, resembling a truncated version of a class II fusion protein. No viral protein has previously been identified with this structure. Our observations were unexpected and contrast with published studies. Hepatitis C virus encodes two envelope glycoproteins, E1 (gp35) and E2 (gp70), both with C-terminal transmembrane anchor domains. Prior studies indicated that another HCV protein, E2, has a class II structure. The structural determinations of the hepacivirus and pestivirus fusion proteins allow the identification of several heretofore unknown features of Flavivirus fusion proteins for drug and vaccine development.
Thus, the instant invention teaches that HCV envelope glycoprotein E1 has a previously unknown structure, a truncated class II fusion protein. This structure identifies regions of HCV E1 and other class II viral fusion proteins important for virus:cell fusion. This invention also teaches that peptides can be designed to inhibit viruses, including HCV and other members of the Flaviviridae family, that have fusion peptides with a class II structure.
Structural features of Flavivirus envelope glycoproteins identified herein provide surprising guidance for the development of vaccines and/or drugs to prevent or treat Flavivirus infections. Prior to the availability of X-ray structural data (Wild, Greenwell, and Matthews, 1993; Wild et al., 1994), several potent HIV-1 TM inhibitors were developed based on the Gallaher HIV-1 TM fusion protein model (Gallaher et al., 1989). DP178 (T20) peptide (FIG. 5A) has been shown to substantially reduce HIV-1 load in AIDS patients in preliminary results from phase III clinical trials. (Hoffman-La Roche and Trimeris, 2002). Peptide drugs should be relatively easy to develop, based on our structures. Once an effective peptide inhibitor is described a non-peptide drug can be developed.
More specifically, the present invention provides for methods of inhibiting viral infection by Flaviviruses and/or fusion between the virion envelope of Flaviviruses and membranes of the target cell (the process that delivers the viral genome into the cell cytoplasm). The invention is related to the discovery, as described herein, that hepacivirus envelope glycoprotein E1 and pestivirus E2 glycoprotein have novel structures. The invention provides for methods that employ peptides or peptide derivatives to inhibit Flavivirus:cell fusion. The present invention provides for methods of treatment and prophylaxis of diseases induced by Flaviviruses.
Various embodiments of the instant invention provide for pharmaceutical compositions comprising one or more peptides selected from one or more of the following groups.

    A) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:36;
    B) Peptides homologous to any one of SEQ ID NO:1 to SEQ ID NO:36, except that they are from a different flavivirus.
    C) Peptides that are functionally equivalent to any one of SEQ ID NO:1 to SEQ ID NO:36, wherein the functionally equivalent peptide is identical to at least one of SEQ ID NO:1 to SEQ ID NO:36 except that one or more amino acid residues has been substituted with a homologous amino acid, resulting in a functionally silent change, or one or more amino acids has been deleted.


Various aspects of this embodiment of the invention provide for compositions that comprise one or more peptides selected from the following.

    A) Peptides having the amino acid sequence one or more of SEQ ID NO:1 to SEQ ID NO:36, wherein the N-terminal “Xaa” is an amino group and the C-terminal “Xaa” is a carboxyl group.
    B) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:36, wherein the N-terminal “Xaa” is not an amino group and/or the C-terminal “Xaa” is not a carboxyl group, wherein the N-terminal “Xaa” is selected from the group consisting of: an acetyl group, a hydrophobic group, carbobenzoxyl group, dansyl group, a t-butyloxycarbonyl group, or a macromolecular carrier group, and/or wherein the C-terminal “Xaa” is selected from the group consisting of an amido group, a hydrophobic group, t-butyloxycarbonyl group or a macromolecular group.
    C) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:36 except that at least one bond linking adjacent amino acid residues is a non-peptide bond.
    D) Peptides having the sequence of any of SEQ ID NO:1 to SEQ ID NO:36, except that at least one amino acid residue is in the D-isomer configuration.
    E) Peptides as in groups “A)” or “B)” except that at least one amino acid has been substituted for by a different amino acid (whether a conservative or non-conservative change).
    F) Peptides that are a functional fragment of a peptide as set out in any of groups “A)” to “E)”, above, where the peptides have at least 3 contiguous nucleotides of any one of SEQ ID NO:1 to SEQ ID NO:36.


The instant invention also provides for substantially purified antibodies that specifically react with one or more of the peptides described above.
The instant invention also provides for methods for treating or preventing viral infections in an animal where the method comprises administering to an animal or human peptides and/or antibodies as described above.
3.1. Abbreviations

    HIV—human immunodeficiency virus
    TBEV—tick-borne encephalitis virus
    DV—dengue virus
    WNV—West Nile virus
    HCV—hepatitis C virus
    GBV—hepatitis GB virus
    CSFV—classical swine fever virus
    BVDV—bovine viral diarrhea virus
    BD—border disease virus
    HSA—human serum albumen





4. BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1. Alignments of tick-borne encephalitis virus E, hepatitis C virus E1 and classical swine fever virus E2 glycoproteins. Panel A: Certain portions of the TBEV E(SEQ ID NO:66), HCV E1 (SEQ ID NO:67), CSFV E2 (SEQ ID NO:68) and HCV E2 (SEQ ID NQ:71) polyproteins are aligned. Bracketed HCV insert sequences (HCV E1. SEO ID NO:69HCV E2, SEQ ID NO:70) are wrapped and do not represent an alignment comparison (FIG. 1A cont.). “(:)” refers to identical amino acids. “(.)” refers to chemically similar amino acids. Panel B: Linear arrangement of the domain structure of TBEV E as determined by Rey et al. (1995). Regions of significant sequence similarities to TBEV E in HCV E1 and E2 and CSFV E2 as determined by the PRSS3 sequence alignment program are indicated. Probabilities (p-values) are based on 1000 shuffles.
FIG. 2. Structures of hepacivirus E1 and pestivirus E2 glycoproteins. Panel A. Structure of TBEV E as determined by Rey et al. (1995) is shown schematically (traced from a RasMac molecular visualization software rendering). Panel B: Structure of HCV E1. HCV E1 sequences with similarity to TBEV E sequences are enclosed in quotation marks. Panel C: Structure of CSFV E2.
FIG. 3. Alignments of the precursor of tick-borne encephalitis virus small membrane protein, prM, and classical swine fever virus El. Panel A: Aligned portions of the TBEV small membrane protein (prM, SEQ ID NO:72) and CSFV E1 (SEQ ID NO:73) protein. Alignments were constructed as detailed in the text. Panel B: Linear arrangement of TBEV prM and CSFV E1 with a region of sequence similarity determined by the PPSS3 algorithm indicted.
FIG. 4. Common order of proteins in Flaviviridae polyproteins. Proteins or portions of proteins with similar functions are located in similar locations along the polyproteins of members of the Flaviviridae. Hydrophobic domains were predicted using TMpred.
FIG. 5. Comparison of human immunodeficiency virus transmembrane glycoprotein (TM) with hepatitis C virus envelope glycoprotein 1 (E1). Presented on the left side of the figure is an updated structure of HIV-1TM sequence (SEQ ID NO:74) from Gallaher et al. (1989) with structural motifs indicated in rainbow order. Amino acids are numbered from the beginning of the Env polyprotein HIV-1TM is truncated after the transmembrane domain. The precise ends of the TM N- and C-helices are unclear because of conflicting structural data. No attempt was made to align the N- and C-helices, although points of contact are solved in the coiled-coil formations. Positions of known neutralizing epitopes on TM are indicated, as well as sequences corresponding to peptides CS3 and DP178 (T20) (Qureshi et al., 1990; Wild et al., 1994) that inhibit HIV-1infectivity. Presented on the right side of the figure is the structure of HCV E1 sequence (SEQ ID NO:75) with motifs that are shared with HIV-1 TM. Boxed arrows are predicted beta sheet structures that are similar to the indicated β sheets of TBEV E. Predicted a helical structures are outlined. Arrows denote directions that the HCV E1 structure could fold in three dimensions.



5. DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to methods of inhibiting Flavivirus infection that comprises inhibiting the fusion between the virion envelope and a cell membrane, the process that delivers the viral genome into the cell cytoplasm. For purposes of clarity of disclosure, and not by way of limitation, the description of the present invention will be divided into the following subsections:

    (i) peptides of the invention
    (ii) utility of the invention

5.1. Peptides of the Invention



Any peptide or protein which inhibits the fusion between the Flavivirus virion envelope and a cell membrane, including those of Flaviviruses which infect human as well as nonhuman hosts, may be used according to the invention. In various embodiments of the invention, these inhibitors may include, but are not limited to peptides related to several membrane-interactive domains of Flavivirus fusion proteins.
Flavivirus inhibitory peptides are, according to the instant invention, identical or homologous to the amino acid sequences HCV Fusion Inhibitory Protein 1, X-YQVRNSSGLYHVTNDCPNSSIVYEAADAIL-Z (SEQ ID NO:1); HCV Fusion Inhibitory Protein 2, X-CSALYWVGDLCGSVFLVGQLFTFSPRRHWTTQDC-Z (SEQ ID NO:2); HCV Fusion Inhibitory Protein 3, X-SPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPT-Z (SEQ ID NO:3); or HCV Fusion Inhibitory Protein 4, X-MMMNWSPTAALLRIPQAIMDMIAGAHWGVLAGIKYFSMVGNW-Z (SEQ ID NO:4), or portions thereof or, alternatively, to a homologous peptide sequence associated with another Flavivirus, including, but not limited to, HGB, DV, JEV, YFV, WNV, CSFV, BVDV, or BDV as provided below in tables 1 through 4.
As used herein the term “homologous peptide” preferably refers to similar peptides from other strains of a given virus or, alternatively from related viruses.
As used herein the term “similar peptides” refers to those peptides having at least 70% identical or chemically similar amino acids. More preferably, it refers to peptides having 75%, 80%, 85%, 90%, 95%, or greater identical and/or chemically equivalent amino acid resides.
As used herein the terms “portion thereof” refers to the peptide resulting from the removal of from one or more amino acids from either or both ends of the listed peptide, i.e. a truncated peptide. The number of amino acids removed may vary from 1-10 so long as the remaining fragment is “functional”. As defined herein the term “functional fragment” refers to a fragment capable of inhibiting virus:cell fusion, inhibiting viral infectivity, capable of eliciting an antibody capable of recognizing and specifically binding to non-truncated peptide and/or interfering with Flavivirus envelope protein-mediated cell infection.







TABLE 1







Flavivirus fusion inhibitory peptide 1








PROTEIN
SEQUENCE





HCV E1
X-YQVRNSSGLYHVTNDCPNSSIVYEAADAIL-Z (SEQ ID NO: 1)


 


HGB E1
X-RVTDPDTNTTILTNCCQRNQVIYCSPSTCL-Z (SEQ ID NO: 5)


 


DV E
X-RDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDF-Z (SEQ ID NO: 6)


 


JEV E
X-RDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDV-Z (SEQ ID NO: 7)


 


YFV E
X-RDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDI-Z (SEQ ID NO: 8)


 


WNV E
X-RDFLEGVSGATWVDLVLEGDSCVTIMSKDKPTIDV-Z (SEQ ID NO: 9)


 


CSFV E2
X-GQLACKEDYRYAISSTNEIGLLGAGGLTTTWKEYN-Z (SEQ ID NO: 10)


 


BVDV E2
X-GHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYS-Z (SEQ ID NO: 11)


 


BDV E2
X-GEFACREDHRYALAKTKETGPLGAESLTTTWTDYQ-Z (SEQ ID NO: 12)
















TABLE 2







Flavivirus fusion inhibitory peptide 2








PROTEIN
SEQUENCE





HCV E1
X-CSALYWVGDLCGSVFLVGQLFTFSPRRHWTTQDC-Z (SEQ ID NO: 2)


 


HGB E1
X-TCDALDIGELCGACVLVGDWLVRHWLIHIDLNET-Z (SEQ ID NO: 13)


 


DV E
X-KRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTC-Z (SEQ ID NO: 14)


 


JEV E
X-SSYVCKQGFTDRGWGNGCGLFGKGSIDTCAKFSC-Z (SEQ ID NO: 15)


 


YFV E
X-GDNACKRTYSDRGWGNGCGLFGKGSIVACAKFTC-Z (SEQ ID NO: 16)


 


WNV E
X-PAFVCRQGVVDRGWGNGCGLFGKGSIDTCAKFAC-Z (SEQ ID NO: 17)


 


CSFV E2
X-KGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPT-Z (SEQ ID NO: 18)


 


BVDV E2
X-RGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMD-Z (SEQ ID NO: 19)


 


BDV E2
X-RGKYNATLLNGSAFQLVCPYEWTGRVECTTISKS-Z (SEQ ID NO: 20)
















TABLE 3







Flavivirus fusion inhibitory peptide 3








PROTEIN
SEQUENCE





HCV E1
X-SPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPT-Z (SEQ ID NO: 3)


 


HGB E2
X-IHIDLNETGTCYLEVPTGIDPGFLGFIGWMAGKVEA-Z (SEQ ID NO: 21)


 


DV E
X-MVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNW-Z (SEQ ID NO: 22)


 


JEV E
X-FYVMTVGSKSFLVHREWFHDLALPWTSPSSTAWRNR-Z (SEQ ID NO: 23)


 


YFV E
X-SYIAEMETESWIVDRQWAQDLTLPWQSGSGGVWREM-Z (SEQ ID NO: 24)


 


WNV E
X-YYVMTVGTKTFLVHREWFMDLNLPWSSAGSTVWRNR-Z (SEQ ID NO: 25)


 


CSFV E2
X-TLRTEVVKTFRRDKPFPHRMDAVTTTVENEDLFY-Z (SEQ ID NO: 26)


 


BVDV E2
X-TLATEVVKIYKRTKRFRSGLVATHTTIYEEDLYH-Z (SEQ ID NO: 27)


 


BDV E2
X-TLATTVVRTYRRSKPFPHRQGAITQKNLGEDLH-Z (SEQ ID NO: 28)
















TABLE 4







Flavivirus fusion inhibitory peptide 4








PROTEIN
SEQUENCE





HCV E1
X-MMMNWSPTAALLRIPQAIMDMIAGAHWGVLAGIKYFSMVGNW-Z (SEQ ID NO: 4)


 


HGB E1
X-WMAGKVEAVIFLTKLASQVPYAIATMFSSVHYLAVGALIYYS-Z (SEQ ID NO: 29)


 


DV E
X-MAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSW-Z (SEQ ID NO: 30)


 


JEV E
X-LAALGDTAWDFGSIGGVFNSIGKAVHQVFGGAFRSLFGGMSW-Z (SEQ ID NO: 31)


 


YFV E
X-LAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNW-Z (SEQ ID NO: 32)


 


WNV E
X-LAALGDTAWDFGSVGGVFTSVGKAVHQVFGGAFRSLFGGMSW-Z (SEQ ID NO: 33)


 


CSFV E2
X-QQYMLKGEYQYWFDLDVTDRHSDYFAEFVVLVVVALLGGRYI-Z (SEQ ID NO: 34)


 


BVDV E2
X-QQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYV-Z (SEQ ID NO: 35)


 


BDV E2
X-QQYMLKGQYQYWFDLEVISSTHQIDLTEFIMLAVVALLGGRYV-Z (SEQ ID NO: 36)









According to the instant invention peptides related to the Flavivirus fusion inhibitory peptides (FIP) preferably comprise at least three contiguous residues of the FIP peptides, or a homologous peptide, more preferably they comprise 4, 5, 6, or 7 contiguous residues. Even more preferably they comprise at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 contiguous residues, and most preferably all residues of these sequences. As used herein the term Flavivirus inhibitory peptides preferably means peptides having a sequence identical to the corresponding portion of the Flavivirus inhibitory protein and peptides in which one or more amino acids are substituted by functionally equivalent amino acids (see infra). The term also refers to derivatives of these peptides, including but not limited to benzylated derivatives, glycosylated derivatives, and peptides which include enantiomers of naturally occurring amino acids. In other embodiments of the invention, the Flavivirus inhibitory peptides, related peptides or derivatives are linked to a carrier molecule such as a protein. Proteins contemplated as being useful according to this embodiment of the invention, include but are not limited to, (human serum albumen). Flavivirus inhibitory peptide-related peptides comprising additional amino acids are also contemplated as useful according to the invention.
Peptides may be produced from naturally occurring or recombinant viral proteins, or may be produced using standard recombinant DNA techniques (e.g. the expression of peptide by a microorganism which contains recombinant nucleic acid molecule encoding the desired peptide, under the control of a suitable transcriptional promoter, and the harvesting of desired peptide from said microorganism). Preferably, the peptides of the invention may be synthesized using any methodology known in the art, including but not limited to, Merrifield solid phase synthesis (Clark-Lewis et al., 1986, Science 231:134-139).
The FIP, or fragments or derivatives thereof, of the invention include, but are not limited to, those containing, as a primary amino acid sequences the amino acid sequence HCV Fusion Inhibitory Protein 1, X-YQVRNSSGLYHVTNDCPNSSIVYEAADAIL-Z (SEQ ID NO:1); HCV is Fusion Inhibitory Protein 2X-CSALYWVGDLCGSVFLVGQLFTFSPRRHWTTQDC-Z (SEQ ID NO:2); HCV Fusion Inhibitory Protein 3, X-SPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPT-Z (SEQ ID NO:3); or HCV Fusion Inhibitory Protein 4, X-MMMNWSPTAALLRIPQAIMDMIAGAHWGVLAGIKYFSMVGNW-Z (SEQ ID NO:4), or a functional portion or functional portions thereof. Also contemplated are homologous peptide sequences associated with another Flaviviruses, including, but not limited to, HGB, DV, JEV, YFV, WNV, CSFV, BVDV, or BDV. Also contemplated are altered sequences (i.e. altered from any of the sequences referred to herein) in which functionally equivalent amino acid residues are substituted for residues within the sequence, resulting in a functionally silent change. For example, one or more amino acid residues within the sequence can be substituted by replacing the original amino acid with another amino acid, of a similar polarity, that acts as a functional equivalent, resulting in a functionally silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. For example, and not by way of limitation, such peptides may also comprise one or more D-amino acids. Furthermore, in any of the embodiments of the instant invention the peptide may comprise an inefficient carrier protein, or no carrier protein at all.
5.3. Utility of the Invention
The Flavivirus inhibitory peptides of the instant invention may be utilized to inhibit Flavivirus virion:cell fusion and may, accordingly, be used in the treatment of Flavivirus infection and also in prophylaxis against Flavivirus infection. The peptides of the invention may be administered to patients in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Methods for administering peptides to patients are well known to those of skill in the art; they include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, oral, and intranasal. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection.
The instant invention provides for pharmaceutical compositions comprising Flavivirus inhibitory peptides, peptide fragments, or derivatives (as described supra) administered via liposomes, microparticles, or microcapsules. Various embodiments of the invention, contemplate the use of such compositions to achieve sustained release of Flavivirus inhibitory peptides. Other embodiments contemplate the administration of the FIP or derivatives thereof, linked to a molecular carrier (e.g. HSA).
Various embodiments of the instant invention provide for administration of the Flavivirus inhibitory peptides and/or antibodies specific for the these peptides to human or animal subjects who suffer from Flavivirus infection (e.g. dengue hemorrhagic fever, West Nile disease, hepatitis C or classical swine fever). In any embodiment the peptides and/or antibodies are typically substantially purified (as used herein the term “substantially purified” refers to a peptide, peptide analog, or antibody that is greater than about 80% pure. More preferably, “substantially purified” refers to a peptide, peptide analog, or antibody that is greater than about 90% or greater than about 95% pure. Most preferably it refers to a peptide, peptide analog, or antibody that is greater than 99% pure. Functionally, “substantially purified” means that it is free from contaminants to a degree that that makes it suitable for the purposes provided herein. Other embodiments provide for the prophylactic administration of the peptides to those at risk for Flavivirus infection.
Other embodiments of the instant invention provide for methods for identifying the structure of truncated Flavivirus fusion proteins which involved in virion:cell fusion by members of the Flaviviridae family and for the structures themselves.
Other embodiments of the invention provide for a peptide having a formula selected from one or more of the following.

    A. Various embodiments of the invention provide for hepatitis C virus Fusion Inhibitory Peptides: hepatitis C virus Fusion Inhibitory Protein 1, X-YQVRNSSGLYHVTNDCPNSSIVYEAADAIL-Z (SEQ ID NO:1); HCV Fusion Inhibitory Protein 2, X-CSALYWVGDLCGSVFLVGQLFTFSPRRHWTTQDC-Z (SEQ ID NO:2); HCV Fusion Inhibitory Protein 3, X-SPRRHWTTQDCNCSIYPGHITGHRMAWDMMMNWSPT-Z (SEQ ID NO:3); or HCV Fusion Inhibitory Protein 4, X-MMMNWSPTAALLRIPQAIMDMIAGAHWGVLAGIKYFSMVGNW-Z (SEQ ID NO:4)
    B. Other embodiments of the invention provide for a peptide or peptide homolog wherein the Flavivirus is member or tentative member of the hepacivirus genus. A preferred embodiment of this invention is drawn to a peptide or peptide analog wherein the tentative member of the hepacivirus genus is hepatitis G virus and peptides are selected from the group consisting of: hepatitis G virus Fusion Inhibitory Peptides: hepatitis G virus Fusion Inhibitory Protein 1, X-RVTDPDTNTTILTNCCQRNQVIYCSPSTCL-Z (SEQ ID NO:5); hepatitis G virus Fusion Inhibitory Protein 2, X-TCDALDIGELCGACVLVGDWLVRHWLIHIDLNET-Z (SEQ ID NO:13); hepatitis G virus Fusion Inhibitory Protein 3, X-IHIDLNETGTCYLEVPTGIDPGFLGFIGWMAGKVEA-Z (SEQ ID NO:21); or hepatitis G virus Fusion Inhibitory Protein 4, X-WMAGKVEAVIFLTKLASQVPYAIATMFSSVHYLAVGALIYYS-Z (SEQ ID NO:29)
    C. Other embodiments of the invention provide for a peptide or peptide homolog from the flavivirus genus. In a preferred aspect of this embodiment, the peptide or peptide analog is from dengue virus and the peptides are selected from the group consisting of: dengue virus Fusion Inhibitory Peptides: dengue virus Fusion Inhibitory Protein 1, X-RDFVEGVSGGSWVDIVLEHGSCVTTMAKNKPTLDF-Z (SEQ ID NO:6); dengue virus Fusion Inhibitory Protein 2, X-KRFVCKHSMVDRGWGNGCGLFGKGGIVTCAMFTC-Z (SEQ ID NO:14); dengue virus Fusion Inhibitory Protein 3, X-MVLLQMEDKAWLVHRQWFLDLPLPWLPGADTQGSNW-Z (SEQ ID NO:22); or dengue virus Fusion Inhibitory Protein 4, X-MAILGDTAWDFGSLGGVFTSIGKALHQVFGAIYGAAFSGVSW-Z (SEQ ID NO:30).
    D. Other embodiments of the invention provide for peptides or peptide homolog from flavivirus genus member, Japanese encephalitis virus. In preferred aspects of these embodiments the peptides and or/peptide analogs are selected from the group consisting of: Japanese encephalitis virus Fusion Inhibitory Peptides: Japanese encephalitis virus Fusion Inhibitory Protein 1, X-RDFIEGASGATWVDLVLEGDSCLTIMANDKPTLDV-Z (SEQ ID NO:7); Japanese encephalitis virus Fusion Inhibitory Protein 2, X-SSYVCKQGFTDRGWGNGCGLFGKGSIDTCAKFSC-Z (SEQ ID NO:15); Japanese encephalitis virus Fusion Inhibitory Protein 3, X-FYVMTVGSKSFLVHREWFHDLALPWTSPSSTAWRNR-Z (SEQ ID NO:23); or Japanese encephalitis virus Fusion Inhibitory Protein 4, X-LAALGDTAWDFGSIGGVFNSIGKAVHQVFGGAFRTLFGGMSW-Z (SEQ ID NO:31).
    E. Other embodiments of the invention provide for peptides and/or peptide homologs wherein the member of the flavivirus genus is yellow fever virus and the peptides are selected from the group consisting of: yellow fever virus Fusion Inhibitory Peptides: yellow fever virus Fusion Inhibitory Protein 1, X-RDFIEGVHGGTWVSATLEQDKCVTVMAPDKPSLDI-Z (SEQ ID NO:8); yellow fever virus Fusion Inhibitory Protein 2, X-GDNACKRTYSDRGWGNGCGLFGKGSIVACAKFTC-Z (SEQ ID NO:16); yellow fever virus Fusion Inhibitory Protein 3, X-SYIAEMETESWIVDRQWAQDLTLPWQSGSGGVWREM-Z (SEQ ID NO:24); or yellow fever virus Fusion Inhibitory Protein 4, X-LAVMGDTAWDFSSAGGFFTSVGKGIHTVFGSAFQGLFGGLNW-Z (SEQ ID NO:32).
    F. Other embodiments of the invention provide for peptides and/or peptide homologs of wherein the member of the flavivirus genus is West Nile virus and the peptides are selected from the group consisting of: West Nile virus Fusion Inhibitory Peptides: West Nile virus Fusion Inhibitory Protein 1, X-RDFLEGVSGATWVDLVLEGDSCVTIMSKDKPTIDV-Z (SEQ ID NO:9); West Nile virus Fusion Inhibitory Protein 2, X-PAFVCRQGVVDRGWGNGCGLFGKGSIDTCAKFAC-Z (SEQ ID NO:17); West Nile virus Fusion Inhibitory Protein 3, X-YYVMTVGTKTFLVHREWFMDLNLPWSSAGSTVWRNR-Z (SEQ ID NO:25); or West Nile virus Fusion Inhibitory Protein 4, X-LAALGDTAWDFGSVGGVFTSVGKAVHQVFGGAFRSLFGGMSW-Z (SEQ ID NO:33).
    G. Other embodiments of the instant invention provide for peptides and/or peptide homologs wherein the Flavivirus is a member of the pestivirus genus. In various aspects of these embodiments the peptides or homologs thereof the member of the pestivirus genus is classical swine fever virus and the peptides are selected from the group consisting of: classical swine fever virus Fusion Inhibitory Peptides: classical swine fever virus Fusion Inhibitory Protein 1, X-GQLACKEDYRYAISSTNEIGLLGAGGLTTTWKEYN-Z (SEQ ID NO:10); classical swine fever virus Fusion Inhibitory Protein 2, X-KGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPT-Z (SEQ, ID NO:18); or classical swine fever virus Fusion Inhibitory Protein 3, X-TLRTEVVKTFRRDKPFPHRMDAVTTTVENEDLFY-Z (SEQ ID NO:26); or classical swine fever virus Fusion Inhibitory Protein 4, X-QQYMLKGEYQYWFDLDVTDRHSDYFAEFVVLVVVALLGGRYI-Z (SEQ ID NO:34).
    H. Other embodiments of the instant invention provide for peptides and peptide homologs wherein the member of the pestivirus genus is bovine viral diarrhea virus and the peptides are selected from the group consisting of: bovine viral diarrhea virus Fusion Inhibitory Peptides: bovine viral diarrhea virus Fusion Inhibitory Protein 1, X-GHLDCKPEFSYAIAKDERIGQLGAEGLTTTWKEYS-Z (SEQ ID NO:11); bovine viral diarrhea virus Fusion Inhibitory Protein 2, X-RGKFNTTLLNGPAFQMVCPIGWTGTVSCTSFNMD-Z (SEQ ID NO:19); or bovine viral diarrhea virus Fusion Inhibitory Protein 3, X-TLATEVVKIYKRTKRFRSGLVATHTTIYEEDLYH-Z (SEQ ID NO:27); or bovine diarrhea virus Fusion Inhibitory Peptide 4, X-QQYMLKGEYQYWFDLEVTDHHRDYFAESILVVVVALLGGRYV-Z (SEQ ID NO:35).
    I. Other embodiments of the instant invention provide for peptides and peptide homologs wherein the member of the pestivirus genus is border disease virus and the peptides are selected from the group consisting of: border disease virus Fusion Inhibitory Peptides: classical swine fever virus Fusion Inhibitory Protein 1, X-GEFACREDHRYALAKTKEIGPLGAESLTTTWTDYQ-Z (SEQ ID NO:12); border disease virus Fusion Inhibitory Protein 2, X-RGKYNATLLNGSAFQLVCPYEWTGRVECTTISKS-Z (SEQ ID NO:20); or border disease virus Fusion Inhibitory Protein 3, X-TLATTVVRTYRRSKPFPHRQGAITQKNLGEDLH-Z (SEQ ID NO:28); or border disease virus Fusion Inhibitory Peptide 4, X-QQYMLKGQYQYWFDLEVISSTHQIDLTEFIMLAVVALLGGRYV-Z (SEQ ID NO:36)


In any of the foregoing groups the amino acids are represented by the single letter code. In various aspects of these embodiments “X” comprises an amino group, an acetyl group, a hydrophobic group or a macromolecular carrier group; “Z” comprises a carboxyl group, an amido group a hydrophobic group or a macromolecular carrier group. In other aspects of this embodiment of the invention, X is a hydrophobic group, a carbobenzoxyl group, a dansyl group, t-butyloxycarbonyl group, a lipid conjugate, a polyethylene glycol group, or a carbohydrate. In any aspect of this embodiment Z may be a t-butyloxycarbonyl group, a lipid conjugate, a polyethylene glycol group, or a carbohydrate.
Moreover, aspects of this embodiment also include peptides wherein at least one bond linking adjacent amino acids residues is a non-peptide bond. In particularly preferred aspects of this embodiment the non-peptide bond is an imido, ester, hydrazine, semicarbazoide or azo bond.
Other aspects of this embodiment provide for peptides wherein at least one amino acid is a D-isomer amino acid.
Additional aspects of this embodiment of the invention provide for peptides wherein compromising at least one amino acid substitution has been made so that a first amino acid residue is substituted for a second, different amino acid residue. These substitutions may be conservative or non-conservative. So long as the peptide is still functional according to the instant invention.
Other aspects of this embodiment of the invention provide for peptides wherein at least one amino acid has been deleted. As noted, supra, the peptides according to this embodiment of the invention must comprise at least 3 contiguous amino acids of one of the SEQ ID NOs indicated above and must be a functional segment.
It is noted that any combination of the modifications listed above is considered as part of the instant invention.
6. EXAMPLE
Hepatitis C Virus E1 is a Truncated Class II Fusion Protein
Proteomics computational tools were used to fit HCV E1 protein to the scaffold of TBEV E, the prototypic class II fusion protein. Because HCV E1 is shorter than TBEV E, we reasoned that the former might contain several “deletions” relative to the latter. The HCV E1 fusion peptide (Flint et al., 1999) was assumed to be located at the end of the molecule furthest from the carboxyl terminal (C-terminal) transmembrane anchor domain, and, like other class II fusion proteins to be comprised mostly of antiparallel β-sheets. This latter assumption was supported by Chou-Fasman (Chou and Fasman, 1974) and Robson-Garnier (Biou et al., 1988) analyses, the most commonly applied secondary structure prediction algorithms.
The fusion peptide of HCV (amino acids [aa] 272 to 281 of the full-length polyprotein) was aligned with the fusion peptide of TBEV E (aa 385-396) (FIG. 1A). Both TBEV E and HCV E1 fusion peptides have cysteine residues at either end and contain a core of mostly aromatic and hydrophobic amino acids (FIG. 1A). Another domain readily identifiable in HCV E1 is the transmembrane domain. Amino acids 361 to 381 of the hydrophobic sequence near the carboxyl terminus of E1 were predicted to form a transmembrane helix by TMpred (transmembrane prediction software, see ch.embnet.org) (TMpred score 1308, >500 is statistically significant).
Several regions of predicted β sheets and α helices in HCV E1 showed similarities to sequences known to assume those secondary structures in TBEV E (FIG. 1A). Beginning from the amino terminus, the first similarity of HCV E1 begins in β sheet Do of TBEV E and extends through the fusion peptide. PRSS3, a sequence alignment algorithm, was used to confirm that there is a significant similarity (p<0.025) between amino acids 246-281 of HCV E1 and amino acids 350-396 of TBEV E (FIG. 1B). The fusion peptide is flanked by β sheets in class II fusion proteins and predicted β sheets with similarities to the b and c β sheets of TBEV E are indeed predicted to be present on either side of the putative HCV E1 fusion peptide by Chou-Fasman and Robson-Garnier analysis. HCV E1 also has an extended region of similarity with the amino acid sequence between the two longest helices in TBEV E, αA and αB. There is a statistically significant (P<0.025) alignment of amino acids 316-356 of HCV E1 with amino acids 496-544 of TBEV E (FIG. 1B).
To determine the plausibility of these alignments, a three-dimensional model of HCV E1 was scaffolded on domain II of TBEV E (FIG. 2A). Similar sequences/structures were drawn in similar locations. Reorienting the “b” sheet in E1 is the only change relative to E required to bring the eight cysteine residues into close proximity. The four dicysteines of HCV E1 potentially form a “zipper” down the center of the molecule like the three dicysteines in domain II of TBEV E (FIG. 2B). This model locates the five HCV E1 glycosylation sites so they are surface accessible. Additionally, most of the hydrophobic residues are present in a region on one side of E1 between the fusion peptide and the transmembrane anchor (see below, FIG. 5).
Each of the HCV E1 structures drawn in FIG. 2B conforms to both Chou-Fasman and Robson-Garnier predictions, with the exception of the region from “i” to “αB”. The structures designated “i” and “j” were predicted to be β sheets by Chou-Fasman analysis, but α helical by Robson-Garnier analysis. The structure designated “αB” was predicted to be a β sheet by Chou-Fasman analysis, but α helical by Robson-Garnier analysis. HCV E1 appears to be missing, relative to TBEV E, much of the portion of the molecule prior to the transmembrane helix (pre-anchor). This region of TBEV E follows the trypsin cleavage site at amino acid 395 used to generate that portion of the ectodomain of E examined by X-ray crystallography, and therefore, the TBEV E pre-anchor (stem) structure is uncertain. The pre-anchor of TBEV E has been predicted to form an amphipathic α helix (Allison et al., 1999). A sequence (aa 693-721) of the pre-anchor domain in TBEV E has the characteristics of a leucine zipper, i.e. leucine or another hydrophobic amino acid in the first and fourth (a and d) positions of a seven amino acid periodicity (FIG. 1A). The pre-anchor sequence of HCV E1 was also predicted to be an α helix with characteristics of a “leucine zipper” (Charloteaux et al., 2002). Because of the significant amino acid sequence similarity with TBEV E, the HCV E1 secondary structures between “αA” and “αB” were depicted as in TBEV E. There are several possible alternatives to the 3D model of HCV E1 drawn in FIG. 2B, and it is possible that the secondary structures change on interaction with membranes.
In contrast to HCV E1, our analyses did not reveal any sequences of HCV E2 with significant similarity to any sequence in domains I or II of TBEV E or any other flavivirus E protein (representatives of each of the four major serogroups were examined). Most of the N-terminal half of HCV E2, which include hypervariable region I (HVR 1), is without any sequence similarity to TBEV E. However, we detected a significant alignment (p<0.025) of the C-terminal half of HCV E2 (aa 549-726) with the region of TBEV E (aa 590-763) from domain III through the first of two predicted transmembrane spanning domains of TBEV E (FIG. 1, TBEV E TM1, amino acids 448-469, TMpred: 1496; TM2, amino acids 474-496, TMpred: 1962). As discussed above, the pre-anchor region of TBEV E has a sequence (aa 693-721) with features of a “leucine zipper”; a similar motif (aa 675-703) is found in the HCV E2 pre-anchor (FIG. 1). In addition, the carboxyl (C) terminus of HCV E2, like that of TBEV E, contains a stretch of hydrophobic amino acids that potentially could span the membrane twice. The transmembrane anchor(s) of HCV E2 (TMpred score: 1364) is interrupted by charged amino acids like TM1 of TBEV E. Thus, by sequence alignments and structural predictions there are demonstrable similarities between the C-terminal portions of HCV E2 and TBEV E.
Significant alignments of E1 of hepatitis GB virus (GBV-B) with HCV E1, indicate that this unclassified member of the Flaviviridae family also encodes a truncated class II fusion protein.
6.1. Materials and Methods
Prototype strains of representatives of the Flaviviridae were used for sequence and structural comparisons. The strains examined include TBEV strain Neudoerf1 (accession number: P14336); and the human prototype strain H (subtype 1a) of hepatitis C virus (P27958), Some comparisons used representatives of the major serogroups of flaviviruses, including Japanese encephalitis virus, strain JaOARS982 (P32886), yellow fever virus, strain 17D-204 (P19901), dengue virus type 2, strain PR-159/S1 (P12823), and West Nile virus, strain NY 2000-crow3356 (AF404756). We also compared HCV sequences to those of GB virus-B virus (AAC54059), an unassigned member of the Flaviviridae.
MACMOLLY®, protein analysis software (Soft Gene GmbH, Berlin), was used to locate areas of limited sequence similarity and to perform Chou-Fasman and Robson-Garnier analyses. PRSS3, a program derived from rdf2 (Pearson and Lipman, 1988), which uses the Smith-Waterman sequence alignment algorithm (Smith and Waterman, 1981), was used to determine the significance of protein alignments. PRSS3 is part of the FASTA package of sequence analysis programs available by anonymous ftp from ftp.virginia.edu. Default settings for PRSS3 were used, including the blosum50 scoring matrix, gap opening penalty of 12, and gap extension penalty of 2. The alignments presented are those that produced the highest alignment scores, rather than the longest sequences that produced significant scores. Chou-Fasman and Robson-Garnier algorithms predict protein structures in an aqueous environment, but they cannot predict protein structures in a lipid bilayer. Domains with significant propensity to form transmembrane helices were identified with TMpred (ExPASy, Swiss Institute of Bioinformatics). TMpred is based on a statistical analysis of TMbase, a database of naturally occurring transmembrane glycoproteins (Hofman and Stoffel, 1993). RasMac, developed by Roger Sayle, was used to render 3D models of TBEV E.
6.2. Results and Discussion
The results indicate that the ectodomain of hepaciviruses is a truncated version of the class II fusion protein structure. The ectodomain of HCV E1 is roughly equivalent to the part of TBEV E from the “hinge” region to the fusion peptide (FIG. 2). Our conclusions contrast with those of Yagnik et al. (2000), who predicted that HCV E2 fits the scaffold of a complete class II fusion protein. These models were not previously described. Yagnik et al. (2000), taught that HCV E2 fits the scaffold of a complete class II fusion protein. Lescar and co-workers (2001) stated that their structural determinations of SFV E1, which established the existence of a second class of fusion proteins, “indeed support the proposed model of the hepatitis C virus envelope protein E2 which was based on the 3D structure of the flavivirus envelope protein E.” In contrast our model indicated that HCV E1 is class II although not similar to that previously described. Although there are sequence and structural similarities between HCV E2 and TBEV E, these similarities are limited to the C-terminal portions of these proteins, and are different than those proposed previously (Yagnik et al., 2000).
7. EXAMPLE
Pestivirus E2 is a Truncated Class II Fusion Protein
To provide additional evidence for the HCV E1 class II fusion protein model, we determined whether the fusion proteins of the third Flaviviridae genus, pestiviruses, might share structural/sequential similarities with fusion proteins of members of the flavivirus and hepacivirus genera. Pestiviruses encode three envelope glycoproteins, Erns, E1 and E2. Erns, a secreted protein with RNAse activity, does not have a hydrophobic transmembrane anchor domain. Erns does possess a C-terminal charged amphipathic segment that can mediate translocation of Erns across bilayer membranes (Langedijk, 2002). Pestivirus E1 and E2 both have C-terminal hydrophobic domains that could function as transmembrane anchors. Therefore, we postulated that either pestivirus E1 or E2 must be the pestivirus fusion protein.
A putative fusion peptide (aa 818-828) is present in CSFV E2, containing a consensus sequence with aromatic and hydrophobic amino acids located between two cysteine residues (FIG. 1). The cysteine residues as well as the sequences in between are highly conserved among pestiviruses, as is true of fusion peptides from other enveloped RNA viruses of class I and II (not shown). Although statistically significant alignments were not detected between the N-terminus of CSFV E2 and TBEV E (or between other flaviviruses), a significant alignment (p<0.01) was detected between CSFV E2 (aa 792-835) and HCV E1 (aa 253-294) in this region (FIG. 1B). Furthermore, sequences flanking the putative fusion peptide were predicted to form β sheets by both Chou-Fasman and Robson-Garnier analyses (supplemental data). A significant alignment (p<0.05) between CSFV E2 (aa 841-913) and HCV E1 (aa 301-383) was also determined. By extension, the central portion of CSFV E2 is predicted to structurally resemble domain II of TBEV E. A significant alignment (p<0.005) was detected between amino acids 914-1018 of CSFV E2 and a sequence in domain III of TBEV E (aa 587-685) (FIG. 1B). There was also a significant similarity (p<0.005) of this region of CSFV E2 (aa 914-1123) with a sequence (aa 549-743) in the region of HCV E2 that aligns with TBEV domain III. In addition, TMpred confirmed that the hydrophobic C-terminal domain of CSFV E2 has a high propensity to span the lipid bilayer (score: 1137). Like the transmembrane domains of HCV E1/E2 and TBEV TM1, the putative transmembrane anchor of CSFV E2 has a central positive charge.
On the basis of the regions of significant sequence similarities between CSFV E2, HCV E1/E2 and TBEV E, coupled with the internal location of a possible fusion peptide, we conclude that relative to TBEV E, CSFV E2 is lacking a portion of domain I including segments corresponding to β sheets Eo through Io. CSFV E2 also appears to contain a somewhat shorter segment relative to TBEV E in the pre-anchor domain, i.e. the sequence between the alignment with TBEV E domain III and the transmembrane domain (FIG. 1B). No leucine zipper is evident in the pre-anchor of CSFV E2. A three dimensional model of CSFV E2 (FIG. 2C) confirms that the alignment in FIG. 1 is plausible. Each of the cysteine residues is in proximity to other cysteine residues and potentially form dicysteine bridges. Like HCV E1, CSFV E2 conforms to the structure of a truncated class II fusion protein, albeit with fewer truncations relative to flavivirus E than HCV E1. Because E2 is conserved among the pestivirus genus, the similarities of CSFV E2 with TBEV E extend to other pestiviruses.
None of the E1 envelope glycoproteins of any pestivirus bear any significant sequence similarities to any sequenced flavivirus E protein. Immature flavivirus virions contain a precursor, prM, to the small membrane protein M. prM is cleaved in the endoplasmic reticulum by furin or by a furin-like protease during virus release to produce the mature M protein localized on the surface of flavivirus virions (Stadler et al., 1997). A sequence (amino acids 173-256) of CSFV E1 has similarity (p=0.030) to amino acids 583-654 of TBEV prM (FIG. 3A). CSFV E1 does not contain the sequence RXR/KR (SEQ ID NO:37), the furin consensus cleavage site. CSFV E1 also does not contain an identifiable fusion peptide, although TMpred predicts a significant transmembrane spanning domain in the first third of CSFV E1. Like the transmembrane domains of TBEV E, HCV E1 and E2 and CSFV E2, and TBEV prM (TMpred score=1828), the C-terminus of CSFV E1 is predicted to form a membrane spanning domain (TMpred score=1884) with a central positive charge.
7.1. Materials and Methods
The Alfort 187 strain of classical swine fever virus, aka hog cholera virus (CAA61161) was used as the prototype of the pestivirus genus of the family Flaviviridae. Type species of other pestiviruses, including bovine viral diarrhea virus (BVDV) genotype 1, aka pestivirus type 1, strain NADL (CAB91847) and border disease virus strain BD31 (AAB37578), were used in other comparisons. Proteomics computational methods were as described in 6.1.
7.2. Results and Discussion
Pestivirus E2 proteins are truncated class II fusion proteins, although with fewer truncations relative to flavivirus E than hepacivirus E1.
8. EXAMPLE
Gene Order of Flaviviridae Genomes
Genes that encode proteins with similar functions may be present in similar locations in genomes of different members of the Flaviviridae family. The positive-polarity single-stranded RNA genomes of all members of the Flaviviridae are translated into a single large polyprotein that is subsequently cleaved by viral and cellular proteases into functional proteins. The order (from N to C terminus) of proteins in the polyproteins of TBEV and other members of the flavivirus genus is C-prM-E-nonstructurals (C: capsid), and the order of proteins in the polyproteins of hepaciviruses is C-E1-E2-p7-nonstructurals (FIG. 4). The 5′ portion of the flavivirus E gene encodes the fusion peptide in domain II of the E protein, whereas the receptor binding domain of E is probably located in domain III encoded by the 3′ portion of the E gene (Crill and Roehrig, 2001; Mandl et al., 2000). Fusion and receptor functions may reside in two different HCV proteins, E1 and E2 respectively, occurring in the same order as the domains of flavivirus E that carry out these functions (FIG. 4). Hepacivirus E1 and E2 may have arisen by insertion of a transmembrane anchor and variable domains, including hypervariable region 1 (HVR-1, FIG. 1), into the ancestral E gene. Alternatively, HCV E1 could have evolved into a separate fusion protein from an ancestral prM, with concurrent lost of the fusion peptide and fusion functions in E2. The sequence similarities between TBEV E and HCV E1 and E2, however, do not favor this latter possibility.
The order of proteins in pestivirus polyproteins is Npro-C-Erns-E1-E2-p7-nonstructurals. Pestiviruses encode two proteins, Npro and Erns, with no obvious homologs among members of the other two Flaviviridae genera. Pestivirus E1 and E2 are similar in sequence to flavivirus M and E, respectively. Like TBEV E, pestivirus E2 may serve both as fusion protein and receptor binding protein. These functions are carried out by TBEV E domains II and III that appear to be represented by similar structures in pestivirus E2 (FIG. 4). TBEV PrM/M functions to protect internal cellular membranes from fusion mediated by E2, and it is possible that pestivirus E1 serves the same function for E2, the fusion/receptor protein. Excepting Npro and Erns, the order of structural proteins with sequence and other similarities is analogous in pestiviruses and flavivirus polyproteins.
TBEV E has two hydrophobic C-terminal transmembrane domains, TM1 and TM2 (FIG. 1). Hepaciviruses and pestiviruses encode a small hydrophobic peptide, “p7”, which could associate with cellular or viral membranes. The cleavage that produces p7 is inefficient and delayed, and therefore much of HCV E2 and pestivirus E2 are present in the cell as uncleaved E2-p7 precursors (Harada, Tautz, and Thiel, 2000). The p7 gene is located in a similar genomic location and could have evolved from the sequence encoding the second transmembrane domain, TM2, of flavivirus E (FIG. 4). The consensus Flaviviridae genome can therefore be represented as X1-C-X2-M-fusion-binding-TM1-TM2-nonstructurals-3′, where X 1 and X2 represents inserted sequences in pestiviruses, Npro and Erns, respectively, M represents flavivirus prM/M-pestivirus E1 and TM2 is the second transmembrane domain of flaviviruses and p7 of hepaciviruses and pestiviruses. These similarities in gene order and functions support the hypothesis that E1 is the fusion protein of HCV.
8.1. Materials and Methods
Prototype strains of representatives of the Flaviviridae as described in 6.1 and 7.1 were used for sequence comparisons.
8.2. Results and Discussion
Hepaciviruses, like alphaviruses, appear to use one envelope protein for attachment (E2) and another for fusion (E1). In contrast, E glycoproteins of TBEV, dengue virus, and other members of the flavivirus genus mediate both receptor binding and membrane fusion functions. E2 functions as one of the pestivirus receptor-binding protein (Hulst and Moormann, 1997), and if the current analysis is correct also carries out the virion:cell fusion function. In addition to E, flaviviruses encode a membrane protein prM whose functions may include shielding of cellular membranes from the fusion peptide of E (Kuhn et al., 2002). Functions of the flavivirus small membrane protein may be vested in E1 of pestiviruses, which has significant sequence similarity with flavivirus prM. Mature flavivirus virions contain prM that has been cleaved to M. Unlike M, pestivirus E1 does not associate with the virion envelope as a precursor protein and lacks a furin cleavage site.
The Flavivirus fusion protein structures and functional domains described here are supported by the observations that envelope glycoproteins with significant sequence similarities, HCV E1/2, TBEV E and pestivirus E2 and TBEV prM and pestivirus E1 are in analogous locations in the polyproteins encoded by the three genera of the Flaviviridae. These results suggest that members of the Flavivirus family may have a common ancestor. Divergence of the genes for the fusion proteins within the three genera of this family may have occurred either through acquisition of sequences and/or lose of sequences in a cassette manner constrained by the domain organization of class II fusion proteins.
9. EXAMPLE
Membrane Interfacial Domains in a Class I Fusion Protein and HCV E1
Although the overall structures of class I and II fusion proteins are distinct, they may share structural/functional characteristics in the parts of the molecules that interact with and disrupt bilayer membranes. It is well established that class I fusion proteins have a fusion peptide at the amino terminus of the molecule that is critical for fusion (Gallaher, 1987; Gallaher, 1996; Gallaher et al., 1989; Gallaher, DiSimone, and Buchmeier, 2001). Class II fusion proteins have an internal fusion peptide that are located after secondary structural folding at distal locations from the transmembrane anchor (Kuhn et al., 2002; Lescar et al., 2001; Rey et al., 1995). To provide further support for the proposed models of HCV E1 and pestivirus E2, we used another proteomics computational tool to compare other potential membrane interactive domains in the proteins with the HIV-1 transmembrane glycoprotein (TM), a class I fusion protein. Besides fusion peptides, another motif in class I fusion proteins that can be important in virus:cell fusion is an aromatic amino acid rich motif proximal to the anchor (FIG. 5A, amino acids 667-683) (Suarez et al., 2000). The pre-anchor domains of class I fusion proteins are not highly hydrophobic according to the Kyte-Doolittle hydropathy prediction algorithm, however, these domains have a tendency to partition into bilayer membranes, as revealed by analyses using the Wimley-White interfacial hydrophobicity scale (Suarez et al., 2000; Wimley and White, 1996). HCV E1 contains three domains that produce significant Wimley-White partition scores using Membrane Protein eXplorer (Jaysinghe, Hristova, and White, 2000). One of these is the transmembrane anchor (aa 361-372). The other two sequences with significant Wimley-White partition scores are located immediately following the fusion peptide (aa 284-300) and at a location (aa 321-340) that the model in FIG. 2B predicts to be near the bilayer membrane (FIG. 5B).
9.1. Materials and Methods
Sequences with a propensity to partition into the lipid bilayer were identified with Membrane Protein eXplorer from the Stephen White laboratory (Jaysinghe, Hristova, and White, 2000) using default settings.
9.2. Results and Discussion
These two HCV E1 domains, in conjunction with the fusion peptide and the transmembrane anchor, potentially form a continuous track of membrane interactive regions that could channel the movement of lipids during virion:cell fusion. These Wimley-White partition analyses thus provide additional support for the proposal that E1 is the fusion protein of HCV.
10. EXAMPLE
Identification of Peptides that Inhibit Fusion/Infectivity Mediated by HCV Envelope Proteins
The membrane fusogenic envelope glycoproteins of Flaviviruses share several common structural features, including “fusion peptides” and globular domain structures consisting mostly of antiparallel β sheets. Furthermore, the E1 protein of HCV and the E proteins of DEN, WNV and YFV each have several motifs with a high propensity to interact with bilayer membranes as revealed by algorithms employing the Wimley-White interfacial hydrophobicity scale. These structural features and membrane interfacial motifs are presumably important in Flavivirus fusion, entry and infection and may represent targets to develop peptide drugs against Flavivirus infection.
10.1. Materials and Methods
To overcome the lack of a conventional cell culture system for the propagation of HCV, infectious pseudotype viruses expressing HCV envelope glycoproteins have been generated (Hsu et al., 2003). Pseudotypes with HIV core proteins and HCV envelope proteins were generated by cotransfection of 293-T cells with equal amounts of plasmids expressing HCV E1 and E2 of strain H77 and the HIV envelope-defective proviral genome, pNL4.3.Luc.R−E− (Pohlmann et al., 2003). Peptides from an 18mer peptide set, overlapping by 7-10 amino acids and representing the entire amino acid sequence of E1 of HCV strain H77, were solubilized in 20% DMSO, diluted (final DMSO concentration <2%). Peptides were incubated on ice for 30 minutes with p24 antigen-normalized HCV pseudotype viral supernatants. The average concentration of peptides was  ̃25 μM, however, actual concentrations of some peptides in solution were 10 μM or less due to low solubility in DMSO (marked by asterisk in Table 5). Supernatants were also treated with DMSO vehicle alone or with a Mab (monoclonal antibody) to HCV E2 known to neutralize pseudotype infectivity. HCV peptides, vehicle, and anti-E2 MAb were also incubated with pseudotypes expressing murine leukemia virus (MLV) envelope proteins and HIV capsid proteins to control for cytotoxicity. Peptide treated and control HCV and MLV pseudotypes were added to cells, which were incubated at 37° C. for 72 h. Cell lysates were then tested for luciferase activity as described (Hsu et al., 2003).
10.2. Results and Discussion
Four HCV E1 peptides demonstrated greater than 70% inhibition of HCV pseudotype infectivity, with one (peptide 54) reducing HCV pseudotype infectivity by >99.9% (Table 5, FIG. 5B). Two of the peptides (66 and 70) correspond to sequences with a high propensity to interact with the surface of bilayer membranes, as determined by application of the Wimley-White interfacial hydrophobicity scale. Peptide 66 also inhibited infection by the HIV(MLV) pseudotype by greater that 50% suggesting either that this peptide is a general inhibitor of viral fusion or that it is cytotoxic. The other two inhibitory peptides (54 and 74) represent sequences of HCV E1 predicted to “fold” over and interact with the portion of E1 displaying high Wimley-White interfacial hydrophobicity scores (FIG. 5B). The postulated folding over of these domains was marked by arrows in the original published figure (FIG. 5 of Garry and Dash, 2003). These results demonstrate the potential of peptides as antiHCV drugs, and indicate that similar strategies can identify peptides that inhibit fusion and infectivity of other Flaviviruses.







TABLE 5







Identification of lead peptides that inhibit infectivity


mediated by HCV envelope proteins. 











Peptide

Percent

Percent


number
‡H77-E1E2†
inhibition
§MLV†
inhibition














52
133,259
−17.16
533,179
−21.4


53
113,469
0.23
443,528
−9.95


54
74
99.93
280,113
36.22


55
112,470
1.12
447,957
−2.00


56
65,612
42.32
433,459
1.30


57
169,860
−49.35
331,852
24.44


58
118,767
−4.42
329,895
24.98


59
91,794
19.29
446,063
−1.57


60
98,766
13.16
340,384
22.49


61
148,796
−30.83
423,925
3.47


62
115,966
−1.96
415,014
5.50


63
57,915
49.08
438,440
0.16


64
113,108
0.55
316,948
27.83


 65*
87,726
22.87
491,789
−11.98


66
23,387
79.46
189,683
56.81


67
64,601
43.20
357,577
28.58


68
79,297
31.28
498,991
−13.62


 69*
196,922
−73.14
354,027
19.39


70
15,717
86.19
553,120
−25.95


71
83,489
26.60
533,765
−21.54


72
75,763
33.39
392,680
10.58


73
100,666
11.49
433,001
1.40


74
32,888
71.09
467,876
−6.54


75
113,359
0.32
420,026
4.36


76
96,283
15.34
473,757
−7.88


77
56,425
50.39
321,076
26.89


 78*
137,700
−21.07
402,953
8.24


79
101,702
10.58
740,034
−68.51


Vehicle
113,733
—
439,158
—


anti-E2
73
99.93
349,113
21.50





‡H77-E1E2 is the pseudotype expressing envelope glycoproteins E1 and E2 of the H77 strain of HCV.


§MLV is a similar pseudotype expressing the envelope glycoprotein of murine leukemia virus and serves as a peptide control.


†The numbers represent the number of luciferase units (lumens) produced after infection by either the HCV or the MLV pseudotype in the presence of the peptide at a concentration of ~25 μM.













TABLE 6







Sequence and Location of peptides shown in Table 5.










Peptide
Peptide




Number
Location
Amino acid sequence
FIP overlap





52
183-200
SCLTVPASAYQVRNSSGL (SEQ ID NO: 38)



 


53
190-207
SAYQVRNSSGLYHVTNDC (SEQ ID NO: 39)
HCV E1 FIP1


 


54
197-214
SSGLYHVTNDCPNSSIVY (SEQ ID NO: 40)
HCV E1 FIP1


 


55
204-221
TNDCPNSSVVYEAADAIL (SEQ ID NO: 41)
HCV E1 FIP1


 


56
211-228
SIVYEAADAILHTPGCVP (SEQ ID NO: 42)



 


57
218-235
DAILHTPGCVPCVREGNA (SEQ ID NO: 43)



 


58
225-242
GCVPCVREGNASRCWVAV (SEQ ID NO: 44)



 


59
232-249
WVAVTPTVATRDGKLPTT (SEQ ID NO: 45)



 


60
239-256
WVAVTPTVATRDGKLPTT (SEQ ID NO: 46)



 


61
246-263
VATRDGKLPTTQLRRHID (SEQ ID NO: 47)



 


62
253-270
LPTTQLRRHIDLLVGSAT (SEQ ID NO: 48)



 


63
260-277
RHIDLLVGSATLCSALYV (SEQ ID NO: 49)



 


64
267-284
GSATLCSALYVGDLCGSV (SEQ ID NO: )50
HCV E1 FIP2


 


65
274-291
ALYVGDLCGSVFLVGQLF (SEQ ID NO: 51)
HCV E1 FIP2


 


66
281-298
CGSVFLVGQLFTFSPRHH (SEQ ID NO: 52)
HCV E1 FIP2/3


 


67
288-305
GQLFTFSPRHHWTTQDCN (SEQ ID NO: 53)
HCV E1 FIP3


 


68
295-312
PRHHWTTQDCNCSIYPGH (SEQ ID NO: 54)
HCV E1 FIP3


 


69
302-319
QDCNCSIYPGHITGHRMA (SEQ ID NO: 55)
HCV E1 FIP3


 


70
309-326
YPGHITGHRMANMMMNW (SEQ ID NO: 56)
HCV E1 FIP3/4


 


71
316-333
HRMANMMMNWSPTAALV (SEQ ID NO: 57)
HCV E1 FIP3/4


 


72
323-340
MMNWSPTAALVVAQLLRI (SEQ ID NO: 58)
HCV E1 FIP4


 


73
330-347
AALVVAQLLRIPQAIMDM (SEQ ID NO: 59)
HCV E1 FIP4


 


74
337-354
LLRIPQAIMDMIAGAHWG (SEQ ID NO: 60)
HCV E1 FIP4


 


75
344-361
IMDMIAGAHWGVLAGIKY (SEQ ID NO: 61)
HCV E1 FIP4


 


76
351-368
AHWGVLAGIKYFSMVGNW (SEQ ID NO: 62)
HCV E1 FIP4


 


77
359-375
GIKYFSMVGNWAKVLVVL (SEQ ID NO: 63)



 


75
365-382
VGNWAKVLVVLLLFAGVD (SEQ ID NO: 64)



 


79
372-389
LVVLLLFAGVDAETHVTG (SEQ ID NO: 65)









The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the claims. Various publications are cited herein, the disclosures of each of which is incorporated by reference in its entirety. Citation or identification of any reference in any section of this application shall not be construed as an admission that such reference is available as prior art to the present invention.
REFERENCES
Each of the following is herein incorporated by reference in its entirety.

    CHAN, D. C., FASS, D., BERGER, J. M., and KIM, P. S. (1997). Core structure of gp41 from the HIV envelope glycoprotein. Cell 89, 263-73.
    FLINT, M., THOMAS, J. M., MAIDENS, C. M., SHOTTON, C., LEVY, S., BARCLAY, W. S., and MCKEATING, J. A. (1999). Functional analysis of cell surface-expressed hepatitis C virus E2 glycoprotein. J Virol 73, 6782-90.
    GALLAHER, W. R. (1987). Detection of a fusion peptide sequence in the transmembrane protein of human immunodeficiency virus. Cell 50, 327-8.
    GALLAHER, W. R. (1996). Similar structural models of the transmembrane glycoproteins of Ebola and avian sarcoma viruses. Cell 85, 1-2.
    GALLAHER, W. R., BALL, J. M., GARRY, R. F., GRIFFIN, M. C., and MONTELARO, R. C. (1989). A general model for the transmembrane proteins of HIV and other retroviruses. AIDS Res Hum Retro 5, 431-40.
    GALLAHER, W. R., DISIMONE, C., and BUCHMEIER, M. J. (2001). The viral transmembrane superfamily: possible divergence of Arenavirus and Filovirus glycoproteins from a common RNA virus ancestor. BMC Microbiol 1, 1.
    GARRY, R. F. and DASH S. (2003). Proteomics computational analysis suggest that hepatitis C virus E1 and pestivirus E2 envelope glycoproteins are truncated class II fusion proteins. Virology 307, 255-65.
    HOFFMAN-LA ROCHE, and TRIMERIS. (2002). Roche and Trimeris announce 24-week results from second pivotal study of HIV fusion inhibitor T-20. trimeris.com/news/pr/2002/020516.html.
    HSU, M., ZHANG, J., FLINT, M., LOGVINOFF, C., CHENG-MAYER, C., RICE, C. M., AND MCKEATING, J. A. (2003). Hepatitis C virus glycoproteins mediate pH-dependent cell entry of pseudotyped retroviral particles. Proc Natl Acad Sci USA 100, 7271-6.
    JAYSINGHE, S., HRISTOVA, K., and WHITE, S. H. (2000). Membrane Protein Explorer. http://blanco.biomol.uci.edu/mpex.
    KUHN, R. J., ZHANG, W., ROSSMANN, M. G., PLETNEV, S. V., CORVER, J., LENCHES, E., JONES, C. T., MUKHOPADHYAY, S., CHIPMAN, P. R., STRAUSS, E. G., BAKER, T. S., and STRAUSS, J. H. (2002). Structure of dengue virus: implications for flavivirus organization, maturation, and fusion. Cell 108, 717-25.
    LESCAR, J., ROUSSEL, A., WIEN, M. W., NAVAZA, J., FULLER, S. D., WENGLER, G., and REY, F. A. (2001). The fusion glycoprotein shell of Semliki Forest virus: an icosahedral assembly primed for fusogenic activation at endosomal pH. Cell 105, 137-48.
    MALASHKEVICH, V. N., SCHNEIDER, B. J., MCNALLY, M. L., MILHOLLEN, M. A., PANG, J. X., and KIM, P. S. (1999). Core structure of the envelope glycoprotein GP2 from Ebola virus at 1.9-A resolution. Proc Natl Acad Sci USA 96, 2662-7.
    QURESHI, N., COY, D., GARRY, R., and HENDERSON L A (1990). Characterization of a putative cellular receptor for HIV-1 transmembrane glycoprotein using synthetic peptides. AIDS 4, 553-558.
    REY, F. A., HEINZ, F. X., MANDL, C., KUNZ, C., and HARRISON, S. C. (1995). The envelope glycoprotein from tick-borne encephalitis virus at 2 A resolution. Nature 375, 291-8.
    SUAREZ, T., GALLAHER, W. R., AGIRRE, A., GONI, F. M., and NIEVA, J. L. (2000). Membrane interface-interacting sequences within the ectodomain of the human immunodeficiency virus type 1 envelope glycoprotein: putative role during viral fusion. J Virol 74, 8038-47.
    WEISSENHORN, W., CARFI, A., LEE, K. H., SKEHEL, J. J., and WILEY, D. C. (1998). Crystal structure of the Ebola virus membrane fusion subunit, GP2, from the envelope glycoprotein ectodomain. Mol Cell 2, 605-16.
    WEISSENHORN, W., WHARTON, S. A., CALDER, L. J., EARL, P. L., MOSS, B., ALIPRANDIS, E., SKEHEL, J. J., and WILEY, D. C. (1996). The ectodomain of HIV-1 env subunit gp41 forms a soluble, alpha-helical, rod-like oligomer in the absence of gp120 and the N-terminal fusion peptide. EMBO J 15, 1507-14.
    WILD, C., GREENWELL, T., and MATTHEWS, T. (1993). A synthetic peptide from HIV-1 gp41 is a potent inhibitor of virus-mediated cell-cell fusion. AIDS Res Hum Retro 9, 1051-3.
    WILD, C. T., SHUGARS, D. C., GREENWELL, T. K., MCDANAL, C. B., and MATTHEWS, T. J. (1994). Peptides corresponding to a predictive alpha-helical domain of human immunodeficiency virus type 1 gp41 are potent inhibitors of virus infection. Proc Natl Acad Sci USA 91, 9770-4.
    WILSON, I. A., SKEHEL, J. J., and WILEY, D. C. (1981). Structure of the haemagglutinin membrane glycoprotein of influenza virus at 3 A resolution. Nature 289, 366-73.
    YAGNIK, A. T., LAHM, A., MEOLA, A., ROCCASECCA, R. M., ERCOLE, B. B., NICOSIA, A., and TRAMONTANO, A. (2000). A model for the hepatitis C virus envelope glycoprotein E2. Proteins 40, 355-66

## Claims

1. An isolated peptide selected from the following:
a) a peptide consisting of SEQ ID NO:25, wherein the peptide's N-terminal chemical moiety is an amino group and the peptide's C-terminal chemical moiety is a carboxyl group;
b) a peptide consisting of SEQ ID NO:25, wherein the peptide's N-terminal chemical moiety is selected from the group consisting of an acetyl group, a hydrophobic group, a carbobenzyloxy group, a dansyl group, a t-butyloxycarbonyl group, and a macromolecular group; or wherein the peptide's C-terminal chemical moiety is selected from the group consisting of a amido group, a hydrophobic group, a t-butyloxycarbonyl group and a macromolecular group;
c) a peptide consisting of SEQ ID NO:25, wherein at least one bond linking adjacent amino acid residues of the peptide is a non-peptide bond; or
d) a peptide consisting of SEQ ID NO:25, wherein at least one amino acid residue of the peptide is in the D-isomer configuration.

2. The isolated peptide of claim 1, wherein the selected peptide consists of SEQ ID NO:25, and wherein the peptide's N-terminal chemical moiety is an amino group and the peptide's C-terminal chemical moiety is a carboxyl group.

3. The isolated peptide of claim 1, wherein the selected peptide consists of SEQ ID NO:25, and the peptide's N-terminal chemical moiety is an acetyl group, a hydrophobic group, a carbobenzyloxy group, a dansyl group, a t-butyloxycarbonyl group, or a macromolecular group; or wherein the peptide's C-terminal chemical moiety is a hydrophobic group, a t-butyloxycarbonyl group or a macromolecular group.

4. The isolated peptide of claim 3, wherein the peptide's N-terminal chemical moiety is a macromolecular group selected from a lipid conjugate, polyethylene glycol, or a carbohydrate; or wherein the peptide's C-terminal chemical moiety is a macromolecular group selected from a lipid conjugate, polyethylene glycol, or a carbohydrate.

5. The isolated peptide of claim 1, wherein the peptide consists of SEQ ID NO: 25, and at least one bond of the peptide is a non-peptide bond selected from the group consisting of an imido bond, an ester bond, a hydrazide bond, a semicarbazide bond and an azo bond.

6. The isolated peptide of claim 1, wherein the peptide consists of SEQ ID NO: 25, and at least one amino acid of the peptide is a D-isomer amino acid.