EP2616478A2 - Anti-heparan sulfate peptides that block herpes simplex virus infection in vivo - Google Patents

Abstract

Provided are anti-heparan sulfate peptides and methods that employ those peptides for the prevention or treatment of viral infections, including herpesviral infections such as α-herpesviral, β-herpesviral, and γ-herpesviral infections, which are exemplified by HSV-1. CMV, and HHV-8 viral infections, respectively. Peptides comprise at least 10 amino acids of the amino acid sequences XRXRXKXXRXRX and/or XXRRRRXRRRXK, wherein X represents any amino acid, which are exemplified by peptides that comprise at least 10 amino acids of the sequence LRSRTKIIRIRH and/or at least 10 amino acids of the sequence MPRRRRIRRRQK. Peptides may be coupled to one or more therapeutic compound(s) to generate peptide-therapeutic compound conjugates, wherein the therapeutic compound may be one or more of a nucleoside analog, an oligosaccharide, and a small molecule.

Description

ANTI-HEPARAN SULFATE PEPTIDES THAT BLOCK HERPES SIMPLEX

VIRUS INFECTION IN VIVO

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application

No. 61/383,520, filed September 16, 2010, the entire disclosure of which is hereby incorporated by reference in its entirety.

GOVERNMENT SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under R01 Grant Nos.

AI057860 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

[0003] The present application includes a Sequence Listing in electronic format as a text file entitled "Sequence_Listing_DA015PCT.txt" which was created on September 9, 201 1 , and which has a size of 2, 129 bytes. The contents of txt file "Sequence_Listing_DA01 5PCT.txt" are incorporated by reference herein.

BACKGROUND OF THE DISCLOSURE

Technical Field

[0004] The present disclosure is directed, generally, to the inhibition of viral infection, including herpes simplex virus (HSV) cellular infection, and to the treatment of diseases associated with HSV and other viral infections. More specifically, the present disclosure provides anti-heparan sulfate and anti-3-0 sulfated heparan sulfate peptides that can block viral infection of a cell both in vitro and in vivo.

Description of the Related Art

[0005] Heparan sulfate (HS) and jts modified form, 3-0 sulfated heparan sulfate

(3-OS HS), when present on a cell surface, provides an attachment site for many human and non-human pathogenic viruses including herpes simplex virus type-1 and -2 (HSV- 1 and HSV-2, respectively) thereby contributing to viral infections. Lycke et ai, J. Gen. Virol. 72: 1 13 1 - 1 137 (1991 ); Shieh et ai, J. Cell Biol. 116: 1273- 1281 ( 1992); WuDunn and Spear, J. Virol. 63:52-58 ( 1989); and Liu and Throp, Med. Res. Rev. 22: 1 -25 (2002). Both wild-type and laboratory strains of HSV bind to HS. Trybala et al., Virology 302:413-419 (2002). In addition to the attachment step, HSV-1 penetration into cells can also be mediated by 3-OS HS, which is produced after a rare enzymatic modification in HS catalyzed by 3-O-sulfortransferases (3-OSTs). Shukla et al, Cell 99: 13-22 (1999); Shukla and Spear, J. Clin. Invest. 108:503-510 (2001 ); Xia et al, J. Biol. Chem. 227:37912-37919 (2002); Tiwari et al, Biochemical and Biophysical Research Communications 338:930-937 (2005); Xu et al, Biochemical Journal 385:451 -459 (2005); and O'Donnell et al, Virolozv 346:452-459 (2006).

[0006] HSV envelope glycoproteins B and C (gB and gC) bind HS and mediate virus attachment to cells. Shieh et al, J. Cell Biol. Π 6: 1273- 1281 (1992); WuDunn and Spear, J. Virol. 63:52-58 ( 1989); and Herold et al, J. Virol. 65: 1090-1098 (1991 ). A third glycoprotein, gD, specifically recognizes 3-OS HS in a binding interaction that facilitates fusion pore formation during viral entry. Shukla et al , Cell 99: 13-22 ( 1999); Shukla and Spear J. Clin. Invest. 108:503-510 (2001 ); and Tiwari et al , J. Gen. Virol. 85:805-809 (2004). Despite the known importance of HS and 3-OS HS during HSV-1 infection in vitro (O'Donnell and Shukla, J. Biol. Chem. 284:29654-29665 (2009) and O'Donnell et al , Virology 397:389-398 (2010)), little is known about the significance of HS and 3-OS during an infection in vivo.

[0007] The versatility of HS to bind multiple microbes and participate in a variety of regulatory phenomena comes from its negatively-charged nature and highly complex structure, which is generated by enzymatic modifications. Liu and Throp, Med. Res. Rev. 22: 1 -25 (2002) and Esko and Lindahl, J. Clin. Invest. 108: 169- 73 (2001 ). Virtually all cells express HS as long un-branched chains often associated with protein cores commonly exemplified by syndecan and glypican families of HS proteoglycans. Esko and Lindahl, J. Clin. Invest. 108: 169- 173 (2001). The parent HS chain, which contains repeating glucosamine and hexuronic acid dimers, can be 100- 150 residues long and may contain multiple structural modifications. Most common among them is the addition of sulfate groups at various positions within the chain, which leads to the generation of specific motifs making HS highly attractive for microbial adherence. Liu and Throp, Med. Res. Rev. 22: 1 -25 (2002); O'Donnell and Shukla, J. Biol. Chem. 284:29654-29665

(2009) ; and O'Donnell et al, Virology 397:389-398 (2010). Emerging evidence suggests that the role of HS in viral infection extends beyond its function as a low-specificity pre- attachment site. For instance, HS mediates HSV-1 transport on filopodia during surfing (Oh et al, Biochem. Biophys. Res. Commun. 391: 176- 181 (2010)) and negatively regulates virus-induced membrane fusion. O'Donnell and Shukla, J. Biol. Chem. 284:29654-29665 (2009). Likewise, for human papilloma virus (HPV), HS proteoglycans play a key role in the activation of immune response, an important aspect for both vaccine development and HPV pathogenesis, de Witte et al, Immunobiology 212:679-691 (2010). Similarly, HS expressed on spermatozoa plays a key role in the capture of human immunodeficiency virus (HIV) and its transmission to dendritic, macrophage, and T-cells. Ceballos et al, J. Exp. Med. 206:2717-2733 (2009). 30S HS also plays a role in hepatitis B virus replication (Zhang et al, Virology 406:280-285

(2010) ) and HS-binding peptides or compounds can be used to prevent genital HPV, HIV, and cytomegalovirus infections. Baleux et al, Nat. Chem. Biol. J_0:743-748 (2009); Donalisio et al, Antimicrob. Agents Chemother. 54:4290-4299 (2010); Luganini et al, Antiviral Res. 85:532-540 (2010); and Copland et al, Biochemistry 47:5774-5783 (2008).

[0008] Despite the promise shown by HS in the development of a herpesvirus therapy, what remains critically needed in the art are anti-HS inhibitors that prevent the cellular infection by viruses, including HSV.

SUMMARY OF THE DISCLOSURE

[0009] The present disclosure achieves these and other related needs by providing peptides, including anti-HS peptides and anti-3-OS HS peptides that significantly inhibit viral infection and/or receptor-mediated cell-to-cell fusion. Exemplified herein are peptides designated G l and G2, which represent two classes of cationic peptides specifically isolated against HS and 3-OS HS, respectively, and which exhibit strong herpesvirus entry-inhibiting activities. The peptides disclosed herein inhibit HSV- 1 spread in corneal keratitis thereby demonstrating both the in vivo significance of HS/3- OS HS in HSV-1 pathogenesis as well as the efficacy of the G l and G2 peptides in the treatment of diseases associated with viral infection.

[0010] Within certain embodiments, the present disclosure provides peptides that comprise at least 10 or at least 12 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R is arginine, and K is lysine. Within certain aspects, these peptides are 10 or 12 amino acids in length. Within other aspects, each X may be independently selected from the group consisting of leucine (L), serine (S), threonine (T), isoleucine (I), and histidine (H).

[0011] Within other embodiments, the present disclosure provides peptides that comprise at least 10 or at least 12 consecutive amino acids of the peptide G l , which has the amino acid sequence LRSRTKIIRIRH (SEQ ID NO: 1 ) wherein L is leucine, R is arginine, S is serine, T is threonine, K is lysine, I is isoleucine, and H is histidine. Within certain aspects, these peptides are 10 or 12 amino acids in length. An exemplary 10 amino acid peptide based upon G l is the peptide RSRTKIIRIR (SEQ ID NO: 5).

[0012] Within further embodiments, the present disclosure provides peptides that comprise at least 10 or at least 12 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4) wherein X is any amino acid, R is arginine, and K is lysine. Within certain aspects, these peptides are 10 or 12 amino acids in length. Within other aspects, X may be independently selected from the group consisting of methionine (M), proline (P), isoleucine (I), and glutamine (Q).

[0013] Within yet further embodiments, the present disclosure provides peptides that comprise at least 10 or at least 12 consecutive amino acids of the peptide G2, which has the amino acid sequence MPRRRRIRRRQK (SEQ ID NO: 3) wherein M is methionine, P is proline, R is arginine, I is isoleucine, Q is glutamine, and K is lysine. Within certain aspects, these peptides are 10 or 12 amino acids in length. An exemplary 10 amino acid peptide based upon G2 is the peptide RRRRIRRRQK (SEQ ID NO: 6).

[0014] The peptides disclosed herein can block the binding of a virus to heparan sulfate or 3-0 sulfated heparan sulfate thereby preventing the viral infection of a target cell, such as a corneal cell. Viruses the binding of which can be blocked by these peptides include herpesviruses, such as a herpesvirus selected from the group consisting of an ot-herpesvirus, a β-herpesvirus, and a γ-herpesvirus. Within certain aspects, the a- herpesvirus is HSV- 1. Within other aspects, the β-herpesvirus is cytomegalovirus (CMV). Within still further aspects, the γ-herpesvirus is human herpesvirus-8 (HHV-8).

[0015] The peptides disclosed herein may be combined into compositions comprising two or more peptides wherein each of the peptides independently comprises at least 10 amino acids of a sequence selected from the group consisting of XRXRXKXXRXRX (SEQ ID NO: 2) and XXRRR XR RXK (SEQ ID NO: 4) wherein X is any amino acid, R is arginine, and is lysine. Within certain aspects of these compositions, at least one of the peptides comprises at least 10 amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ), such as the peptide RSRTKIIRIR (SEQ ID NO: 5). Within other aspects of these compositions, at least one of the peptides comprises at least 10 amino acids of the sequence MPRRR IRRRQ (SEQ ID NO: 3), such as the peptide RRRRIRRRQK (SEQ ID NO: 6).

[0016] Still further embodiments of the present disclosure provides methods for blocking the binding of a virus to a target cell wherein the methods comprise the step of contacting the target cell with a peptide comprising at least 10 or at least 12 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R is arginine, and K is lysine. Within certain aspects of these methods, the peptide comprises at least 10 or at least 12 consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ), such as the peptide RSRTKIIRIR (SEQ ID NO: 5).

[0017] Yet further embodiments of the present disclosure provides methods for blocking the binding of a virus to a target cell wherein the methods comprise the step of contacting the target cell with a peptide comprising at least 10 or at least 12 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4) wherein X is any amino acid, R is arginine, and K is lysine. Within certain aspects of these methods, the peptide comprises at least 10 or at least 12 consecutive amino acids of the sequence MPRRRR1RRRQK (SEQ ID NO: 3), such as the peptide RRRRIRRRQK (SEQ ID NO: 6).

[0018] Viruses the target cell binding of which can be blocked according to these methods include one or more herpesviruses wherein each herpesvirus is selected from the group consisting of an a-herpesvirus, a β-herpesvirus, and a γ-herpesvirus. Within certain aspects, the a-herpesvirus is HSV-1. Within other aspects, the β-herpesvirus is cytomegalovirus (CMV). Within yet other aspects, the γ-herpesvirus is human herpesvirus-8 (HHV-8).

[0019] Still further embodiments of the present disclosure provide methods for the treatment of a patient who is susceptible to a viral infection wherein the methods comprise the step of administering to the patient a peptide comprising at least 10 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R is arginine, and K is lysine. Within certain aspects of these methods, the peptide comprises at least 10 or at least 12 consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ), such as the peptide RSRTKIIRIR (SEQ ID NO: 5).

[0020] Related embodiments of the present disclosure provide methods for the treatment of a patient who is susceptible to a viral infection wherein the methods comprise the step of administering to the patient a peptide comprising at least 10 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4) wherein X is any amino acid, R arginine, and is lysine. Within certain aspects of these methods, the peptide comprises at least 10 or at least 12 consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3), such as the peptide RRRRIRRRQK (SEQ ID NO: 6).

[0021] Yet further embodiments of the present disclosure provide HS and 3-OS

HS binding peptide-therapeutic compound conjugates that comprise an HS or a 3-OS binding peptide, as summarized above and as described in greater detail below, that is coupled to a therapeutic compound, such as an antiviral compound selected from a nucleoside analog, an oligosaccharide, and a small molecule. [0022] Within certain aspects of these embodiments, nucleoside analogs that may be used in these HS and 3-OS HS binding peptide-therapeutic compound conjugates include guanosine analogs such as acyclovir (Formula I) and valacyclovir.

Formula 1

[0023] Within other aspects of these embodiments, oligosaccharides that may be used in these HS and 3-OS HS binding peptide-therapeutic compound conjugates include oligosaccharides such as tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides that are capable of binding to one or more of HSV-1 glycoproteins gB, gC, and gD. For example an oligosaccharide can be an HS octasaccharide 1 having the structure of Formula II:

Formula II

[0024] Within still further aspects of these embodiments, small molecules that may be used in these HS and 3-OS HS binding peptide-therapeutic compound conjugates include Bis-2-methyl-4-amino-quinolyl-6-carbamide (Surfen) having the structure of Formula III: Formula III

BRIEF DESCRIPTION OF THE FIGURES

[0025] Figure 1 demonstrates that the - inhibition of HSV-1 entry by 12-mer synthetic peptides is not specific to any particular gD receptor.

[0026] Figure 2 demonstrates that G l and G2 peptides block HSV-1 entry into human target cells.

[0027] Figure 3 demonstrates that HSV- 1 entry blocking activity of G2 peptide is not HSV-1 strain specific.

[0028] Figure 4 presents the results of deletion analysis and alanine scanning mutagenesis, which reveal the significance of positively charged residues in HSV-1 entry inhibition.

[0029] Figure 5 demonstrates that G2 blocks cellular entry by representative members of beta and gamma herpesvirus subfamilies (CMV and HHV-8).

[0030] Figure 6 demonstrates that G2 functions by preventing HSV- 1 attachment to cells, which results in loss of binding and viral replication.

[0031] Figure 7 demonstrates the activity of G2 against HSV-1 glycoprotein induced cell-to-cell fusion and spread.

[0032] Figure 8 demonstrates that G l or G2 effectively block infection by HSV- 1 in a mouse model of corneal keratitis. DETAILED DESCRIPTION OF THE DISCLOSURE

[0033] The present disclosure is based upon the unexpected discovery that certain peptides, including certain 10-mer and 12-mer peptides, can specifically bind to HS and 3-OS HS and can block the entry of a virus, such as a herpes simplex virus {e.g., HSV-1 ), into a target cell.

[0034] The results of the viral entry and gD-binding assays and the fluorescent microscopy data presented herein demonstrate that both G l and G2 are potent in blocking viral entry, in particular HSV-1 entry, into primary cultures of human corneal fibroblasts (CF) and CHO-K 1 cells transiently expressing different gD-receptors. Moreover, the G2 peptide, which was isolated against 3-OS HS, displays a wider ability to inhibit the entry of clinically relevant strains of HSV-1 , and some divergent members of herpesvirus family including cytomegalovirus (CMV) and human herpesvirus-8 (HHV-8). The entry blocking activity of the peptides disclosed herein is independent of gD receptor, virus strain, or cell-type. Without being bound by theory, it is believed that the G l and G2 peptides function by interfering with viral binding to a cell.

Utility of Anti-HS andAnti-3-OS HS Peptides

[0035] As described in greater detail herein, the anti-HS and anti-3-OS peptides of the present disclosure will find broad utility in preventing viral infection of a target cell, both in vivo and in vitro. Thus, for example, anti-HS and anti-3-OS peptides will find therapeutic utility as efficacious compounds for the treatment of a viral disease, such as a herpesvirus-mediated disease including an α-herpesvirus-, a β-herpesvirus-, and/or a γ-herpesvirus-mediated disease.

[0036] The anti-HS and anti-3-OS peptides disclosed herein will also find utility in studies seeking to demonstrate the significance of HS during in vivo viral infection, such as HSV-1 infection. HS has been well studied as an attachment receptor (Shukla and Spear J. Clin. Invest. 108:503-510 (2001 )), but little has been reported on its function in vivo. The experiments presented herein, including experiments with a mouse corneal infection model, demonstrate the efficacy of the G l and G2 peptides in blocking infection in vivo and that HS is an important HSV- 1 co-receptor both in vitro and in vivo. [0037] The ability the presently disclosed peptides to act specifically on HS/3-OS

HS is significant because HS is widely expressed on all cells and tissues and it is known to regulate many important biological phenomena. Esko and Lindahl, J. Clin. Invest. 108: 169- 173 (2001 ) and Bishop et al, Nature 446: 1030- 1037 (2007). Thus, the presently disclosed peptides can be used to prevent the infection of a wide range of cells and tissues, both in vitro and in vivo, and as probes to study HS functions in a wide variety of biological contexts.

[0038] Additionally, HS moieties are frequently up-regulated during pathological conditions and may contribute to inflammation. Esko and Lindahl, J. Clin. Invest.

108: 169- 173 (2001 ). Thus, the presently disclosed peptides may also find utility in blocking the pathological effects of HS and in regulating inflammation.

[0039] The complex enzymatic regulation of HS chain gives HS a complex ability (and affinity) to bind many proteins to perform new functions. Esko and Lindahl, J. Clin. Invest. 108: 169- 173 (2001 ) and Bishop et al., Nature 446: 1030- 1037 (2007). The 3-OS HS binding peptides will, therefore, find utility as a probe and/or as a diagnostic tool to assess structural alterations within HS or its turnover on cell surfaces.

[0040] Because many unrelated viruses bind HS (Liu and Throp, Med Res. Rev.

22: 1 -25 (2002)), the G l and G2 peptides will find broad utility in those applications where it is desired to block infection by a wide variety of viruses that utilize HS and/or 3- OS HS binding to facilitate target cell binding and infection.

[0041] These and other utilities are contemplated by the presently disclosed anti-

HS and anti-3-(9S peptides, including the Group 1 and Group 2 peptides G l and G2, which are described in substantial detail herein.

Anti-Heparan Sulfate and Anti-3-O Sulfated Heparan Sulfate Peptides

[0042] As summarized above, the present disclosure provides peptides that were identified by the screening of a random M 13-phage display library with heparan sulfate and 3-0 sulfated heparan sulfate and the subsequent isolation of HS- and 3-0 sulfated HS binding phages. The peptides disclosed herein, which are exemplified by those peptides that are presented in Table 1 , are characterized by the presence of the positively charged amino-acid residues arginine and/or lysine, the unique arrangement of which is important for blocking virus-cell binding and/or virus-induced membrane fusion.

Table 1

Amino acid (AA) Sequences of Phase-displayed Peptides Isolated by Three Round

Screening against HS and 3-OS HS

G1 peptide Isolated against HS

G2 peptide isolated against 30S HS

Frequency/ number of times peptide sequences isolated; ** P<0.001, Frequently isolated peptides.

[0043] The peptides that are described herein are enriched in basic amino acid residues and classified into two major groups: (1 ) Group 1 , which includes a class of peptides having alternating charges (XRXRXKXXRXRX; SEQ ID NO: 2) and is represented herein by the G l peptide, which has the amino acid sequence LRSRTKIIRIRH (SEQ ID NO: 1 ) and (2) Group 2, which includes a class of peptides having repetitive charges (XXRRRRXRRRXK: SEQ ID NO: 4) and is represented herein by the G2 peptide, which has the amino acid sequence MPRRRRIRRRQK (SEQ ID NO: 3).

[0044] Group 1 peptides of the present disclosure comprise at least 10 or at least

12 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R is arginine, and is lysine. Within certain Group 1 peptides, X may be independently selected from the group consisting of leucine, serine, threonine, isoleucine, and histidine.

[0045] Exemplified herein are Group 1 peptides that are 10, 1 1 , or 12 amino acids in length such as peptides that comprise at least 10 or at least 12 consecutive amino acids of the sequence LRSRTKIIR1RH (G l ; SEQ ID NO: 1) wherein L is leucine, R is arginine, S is serine, T is threonine, K is lysine, I is isoleucine, and H is histidine.

[0046] Group 2 peptides of the present disclosure comprise at least 10 or at least

12 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4) wherein X is any amino acid, R is arginine, and K is lysine. Within certain Group 2 peptides, X may be independently selected from the group consisting of methionine, proline, isoleucine, and glutamine.

[0047] Exemplified herein are Group 2 peptides that are 10, 1 1 , or 12 amino acids in length such as peptides that comprise at least 10 or at least 12 consecutive amino acids of the sequence MPRRRRI RRRQK (SEQ ID NO: 3) wherein M is methionine, P is proline, R is arginine, I is isoleucine, Q is glutamine, and is lysine.

[0048] The peptides disclosed herein can block binding of a virus to heparan sulfate or 3-0 sulfated heparan sulfate and/or can prevent a viral infection of a target cell, such as a corneal cell. Viruses the binding of which can be blocked by these peptides include herpesviruses, such as cc-herpesviruses, β-herpesviruses, and γ-herpesviruses. Within certain aspects, the ct-herpesvirus is HSV-1 . Within other aspects, the β- herpesvirus is cytomegalovirus (CMV). Within still further aspects, the γ-herpesvirus is human herpesvirus-8 (HHV-8).

[0049] While the G l and G2 peptides represent specific examples of Group 1 and

Group 2 peptides, respectively, it will be understood that alternative Group 1 and Group 2 peptides may be identified by the identification of alternative functional amino acids. For example, alternative functional amino acids within Group 1 and Group 2 peptides can be identified through the generation of point mutations and/or via alanine scanning mutagenesis.

[0050] It is disclosed herein that, while both G l and G2 peptides are capable of blocking HSV-1 -mediated cell binding and infection, G2 exhibits the additional capacity to block the entry of divergent herpesviruses such as, for example, CMV and HHV-8. Shukla and Spear J. Clin. Invest. 108:503-510 (2001 ). Without being limited by theory, because the G2 peptide can block membrane fusion it is believed that the G2 peptide can interfere with gD's interaction with its receptor, 3-OS HS. Akhtar and Shukla FEBS J. 276:7228-7236 (2009); Clement et al., J. Cell. Biol. 174: 1009- 1021 (2006); and Spear and Longnecker J. Virol. 77: 10179- 10185 (2003).

[0051] Among the structural differences between G l and G2, it appears that G2 shows more dependence on the positively charged residues than G l , which may depend upon the presence of a lysine residue at the N-terminus. In general, arginine has been found important for charge-charge interaction with HS. Knappe et al , J. Biol. Chem. 282:27913-22 (2007).

[0052] The peptides disclosed herein can be combined into compositions comprising one, two, or more peptides wherein each of the peptides independently comprises at least 10 amino acids of the amino acid sequences XRXRX XXRXRX (SEQ ID NO: 2) and/or XXRRRRXRRRX (SEQ ID NO: 4). Exemplified herein are compositions comprising one or more peptides each of which includes at least 10 amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ) and/or at least 10 amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3).

[0053] The peptides of the present disclosure can be provided to a patient as part of a pharmaceutical composition where it is mixed with a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutical composition" refers to a preparation of one or more of the peptides described herein with one or more other chemical component(s) such as a physiologically suitable carrier or excipient. The purpose of a pharmaceutical composition is to facilitate administration of a compound to the patient. Techniques for formulation and administration of compositions can be found in "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, Pa.

[0054] As used herein, the phrases "physiologically acceptable carrier" and

"pharmaceutically acceptable carrier" refer to a carriers, diluents, and/or adjuvants that do not cause significant irritation to a patient and do not abrogate the biological activity and properties of the administered peptide. Suitable carriers may include polyethylene glycol (PEG), a biocompatible polymer with a wide range of solubility in both organic and aqueous media.

[0055] As used herein, the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active peptide. Exemplary excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0056] Pharmaceutical compositions can be manufactured by processes well known in the art such as, for example, by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions can be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0057] For injection, the active peptides of the disclosure may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0058] Compositions of the present disclosure that are suitable for oral administration may be prepared as capsules, cachets, tablets or lozenges, each containing a predetermined amount of the peptide of the invention, or which may be contained in liposomes or as a suspension in an aqueous liquor or non-aqueous liquid such as a syrup, an elixir, or an emulsion. An exemplary tablet formulation includes corn starch, lactose, and magnesium stearate as inactive ingredients. An exemplary syrup formulation includes citric acid, coloring dye, flavoring agent, hydroxypropylmethylcellulose, saccharin, sodium benzoate, sodium citrate and purified water.

[0059] For oral administration, the compounds can be formulated readily by combining the active peptides with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0060] Peptide compositions may also contain one or more pharmaceutically acceptable carriers, which may include excipients such as stabilizers (to promote long term storage), emulsifiers, binding agents, thickening agents, salts, preservatives, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such agents for pharmaceutically active compounds is well known in the art.

[0061] The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Methods typically include the step of bringing the active ingredients of the invention into association with a carrier that constitutes one or more accessory ingredients. [0062] ' Compositions of the present disclosure that suitable for inhalation can be delivered as aerosols or solutions. An exemplary aerosol composition includes a peptide suspended in a mixture of trichloromonofluoromethane and dichlorodifluoromethane plus oleic acid. An exemplary solution compositin includes a peptide dissolved or suspended in sterile saline (optionally about 5% v/v dimethylsulfoxide ("DMSO") for solubility), benzalkonium chloride, and sulfuric acid (to adjust pH).

[0063] Compositions of the present disclosure that are suitable for parenteral administration include sterile aqueous preparations of the peptides of the present disclosure and are typically isotonic with the blood of the patient to be treated. Aqueous preparations may be formulated according to known methods using those suitable dispersing or vetting agents and suspending agents. Sterile injectable preparations may also be sterile injectable solutions or suspensions in a non-toxic parenteral ly-acceptable diluent or solvent, for example as a solution in 1 ,3-butane diol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In aqueous solutions, up to about 10% v/v DMSO or Trappsol can be used to maintain solubility of some peptides. Also, sterile, fixed oils may be conventionally employed as a solvent or suspending medium such as, for example, synthetic mono- or di-glycerides. In addition, fatty acids (such as oleic acid or neutral fatty acids) can be used in the preparation of injectibles. Further, Pluronic block copolymers can be formulated with lipids for time release from solid form over a period of weeks or months.

[0064] Compositions suitable for topical administration may be presented as a solution of the peptide in Trappsol or DMSO, or in a cream, ointment, or lotion. Typically, about 0.1 to about 2.5% active ingredient is incorporated into the base or carrier. An example of a cream formulation base includes purified water, petrolatum, benzyl alcohol, stearyl alcohol, propylene glycol, isopropyl myristate, polyoxyl 40 stearate, carbomer 934, sodium lauryl sulfate, acetate disodium, sodium hydroxide, and optionally DMSO. An example of an ointment formulation base includes white petrolatum and optionally mineral oil, sorbitan sesquioleate, and DMSO. An example of a lotion formulation base includes carbomer 940, propylene glycol, polysorbate 40, propylene glycol stearate, cholesterol and related sterols, isopropyl myristate, sorbitan palmitate, acetyl alcohol, triethanolamine, ascorbic acid, simethicone, and purified water.

Conjugated Anti-Heparan Sulfate and Anti-3-0 Sulfated Heparan Sulfate Peptides for Intracellular Delivery of Therapeutic Compounds

As part of the present disclosure, it was discovered that the heparan sulfate and 3-0 sulfated heparan sulfate binding peptides, upon binding to HS and 3-OS HS, cross the plasma membrane and are transported into the cytoplasm and nucleus.

Accordingly, the HS and 3-OS HS binding peptides described herein may be conjugated to one or more therapeutic compound to affect the intracellular and/or intranuclear delivery of the therapeutic compounds. Such conjugated HS and 3- OS HS peptides will find utility, for example, (1 ) in the preferential delivery of one or more compound(s) to an infected cell and/or (2) as a multivalent drug therapy wherein the HS and 3-OS HS peptide can (a) block viral infection (as described herein) and (b) cross the plasma membrane in an HS- and/or a 3-OS-dependent manner thereby delivering the compound(s) to affect intracellular and/or intranuclear clearance of virus- infected cells.

Nucleoside Analogs

A wide variety of therapeutic compounds can be conjugated to the HS and

3-OS HS binding peptides, which are described herein, to generate the presently disclosed therapeutic compound HS and 3-OS HS binding peptide conjugates. Exemplified herein are HS and 3-OS HS binding peptide conjugates that employ the guanosine analog acyclovir, or its prodrug valacyclovir, which can be metabolized and incorporated into the viral DNA within a virally infected cell. Acyclovir is depicted in the following Formula I:

Formula I

Acyclovir serves as a chain terminator for viral DNA synthesis and an inhibitor for viral DNA polymerase. Piret and Boivin, Antimicrob. Agents Chemother. 55£2}:459-72 (201 1 ). Acyclovir differs from other nucleoside analogues in that it contains a partial nucleoside structure wherein the sugar ring is replaced with an open- chain structure. Acyclovir is converted into acyclo-guanosine monophosphate by viral thymidine kinase. Cellular kinases subsequently convert the monophosphate form of acyclo-guanosine into acyclo-guanosine triphosphate, which is the high affinity substrate viral DNA polymerase. Because acyclovir has no 3' end, its incorporation into a nascent DNA strand results in its chain termination activity.

Acyclovir is active against a number of herpesvirus species, including herpes simplex virus type I (HSV-1 ), herpes simplex virus type II (HSV-2), and varicella zoster virus (VZV). Acyclovir is less active against Epstein-Barr virus (EBC) and cytomegalovirus (CMV).

Oligosaccharides

In addition to the use of nucleoside analogs, such as the guanosine analog acyclovir in generating HS and 3-OS HS binding peptide therapeutic compound conjugate, the present disclosure further contemplates conjugates that comprise one or more oligosaccharide such as a herpesvirus HS oligosaccharide. Exemplified herein are HS and 3-< S HS binding peptides that are conjugated to the HS octasaccharide 1 that has been shown to bind to the HSV-1 glycoprotein gD. Copeland et al., Biochemistry 47(21 ):5774-83 (2008) and Liu et al, J. Biol. Chem. 277(36 :33456-67 (2002). HS octasaccharide 1 is presented herein by Formula II.

Formula II

Other HS oligosaccharides, including HS octasaccharides, that are capable of binding to HSV-1 glycoproteins gB, gC, and gD can also be employed in the HS and 3-OS HS conjugates disclosed herein. HS structural elements that are critical to gB, gC, and gD binding can be identified by digesting heparan polysaccharides into oligosaccharides with heparan lyase. The resulting oligosaccharides can then be subjected to size exclusion chromatography to separate into tetrasaccharides, hexasaccharides, octasaccharides, and decasaccharides. Oligosaccharide fractions can be incubated with gB, gC, and/or gD and their binding affinities assessed using affinity co- electrophoresis. Oligosaccharide structures can be determined by immunoprecipitating gB-, gC-, and/or gD-oligosaccharide complexes, eluting bound oligosaccharides, and separating by anion exchange chromatography as described in Liu et al, J. Biol. Chem.

277(36):33456-67 (2002). The precise structure of an HS oligosaccharide having high gB, gC, and/or gD affinity can be determined through a combination ofdisaccharide compositional analysis and nanoelectrospray ionization (nESl) mass spectropmetry. Pope et al, Glycobiology 1 1 (6):505-13 (2001 ) and Liu et al, J. Biol. Chem.

285(44):34240-9 (2010).

Structurally well defined oligosaccharides can be synthesized by employing enzymatic synthetic methodology known in the art such as those described by Liu et al, J. Biol. Chem. 285(44):34240-9 (2010) and Linhardt et al , Semin. Thromb. Hemost. 33(5):453-65 (2007) and as presented in the following Synthetic Pathway I for HS oligosaccharides:

Synthetic Pathway I

Starting from a disaccharide primer prepared from nitrous acid-degraded heparosan, backbone elongation can be achieved by altering KifA and pmHS2 treatment with UDP-GIcNTFA and UDP-GlcUA as donor substrates, which can be followed by modification with specific sulfotransferases. To generate different HS sequences, the enzymatic steps can be varied. For example, the Cs-epi can be removed and 2-0 sulfotransferase R189A mutant can be used for step c of the synthetic pathway shown above. Unlike wild type 2-OST, 2-OST R189A specifically sulfates the GlcUA, not IdoUA. Bethea et al., Proc. Natl , Acad. Sci. U.S.A. 105(48): 18724-9 (2008). As a result, the desired octasaccharide can have GlcUA2S instead of the IdoUA2S units. Similar enzymatic variations including HS oligosaccharides prepared without 6-O-sulfation by skipping the 6-0 sulfotransferase (6-OST) treatment step (step d) will yield a number of unique octasaccharides for the generation of HS and 30S HS binding peptide conjugates.

Suitable oligosaccharides, including octasaccharides, can be tested for their ability to inhibit multiple steps during a viral lifecycle, such as a herpesvirus lifecycle {e.g., HSV-1 ). Attachment inhibition can be determined by a flow cytometry binding assay. O'Donnell et al, Virology 397(2):389-98 (2010). Green HSV-1 (K26GFP) can be tested for bining to HeLa cells at 4oC to prevent penetration. HeLa eels can be preincubated with an octasaccharide or control followed by the addition of green virus. Unbound virions are washed away and flowcytometry performed to quantify the presentee of a green signal on a cell. Desai and Person, J. Virol. 72(9):7563-8 ( 1998).

Small Molecules

The present disclosure further contemplates HS and 30S HS binding peptides that are conjugated to one or more small molecule(s). Small molecule inhibitors are well known, as exemplified by the HS binding small molecule Bis-2-methyl-4-amino- quinolyl-6-carbamide (Surfen; see Formula III), and can be readily identified by methodology that is known in the art. Formula III

For example, robotic screening of small molecule libraries can be performed to identify new inhibitors of HSV-1 gB, gC, and/or gD functions. Baculovirus-expressed gB, gC, and/or gD can be affinity purified and screened against one or more drug-like small molecule libraries that provide: (1 ) diversity screening compounds; (2) kinase targeted compounds; and (3) LOPAC (Library of Pharmaceutically Active Compounds). Cytotoxicity of the hit compounds can be tested as described in Bacsa et al, J. Gen. Virol. 92(Pt 4):733-43 (201 1 ). Potential gB, gC, and/or gD binders/inhibitors can be analyzed by surface SPR and/or Bioforte OCTET for affinity determinations as described in Tong et al. , Cell Res. (in press) (201 1 ) and Abdiche et al., Anal. Biochem. 41 1 (1 ): 139-51 (201 1 ).

Coupling of Therapeutic Compounds to HS and 3-OS HS binding peptides HS and 3-OS HS binding peptides can be coupled to one or more therapeutic compound to generate HS and 3-OS HS binding peptide-therapeutic compound conjugates by methodology that is well know to those of skill in the art.

HS and 3-OS HS binding peptides can be coupled to HS oligosaccharides to generate glycopeptides having enhanced affinity through a multivalent effect through the binding to multiple viral surface molecules, such as herpesvirus surface molecules. Peptides can be linked to HS oligosaccharides via hydrazide/aldehyde chemistry as presented in the following Synthetic Pathway II : Synthetic Pathway II

pep e on res n 1) N-terminal capping and

mild acid cleavage from resin

LIRSRKNPIISR-— NHNH₂

I

Fully protected Linker

peptide 9 on resin C-terminal functionalized

3) TFA deprotection HSV-1 binding peptide

Both C- and N-terminal hydrazide functional ization of the peptides can ge employed to achieve optimal linkage. Carboxylic acid 17 is added to functionalize the peptide's N-terminus. Upon cleavage from the resin and global deprotection, the peptide with N-terminal hydrazide is obtainied, wich can undergo chemoselective ligation reation with an aldehyde functionalized HS oligosaccharide followed by reduction to generate a glycopeptides 18. Alternatively, the peptide can be functionalized at its C-terminus with amine 19 to introduce a hydrazide moiety, which can subsequently be coupled with HS oligosaccharide to form a glycopeptides in a similar manner as the formation of 18. HS and/or 30S HS binding peptide-oligosaccharide conjugates can, optionally, employ a linker of varying length to optimize binding affinity. The binding of a multivalent glycopeptides to a virion can be determined by using a Bioforte OCTET system as described in Abdiche et ai, Anal. Biochem. 41 1 (0: 139-51 (201 1 ).

HS and 3-OS HS binding peptides can be coupled to one or more nucleoside analog by methodology that known in the art. For example, acyclovir can be modified with an HS or a 3-OS HS binding peptide by esterification of acyclovir with a protected peptide followed by acid promoted deprotection as presented in the following Synthetic Pathway III:

Synthetic Pathway III

cyclovir

1 ) EDCI, DMAP, acyclovir

HS-targeting peptide

2) TFA (peptide deprotection)

acyclovir conjugate

Acyclovir has been shown to be stable under peptide deprotection conditions. Friedrichsen et al, Eur. J. Pharm. Sci. 16(l -2): l - l 3 (2002). The attachment of a therapeutic compound to an HS and 3-OS HS binding peptide will significantly enhance the cellular uptake of the therapeutic compound. Once inside a cell, the intracellular carboxyl esterases cleave the ester linkage thereby releasing the therapeutic compound. De Clercq and Field, Br. J. Pharmacol. I 47(l ): l - 1 1 (2006). Depending upon the precise application contemplated, and the nature of the therapeutice compound, a linker may be employed between the HS and 3-OS HS binding peptide and the therapeutic compound.

Methods for Blocking Viral Binding to and Viral Infection of a Target Cell and for Treating Virus-mediated Disease in a Patient

[0065] In addition to the Group 1 and Group 2 peptides, and compositions thereof, that are described above, the present disclosure also provides methods for blocking the binding of a virus to a target cell and/or the infection of a target cell by a virus. By these methods, a target cell is contacted, either in vitro or in vivo, with a Group 1 and/or a Group 2 peptide that comprises at least 10 or at least 12 consecutive amino acids of the sequences XRXRX XXRXRX (SEQ ID NO: 2) and XXRRRRXRRRXK (SEQ ID NO: 4). [0066] Within certain aspects of these methods, the target cell can be contacted with a peptide that comprises at least 10 or at least 12 consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ) and/or at least 10 or at least 12 consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3).

[0067] Also disclosed herein are methods for the treatment of a patient who is either infected with a virus or who is susceptible to a viral infection. By these methods, a Group 1 and/or a Group 2 peptide that comprises at least 10 or at least 12 consecutive amino acids of the sequences XRXRXKXXRXRX (SEQ ID NO: 2) and XXRRRRXRRRXK (SEQ ID NO: 4) is administered to a patient subsequent or prior to exposure and/or infection with a virus, such as a herpesvirus (e.g., an a-herpesvirus, a β- herpesvirus, and/or a γ-herpesvirus). Within certain aspects, the ct-herpesvirus is HSV- 1 . Within other aspects, the β-herpesvirus is cytomegalovirus (CMV). Within yet other aspects, the γ-herpesvirus is human herpesvirus-8 (HHV-8).

[0068] · Within certain aspects of these methods, a peptide that comprises at least 10 or at least 12 consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ) and/or at least 10 or at least 12 consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3) can be administered to the patient.

[0069] As shown herein, the in vivo administration of a G 1 and/or G2 a peptide(s) prevents HSV-1 spread in the cornea. These findings highlight the in vivo significance of HS and 30S HS during viral infection, such as during an ocular herpes infection. Thus, G l and G2 represent exemplary 12-mer peptides that exhibit a unique ability to bind to critical domains within HS and/or 30S HS, respectively, which domains are believed to be required for viral entry. Thus, the peptides presented herein will find broad application methods for the treatment of diseases associated with HS and/or 30S HS- mediated viral infections, such as herpesvirus infections.

[0070] Suitable routes of in vivo administration may, for example, include oral, rectal, transmucosal, transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intravenous, inrtaperitoneal, intranasal, or intraocular injections. Alternately, peptide compositions may be administered in a local rather than a systemic manner such as, for example, via injection of the preparation directly into a specific region of a patient's body.

[0071] More specifically, a therapeutically effective amount means an amount of active ingredients effective to prevent, alleviate or ameliorate symptoms of disease or prolong the survival of the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art.

[0072] For any peptide composition used in the methods of the present disclosure, the therapeutically effective amount and toxicity can be estimated initially from in vitro assays and cell culture assays. A suitable dose can be determined in animal models and such information can be used to more accurately determine useful doses in humans.

[0073] Toxicity and therapeutic efficacy of the peptides described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures, and/or in experimental animal models. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in a human patient. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration, and dosage can be chosen by the individual physician in view of the patient's condition. See, e.g. , Fingl el al, "The Pharmacological Basis of Therapeutics", Ch. 1 p. 1 (1975).

[0074] Depending on the severity and responsiveness of the viral infection to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the viral infection is achieved. The amount of a peptide composition to be administered will depend upon the patient being treated, the severity of the affliction, the manner of administration, and the judgment of the prescribing physician.

[0075] Doses of the pharmaceutical compositions will vary depending upon the patient and upon the particular route of administration used. Dosages can range from 0.1 to 100,000 μg/kg a day, more typically from 1 to 10,000 μg kg or from 1 to 100 μg kg of body weight or from 1 to 10 μg kg. Doses are typically administered from once a day to every 4-6 hours depending on the severity of the condition. For acute conditions, it is preferred to administer the peptide every 4-6 hours. For maintenance or therapeutic use, it may be preferred to administer only once or twice a day. Preferably, from about 0.18 to about 16 mg of peptide are administered per day, depending upon the route of administration and the severity of the condition. Desired time intervals for delivery of multiple doses of a particular composition can be determined by one of ordinary skill in the art employing no more than routine experimentation.

* * * * *

[0076] All patents, patent application publications, and patent applications, whether U.S. or foreign, and all non-patent publications referred to in this specification are expressly incorporated herein by reference in their entirety.

EXAMPLES

Example Γ

Experimental Procedures

Selection of Phases against HS and 3-OS HS by Library Panning

[0077] A phage display library (PhD,™-12) expressing 12-mer peptides fused to a minor coat protein (pill) of a non-lytic bacteriophage (M l 3) was purchased from New England Biolabs (Cambridge, MA). A purified form of HS isolated from bovine kidney was purchased from Sigma. Soluble 30S HS modified by 3-0ST-3 was prepared as previously described. Tiwari et ah, J. Gen. Virol. 88: 1075- 1079 (2007).

[0078] Screening of the phage display library was accomplished by an affinity selection (or bio-panning) process during which phage populations were selected for their ability to bind HS and 3<9S HS (modified by 3-0ST-3). Both targets at a concentration of 10μg/ml were used for overnight coating of wells of 96 well plates (Nalge Nunc International, Naperville, IL) in a humidifier chamber at 4°C. The following day, the plates were blocked for 1 hr at room temperature with 5 mg/ml bovine serum albumin (BSA) in 0.1 M NaHC0₃ (pH 8.6) buffer. The plates were then washed six times with TBST (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.1 % [vol/vol] Tween-20). The phage library was added to the plate at a concentration of 2 * 10" in 100 μΐ in TBST. The plate was gently rocked for 1 hr at room temperature. Unbound phages were removed by washing plates 10 times with 1 ml of TBST. Bound phages were eluted by adding 100 μΐ of Tris-HCl at pH 3.0. The eluate containing bound phages was removed and the phages were amplified in Escherichia coli ER2738 bacteria and partially purified by polyethylene glycol (PEG) precipitation. The binding, elution, and amplification steps were repeated using HS and 3-0ST-3 modified HS as targets. Three rounds of selection were carried out to select for binders of progressively higher specificity. Low concentrations of detergent (Tween-20) in the early rounds resulted in high eluate titers, and the stringency was gradually increased with each successive round by raising Tween- 20 concentration stepwise to a maximum of 0.5%. This allowed selection of high affinity binding phages. For final selection, the eluted phages were plaque purified and titered on soft-agar plates.

Nucleotide sequencing and analysis

[0079] Automated nucleotide sequencing was performed to determine the sequences of the peptides encoded by the phages (Research Resource Center (RRC), University of Illinois at Chicago). Phage DNA was purified according to the manufacture's protocol using QIAGEN mini-prep kit (Valencia, CA). DNA sequencing was initiated using ABI prism BigDye Terminator Kit (Applied Biosystem, Foster City, CA) and the -96 gill sequencing primer (New England Biolabs, Cambridge, MA). Sequencing was performed on a Hitachi 3100 gene analyzer (Applied Biosystems, Foster City, CA) and the 36 nucleotide long' DNA regions encoding the 12-mer peptides were identified and used for peptide synthesis. The synthetic peptides were resuspended at a concentration of 10 mM in phosphate buffer saline (PBS) at pH 7.4, and stored at -80°C until use. The purity of the peptides was >95% as verified by high-performance liquid chromatography. The correct mass of the peptides was confirmed by mass spectrometry.

Cell culture and viruses

[0080] The presently described examples employed a variety of cell types, including wild-type Chinese hamster ovarian (CHO-K 1 ), mutant CHO-745, and CHO- Ιβ8 cells. In addition, primary cultures of human corneal fibroblasts (CF), retinal pigment epithelial (RPE), human conjunctival (HCjE), Vero, and HeLa cells were also used.

[0081 ] CHO cell lines were grown in Ham's F- 12 medium (Gibco BRL, Carlsbad,

CA, USA) supplemented with 10% fetal bovine serum (FBS), penicillin, and streptomycin (P/S) (Gibco/BRL). CF, HeLa, and RPE cells were grown in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% FBS and P/S (Tiwari et al, J. Virol. 80:8970-8980 (2006) and Tiwari et al, FEBS J. 275 :5272-5285 (2008)), and Vero cells were grown in DMEM with 5% FBS and P/S. Cultured HCjE cells were grown as described in Akhtar et al, Invest. Ophthalmol. Vis. Sci. 49:4026-4035 (2008).

[0082] HSV-1 strains, including the β-galactasidase expressing recombinant

HSV-1 (KOS) gL86 virus strain, the HSV-2 G strain, and the HfM, F, MP and KOS strains were provided by P.G. Spear at Northwestern University. Oh et al, Biochem Biophys Res Commun 391 , 176-81 (2010). Green fluorescent protein (GFP) expressing HSV- 1 (K26GFP), GFP expressing HHV-8 were provided by Drs. Prashant Desai at Johns Hopkins University and J. Viera at University of Washington. Desai and Person, J. Virol. 72:7563-7568 ( 1998). The β-galactasidase-expressing recombinant cytomegalovirus (CMV) was obtained from ATCC.

HSV-1 Entry Assay

[0083] HSV-1 entry was assayed as described in Shukla et al , Cell 99: 13-22

(1999). CHO- l cells were grown in 6-well plates to subconfluence and then transfected with 2.5 g of expression plasmids for gD receptors nectin-1 (pBG38), HVEM (pBec l O), 3-OST-3 isoform (pDS43), or pCDNA3.1 (empty vector) using LipofectAMINE (Gibco/BRL). At 16 h post-transfection, the cells were replated into 96-well dishes for pre-incubation with peptides at different concentrations for 60 min at room temperature. In parallel, natural target cells (HeLa, CF, and RPE) were also pre-treated with the peptides for the same duration. In all cases, unbound peptides were removed after washing 3X with PBS. Thereafter, cells were infected for 6 h with a recombinant virus, HSV- 1 (KOS) gL86, at multiple plaque forming units (PFUs) and β-galactosidase assays were performed using either a soluble substrate o-nitrophenyl-p-D-galactopyranoside (ONPG at 3.0 mg/ml; ImmunoPure, Pierce) or X-gal (Sigma). For the soluble substrate, the enzymatic activity was measured at 410 nm using a micro-plate reader (Spectra Max 190, Molecular Devices, Sunnyvale, CA). For the X-gal assay, cells were fixed (2% formaldehyde and 0.2% glutaraldehyde) and permeabilized (2 mM MgCl₂, 0.01% deoxycholate, and 0.02% nonidet NP-40 (Sigma)). 1 ml of β-galactosidase reagent ( 1.0 mg ml X-gal in ferricyanide buffer) was added to each well and incubated at 37°C for 90 min before the cells were examined using bright field microscopy under the 20* objectives of the inverted microscope (Zeiss Axiovert 100 M).

Fluorescent Microscopy o f Viral Entry

[0084] Cultured monolayers of HeLa and CF (approximately 10⁶ cells/well) were grown overnight in DMEM media containing 10% FBS on chamber slides (Lab-Tek). One pool of each cell-type was pre-treated with G l , G2, or a control peptide for 60 min. Cells were then infected with HSV- 1 26GFP (Desai and Person J. Virol. 72:7563-7568 ( 1998)) at 50 PFU in serum-free media OptiMEM (Invitrogen), which was followed by fixation of cells at 90 min post-infection using fixative buffer (2% formaldehyde and 0.2% glutaradehyde). The cells were then washed and permeabilized with 2 mM MgCl₂, 0.01 % deoxycholate, and 0.02% Nonidet NP-40 for 20 min. After rinsing, 10 nM rhodamine-conjugated phalloidin (Invitrogen) was added for F-actin staining at room temperature for 45 min. The cells were washed and the images of labeled cells were acquired using a confocal microscope (Leica, Solms, Germany) and analyzed with MetaMorph software (Molecular Devices, Sunnyvale, CA).

CMV Entry Assay

[0085] Natural target RPE cells were incubated with G l and G2 peptides for 60 min at room temperature before the cells were infected with β-galactosidase expressing CMV (ATCC) for 8 h. β-galactosidase assays were performed using either a soluble substrate o-nitrophenyl-P-D-galactopyranoside (ONPG at 3.0 mg/ml; ImmunoPure, Pierce) or X-gal (Sigma). HHV-8 Infection Assay

[0086] HCjE cells grown in chamber slides (Labteck) or in a 96 well plate were pre-treated with G l , G2, or control peptides for 60 min at room temperature followed by inoculation with recombinant rHHV-8.152, expressing the green fluorescent protein (GFP). Viera et al, J. Virol. 72:5182-5188 (2000). 48-hr post-infection, GFP-positive cells were visualized under microscope (Zeiss Axioverst 100M). HHV-8 infection was determined as relative fluorescence units (RFU) using GENios Pro plate reader (TECAN) at 480-nm excitation and 520-nm emission frequencies. Five measurements of negative control, positive control, and the test samples were performed. Data were expressed as mean ± SD.

HSV-1 Glycoprotein Induced Cell-to-cell Fusion Assay

[0087] CHO- 1 "effector" cells (grown in F- 12 Ham, Invitrogen) were co- transfected with plasmids expressing four HSV- 1 (KOS) glycoproteins: pPEP98 (gB), pPEP99 (gD), pPEPl OO (gH), and pPEP l O l (gL), along with the plasmid pT7EMCLuc that expresses firefly luciferase gene under transcriptional control of the T7 promoter. Pertel et al, Virology 279:3 13-324 (2001 ). Wild-type CHO- 1 cells, which express cell- surface HS but lack functional gD receptors, were transiently transfected with HSV- 1 entry receptors. Wild-type CHO-K1 cultured cells expressing HSV- 1 entry receptors or naturally susceptible cells (human CF) considered as "target cells" were co-transfected with pCAGT7 that expresses T7 RNA polymerase using chicken actin promoter and CMV enhancer. Tiwari et al, FEBS Letters 581:4468-4472 (2007). Untreated effector cells expressing pT7EMCLuc and HSV-1 essential glycoproteins, and target cells expressing gD receptors transfected with T7 RNA polymerase, were used as positive controls. G2 or control peptide-treated target cells were then co-cultivated (1 : 1 ratio) for 18 h with effector cells for fusion. Activation of a reporter luciferase gene — as a measure of cell fusion— was examined using reporter lysis assay (Promega) at 24 hr post mixing. Flow Cytometry Analysis

[0088] Flow cytometry was performed to detect the effect of G2 peptide on GFP-

HSV-1 binding to human CF. Monolayers of approximately 5 ^χ 10⁶ CF were pre-treated with G2 peptide for 60 min followed by incubation with GFP-expressing HSV- 1 ( 26GFP) at 4°C. A control peptide treated and untreated cells were similarly incubated with HSV-1 GFP virions. Uninfected CF were used as background negative control. GFP expression from the viral capsid on cell surface was examined by a flowcytometer (MoFlo).

Immunohistochemistry

[0089] BALB/c mice with pre-scarred corneal upper surface were treated with

PBS-based eye drops containing 0.5 mM G l , G2, or control peptide followed by inoculation of HSV-1 ( OS). Mice were sacrificed after 4 and 7 days for HSV- 1 detection. Immunohistochemistry was performed as described by Akhtar et al, Invest. Ophthalmol. Vis. Sci. 49:4026-4035 (2008). Briefly, tissue sections were hydrated with distilled water and antigen retrieval was performed using DAKO Target Retrieval Solution 10x concentrate (DAKO, Carpinteria, CA). Nonspecific staining was blocked using H₂0₂ solution for 10 minutes followed by a protein block for 10 minutes. Sections were incubated with HSV- 1 gD specific antiserum ( 1 : 100 dilution) at room temperature for 1 hr followed by a 40-minute incubation with the secondary antibody (HRP- conjugated goat anti-rabbit IgG, 1 :500; Sigma, St. Louis, MO). Expression of gD was detected using the DAKO Envision⁺ kit. Confocal and differential interference contrast (DIC) image acquisition was conducted with an SB2-AOBS confocal microscope (Leica, Solms, Germany).

Statistics

[0090] The data presented herein are means ± SD of triplicate measures of three or more experiments each performed independently. Error bars represent one standard deviation (SD). Statistical significance was calculated using Student's t-test. A p-value <0.05 was considered statistically significant. Example 2

Identification ofHS and 3-OS HS Binding Peptides that Block HSV-1 Entry

[0091] Three rounds of screening of phages from a 12-mer peptide phage display library resulted in the enrichment of phages that specifically-bound to HS and/or to 3-OS HS. Peptide sequences from individual plaques were deduced by determining the nucleotide sequences of the portion of the phage genome that encoded them. The predicted peptide sequences of about 200 plaques were determined and sorted into two groups on the basis of their targets. A frequently repeating peptide sequence from each group was subsequently selected for further characterization. The two most frequently isolated peptide sequences LRSRTKIIRIRH (designated G l for HS binding group 1 ) and MPRRRR1RRRQ (designated G2 for 3-OS HS binding group 2) were synthesized and examined for each peptide's ability to inhibit HSV-1 infection of 3-OST-3 (Figure 1 A), nectin-1 (Figure I B), and HVEM (Figure 1 C) expressing CHO-K 1 cells. Cells were pre- incubated with Gl , G2, or control peptide (Cp) at indicated concentration in mM or mock treated (C) with 1 χ phosphate saline buffer for 60 min at room temperature. After 60 min, a β-galactosidase-expressing recombinant virus HSV-1 ( OS) HSV-1 gL86 (25 pfu/cell) virus was used for infection. After 6 hr, the cells were washed, permeabilized, and incubated with ONPG substrate (3.0 mg/ml) for quantitation of β -galactosidase activity expressed from the input viral genome. The enzymatic activity was measured at an optical density of 410 nm (OD 410). Each value shown is the mean of three or more determinations (± SD).

[0092] Both of the G l and G2 peptides were able to block HSV-1 entry into

CHO- 1 cells expressing one of the three gD receptors {i.e., 3-OS HS, nectin-1 , and HVEM). Viral entry blockage occurred in a dose-dependent manner and was independent of gD receptor used. The concentration of each peptide that produced 50% of its maximum potential inhibitory effect (IC50) ranged from 0.02 to 0.03 mM. A control phage bearing the sequence RVCGSIGKEVLG (designated Cp) did not inhibit HSV- 1 entry. None of the peptides exhibited significant cytotoxicity (MTS assay, Promega) at <5 mM. The highest concentration of peptides in the experiments presented herein was 0.5 mM.

[0093] The ability of the G l and G2 peptides to block HSV-1 entry into natural target cells (HeLa and primary cultures of human CF) was compared. A similar dosage response curve was generated when HeLa (Figure 2A) or CF (Figure 2B) were pre- treated with G l or G2 peptides during HSV-1 entry. The control peptide treated cells had no effect on HSV-1 entry (Figure 2). HeLa cells (Figure 2A) and primary cultures of human corneal fibroblasts (CF) (Figure 2B) were tested. Cells in 96-well plates were pre-treated for 60 min with indicated mM concentrations of G l , G2, or Cp peptides. Mock-treated cells (abbreviated as C) served as a control. Pretreated cells were infected with a β galactosidase-expressing recombinant virus HSV-1 ( OS) HSV-1 gL86 (25 pfu/cell) for 6 hr. Viral entry was quantitated as described above in reference to Figure 1 . Confirmation of HSV-1 entry blocking activity of G l , G2, and control (Cp) peptides on a per cell basis was obtained after cells were infected as described above followed by X-gal (1 .0 mg/ml) staining (Right panels), which yields an insoluble blue product upon hydrolysis by β-galactosidase expressed from the input viral genomes. Dark (blue) cells represent infected cells, uninfected cells do not show any color. Microscopy was performed using a 20^χ objective of Zeiss Axiovert 100.

[0094] Use of insoluble blue cell assay (X-gal as the substrate for β- galactosidase) further confirmed that the peptides were effective in blocking infection of individual cells (Figures 2A and 2B, right panels). In virtually all cases, G2 peptide was slightly more effective in blocking entry than G l .

Example 3

The Peptide Inhibitors are also Active against a Variety of HSV-1 Strains

[0095] This Example demonstrates that the inhibitory effect of the G2 peptide is not limited by viral strain or serotype.

[0096] The anti-HSV properties of the G l and G2 peptides were evaluated against common strains of HSV- 1 and HSV-2 (i.e., strains F, G, Hfem, MP, KOS, and 17). Dean et al, Virology 199:67-80 (1994). 3-0ST-3 expressing CHO lg8 reporter cells were used that express β-galactosidase upon viral entry. Montgomery et al, Cell 87:427- 436 ( 1996). Cells were pre-incubated with G l , G2, or control peptide (Cp) and subsequently infected with the viruses. G2 or Cp control at 0.5 mM concentration was incubated for 60 min at room temperature with a reporter CHO-Ig8 cells that express β- galactosidase upon HSV-1 entry. After incubation, the cells were infected with HSV- 1 (Pal, 17, Hfm, F, KOS, and MP) and HSV-2 (G) strains at 25 pfu/cell for 6 hr at 37°C. Blockage of viral entry was measured by ONPG assay as described in Example 2 and as presented in Figure 1. These results, which are presented in Figure 3, demonstrated that G l and G2 blocked entry of various HSV- 1 strains by 70-80 % at 0.5 mM concentration.

Example 4

Structural Aspects of Gl and G2 Peptides:

G2 Shows More Dependence on Charged Residues

|0097] To better understand the inhibitory potential of G l and G2 peptides, synthetic short variants (10-mer) were synthesized that lacked the terminal non-positively charged amino acids. In case of G l , N-terminus L and C-terminus H residues were removed. For G2, the N-terminus flanking residues (M and P) were removed. Without being bound by theory, it is believed that 12-mer peptides are too short to adopt substantial secondary or tertiary structures and that the primary structure of those peptides, which includes a defined sequence or groupings of positively charged residues, plays a critical role in mediating inhibition of HSV-1 entry. Synthetic 10-mer versions of G l and G2 (shown above) were tested for blocking HSV-1 (KOS)gL86 entry into cultured CF. After 6 h, the viral entry was measured by ONPG assay as described in Figure 1 .

[0098] As shown in Figure 4A, the 10-mer version of G2 was very similar to the

12-mer in its ability to block HSV-1 entry into CF. In contrast, the 10-mer version of G l peptide almost completely lost its ability to block HSV- 1 entry into target cells. Thus, it is terminal L and H residues appear to be required for the anti-HSV- 1 activity of G l . G2, on the other hand, relies more on its charged residues for its functional activity.

[0099] Alanine (A) scanning mutagenesis was performed to identify specific amino acid residues responsible for each peptide's function, stability, and conformation. O'Nuallain et al, Biochemistry 46: 13049-13058 (2007). Twelve synthetic peptides were made whereby each residue of G2 was sequentially replaced with an alanine residue and corresponding changes in the G2 peptide were evaluated for their ability to affect viral entry (Figure 4B). The location of alanine in the peptide is denoted by a number next to it. Cp represents the control peptide and the oligomeric G2 is listed as G2-0. Figure 4B depicts the relative loss of inhibitory potential upon substitution of a residue within G2 by an alanine. Activity of each peptide was normalized against the wild-type G2, which was kept at 1 .00. Numbers higher than 1 show loss of activity whereas a lower number represents gain of activity.

[00100] This Example demonstrates that the first four arginine (R) residues and the last R and lysine ( ) residue were essential. The middle two amino acids could be substituted with only a moderate loss of activity. The uncharged amino acids each tolerated substitution with alanine. Under similar experimental conditions, G2 oligomers (G2-0) were also examined and it was evident that they blocked infection about 2-fold better than G l (Figure 4B). These mutagenesis results demonstrate that the presence of positively charged amino acid residues plays an important role in HSV- 1 entry blocking activity shown by G2.

Example 5

G2 Represents a Class of Broad Spectrum Anti-HS Peptides with Activity against Multiple Herpesviruses

[00101] This Example demonstrates that G2, but not G l , is effective in blocking viral entry of herpesviruse family members in addition to a-herpesviruses {e.g., HSV-1 ).

[00102] Many infectious viruses, including many herpesviruses, utilize cell surface HS moieties during viral binding and entry. Shukla and Spear, J. Clin. Invest. 108:503- 10 (2001 ). As with HSV- 1 (an cc-herpesvirus), β-herpesvirus (cytomegalovirus; CMV) and γ-herpesvirus (human herpesvirus-8; HHV-8) also use HS during cell entry and fusion. Liu and Throp, Med. Res. Rev. 22: 1 -25 (2002) and Shukla and Spear, J. Clin. Invest. 108:503-510 2001 . [00103] In order to detect each peptide's effect on viral entry, Lac Z-expressing reporter CMV and GFP-expressing HHV-8 viruses (Viera et al., J. Virol. 72:5182-51 88 (2000)) and their natural target cells were employed for entry measurements. G2 peptide, but not G l peptide, showed clear effects against CMV and HHV-8 (Figure 5). A monolayer of cultured RPE cells grown in a 96 well plate were pre-treated with G l , G2, or control peptide (Cp) at 0.5 mM concentration (Figure 5A). A mock-treated population of RPE cells served as positive control (abbreviated as C). After 60 min of incubation at room temperature, the cells were infected with β-galactosidase expressing CMV reporter virus. After 8 h, viral entry was measured as described for Figure 1.

[00104] The effect of CMV entry blocking activity of G l , G2 or Cp peptide for individual RPE cells was determined by X-gal staining, which yields an insoluble blue product upon hydrolysis by β-galactosidase (Figure 5B). Individual cells were examined using a Zeiss Axiovert 100 microscope at 20x magnification. Infected cells turn blue. These data demonstrate that G2 peptide was effective in blocking CMV entry into RPE cells, whereas G l peptide had no effect on viral entry. The effect of G l was similar to the control peptide (cp) or peptide untreated cells and the same pattern was repeated when the effects of the peptides were examined on a per cell basis by an X-gal assay.

[00105] The ability to block HHV-8 infection was examined in human conjunctival epithelial (HCjE) cells, a natural target for HHV-8 infection. Human conjunctival (HCjE) cells were pre-incubated with G l , G2 or control peptide (Cp) were infected with HHV-8 virions for 48 h at 37°C. After incubation the cells were washed thoroughly to remove unbound viruses. GFP-expression of HHV-8 was quantitated by determining relative fluorescence units (RFU) using a 96-well fluorescence reader (TECAN). Emission of fluorescence indicates virus infection. These data, which are presented in Figure 5C, are the means of triplicate measures and are representative of 3 independent experiments. Compared to G l or Cp treated cells, G2 treated HcjE cells had relatively low GFP-expression. This suggested that G2 was able to block infection. While G l also demonstrated a reduction in fluorescence in the representative case shown in Figure 5C, it was not found to be statistically significant upon repeated experiments. This was confirmed by examination of individual cells by fluorescence microscopy. [00106] Viral replication in HCjE cells was visualized under fluorescent microscope (Zeiss Axiovert 100) in cells that were pretreated with G l , G2 or Cp, as described above (Figure 5D). Asterisks indicate significant difference from controls and/or treatments (P < 0.05, / test) and error bars represent SD. Cells treated with G2 did not show fluorescence originating from GFP virus replication. However, the fluorescence was more easily seen with G l or Cp treated cells. The results suggest that G2 is more effective than G l in blocking entry of divergent herpesviruses. G l may have some activity but the virus can possibly overcome it easily.

Example 6

Mechanism for HSV-1 Entry Inhibition by the Peptides

[00107] This Example demonstrates that G2 peptide prevents target cell infection by herpesviruses by blocking viral HS binding sites and, hence, viral attachment.

[00108] Cultured CF were pre-incubated with 0.5 m G2 peptide or control- peptide (cp) and then infected with a GFP-tagged HSV- 1 ( 26GFP) virus. Cells were fixed at 60 min post-infection and stained for F-actin and the nucleus (DAPI). GFP- expressing HSV- 1 ( 26GFP) binding to CF in presence and absence of G2 peptides was examined by fluorescence microscopy (Figure 6A). CF were grown in collagen coated chamber slides and incubated at room temperature for 60 min with G2 (+) or control Cp . (G2 (-)) peptide. This was followed by the incubation of the cells in cold with GFP- expressing HSV-1 (K26GFP) for 30 min and washing of unbound virioins with PBS. Cells were fixed, stained with phalloidin for F-actin and DAPI for nuclei and examined by a fluorescence microscope (Leica, SP2). The presence of the virus was shown by detecting GFP. These data demonstrated that G2-treated cells resisted virus attachment as compared to the control peptide treated cells.

[00109] To examine this effect on a population of 10^s cells, GFP intensity as an indicator of virus binding, was measured after incubation with the virus in cold and rigorous washing of the cells afterwards (Figure 6B). Relative virus binding to CF was estimated by fluorescence measurements. Cultured CF were pre-incubated with G2 and control peptide (Cp) for 60-min before ice-cold incubation with GFP-expressing HSV- 1 virus for 30 min. Cells were washed 3 times and viruses remaining on cell surfaces were assayed for GFP fluorescent intensity using a fluorescence reader (Tecan). Clearly, the binding was significantly higher in Cp-treated compared to G2 peptide treated cells.

[00110] GFP-expressing HSV-1 (K26GFP) intensity as a surrogate for virus binding was quantified in presence G2 or control peptide (abbreviated as C) by flowcytometry (Figure 6C). The cell/virus incubation was performed as described above. G2 peptides block HSV-1 replication into cultured human corneal fibroblasts (CF) (Figure 6D). Cultured CF were pre-incubated with G2 or mock-treated (Cp) before infection with HSV-1 ( 26GFP) virus for 6 h. Viral replications in CF were quantified 0- 36 h post-infection by measuring GFP fluorescent intensity using a fluorescence reader (Tecan). The data shown are the means of triplicate measures and are representative of three independent experiments. Asterisks indicate significant difference from other treatments (P<0.01 , / test), error bars represent standard deviation (SD).

Example 7

G2 Peptide Acts by Inhibiting HSV-1 Binding to HS

[00111] This Example demonstrates, via flow cytometry detection, a significant reduction of GFP reporter virus binding to cells pretreated with G2.

[00112] Primary cultures of human CF pretreated with G2 peptide or control peptide (C) were analyzed for HSV- 1 (K26GFP) binding. The peptide untreated CF incubated with the virus served as a positive control and uninfected CF served as a negative background control. The results presented in Figure 6 demonstrate that the virus failed to bind G2 peptide treated CF (Figure 6C). While the control peptide exhibited a low level inhibitory activity, the activity of the G2 peptide was far more robust. This result confirms that G2 has the ability to block virus attachment to cells.

[00113] To further confirm that blocking of viral attachment results in a reduction of viral replication, GFP fluorescence was measured as a function of time ( 26GFP) (Desai and Person J. Virol. 72:7563-7568 (1998)) in both G2- and mock-treated cells. GFP intensity (which reflects the degree of virus production) increased significantly over time (Figure 6D) in mock-treated cells but not when the cells were treated with G2. These results demonstrate that G2 blockage of virus binding results in a substantial reduction of viral replication.

Example 8

Pretreatment of G2 Peptide to the Target Cell

Significantly Affect Cell-to-cell Fusion and Viral Spread

[00114] This Example demonstrates that G2 not only blocks viral attachment to a target cell, but it also inhibits viral penetration by blocking membrane fusion.

[00115] Since G2 was isolated against 3-OS HS, which can mediate viral penetration by promoting membrane fusion (Tiwari et al., J. Gen. Virol. 85:805-809 (2004)), the ability of G2 to block HSV- 1 glycoprotein-mediated membrane fusion was tested. Pertel et al., Virology 279:313-324 (2001 ). The same membrane fusion is used during polykaryocytes formation and cell-to-cell spread. Tiwari et al. , FEBS Letters 581:4468-4472 (2007) and Tiwari et al, Biochemical and Biophysical Research Communications 390:382-387 (2009). 3-0ST-3 expressing CHO- 1 cells and primary cultures of human CF were pre-incubated with G2 peptide followed by co-culture with effector CHO- 1 cells expressing HSV-1 glycoproteins. The membrane fusion that ensues upon co-culturing the cells can be estimated by a Luciferase based reporter assay. Pertel et al, Virology 279:313-324 (2001 ). Likewise, polykaryocyte formation can be visualized by Giemsa staining.

[00116] Figure 7 shows "effector" CHO- 1 cells expressing HSV- 1 glycoproteins

(gB, gD, gH-gL and T7 polymerase) that were pre-incubated with G2 peptide (black bar) or 1 x PBS (white bar) (+) for 90 min. Control effector cells (T7 polymerase and gD, gH- gL only) (-) were also pre-incubated with G2 for the same duration. The effector cells were then mixed with primary cultures of human corneal fibroblasts (CF; Figures 7A and 7B) or 3-OST-3-expressing CHO-K 1 cells (Figures 7C and 7D) transfected with Luciferase gene under T7 control. Membrane fusion as a surrogate for viral spread was detected by monitoring luciferase activity (Figures 7A and 7C). Relative luciferase units (RLUs) were determined using a Sirius luminometer (Berthold detection systems). Error bars represent standard deviations. * P< 0.05, one way ANOVA. Microscopic images of Gimesa (Fluka) stained polykaryocytes show the preventative effect of G2's on cell fusion (Figures 7B and 7D). Shown are 40x magnified photographs of cells undergoing membrane fusion (Zeiss Axiovert 200).

[00117] These data show that prior treatment with G2 was very effective in blocking membrane fusion (Figures 7A and 7C) and that this ability translates into the loss of syncytia formation (Figures 7Ba and 7Da) compared to mock-treated cells (Figures 7Bb and 7Db).

Example 9

Gl and G2 Peptides Show Protective Effects against

HSV-1 Infection of the Mouse Cornea

[00118] This Example demonstrates that anti-HS and anti-3(9S HS peptides exhibit efficacy as anti-HSV prophylactic agents and that HS is an important co-receptor for an ocular HSV-1 infection in vivo.

[00119] The abilities of G l and G2 peptides against HSV- 1 infection was tested in a mouse cornea model. The cornea is known to express many gD receptors including 30S HS. Tiwari et al, J. Virol. 80:8970-8980 (2006) and Tiwari et al., FEBS Letters 581:4468-4472 (2007). The cornea is also an attractive target for HSV- 1 infection leading to the development of herpetic stromal keratitis (HSK), a potential blinding disease common in developed countries including United States. Liesegang, Cornea 20: 1 - 13 (2001 ).

[00120] Immunohistochemistry was used to locate HSV-1 glycoprotein D (gD) expression in the cornea pre-treated with either a control peptide, G l or G2 followed by HSV-1 infection. 100 μΐ of G l , G2, or Cp (control) peptide at 0.5 mM concentration was poured into the mouse cornea as a prophylactic "eye drop" followed by an infection with HSV-1 (KOS) at 10⁶ PFU. At 4 or 7 days post infection, immunohistochemistry was performed using anti-HSV- 1 gD polyclonal antibody. In sections of cornea of mice euthanized at 4^th and 7^th day following pretreatment with Cp-peptide (control) followed by virus inoculation, severe chronic inflammation combined with significant staining for HSV- 1 gD was demonstrated on day 4 (Figure 8A). HSV- 1 staining was gone by day 7, which is typical with normal mice; however, damage to the corneal epithelium was still evident (Figure 8B). In contrast, virtually no HSV-1 protein expression was detected in corneas treated with G l or G2 peptide and the epithelium remained intact at both 4 and 7 days post infection (Figures 8B, 8C, 8E, and 8F).

Claims

What is claimed is:

1. A peptide comprising at least 10 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R is arginine, and K is lysine.

2. The peptide of claim 1 wherein said peptide comprises at least 12 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2).

3. The peptide of claim 2 wherein said peptide is 12 amino acids in length.

4. The peptide of claim 1 wherein each X is independently selected from the group consisting of leucine, serine, threonine, isoleucine, and histidine.

5. The peptide of claim 1 wherein said peptide comprises at least 10 consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ).

6. The peptide of claim 5 wherein said peptide comprises at least 12 consecutive amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ).

7. The peptide of claim 6 wherein said peptide is 12 amino acids in length.

8. The peptide of claim 1 wherein said peptide can block binding of a virus to heparan sulfate or 3-0 sulfated heparan sulfate.

9. The peptide of claim 8 wherein said peptide can prevent a viral infection of a target cell.

10. The peptide of claim 9 wherein said target cell is a corneal cell. 1 1. The peptide of claim 8 wherein said virus is a herpesvirus.

12. The peptide of claim 1 1 wherein said a-herpesvirus is HSV- 1 .

13. A peptide comprising at least 10 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4) wherein X is any amino acid, R is arginine, and K is lysine.

14. The peptide of claim 13 wherein said peptide comprises at least 12 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4).

15. The peptide of claim 14 wherein said peptide is 12 amino acids in length.

16. The peptide of claim 13 wherein each X is independently selected from the group consisting of methionine, proline, isoleucine, and glutamine.

17. The peptide of claim 13 wherein said peptide comprises at least 10 consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3).

18. The peptide of claim 17 wherein said peptide comprises at least 12 consecutive amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3).

19. The peptide of claim 18 wherein said peptide is 12 amino acids in length.

20. The peptide of claim 13 wherein said peptide can block binding of a virus to heparan sulfate or 3-0 sulfated heparan sulfate.

21. The peptide of claim 13 wherein said peptide can prevent a viral infection of a target cell.

22. The peptide of claim 21 wherein said target cell is a corneal cell.

23. The peptide of claim 20 wherein said virus is a herpesvirus.

24. The peptide of claim 23 wherein said herpesvirus is selected from the group consisting of an a-herpesvirus, a β-herpesvirus, and a γ-herpesvirus.

25. The peptide of claim 24 wherein said a-herpesvirus is HSV- 1.

26. The peptide of claim 24 wherein said β-herpesvirus is cytomegalovirus (CMV).

27. The peptide of claim 24 wherein said γ-herpesvirus is human herpesvirus-8 (HHV-8).

28. A composition comprising two or more peptides wherein each of said peptides independently comprises at least 10 amino acids of a sequence selected from the group consisting of XRXRXKXXRXRX (SEQ ID NO: 2) and XXRRRRXRRRXK (SEQ ID NO: 4) wherein X is any amino acid, R is arginine, and K is lysine.

29. The composition of claim 28 wherein at least one of said peptides comprises at least 10 amino acids of the sequence LRSRTKI1RIRH (SEQ ID NO: 1 ).

30. The composition of claim 28 wherein at least one of said peptides comprises at least 10 amino acids of the sequence MPRRRRIRRRQK (SEQ ID NO: 3).

31 . A method for blocking the binding of a virus to a target cell, said method comprising the step of contacting said target cell with a peptide comprising at least 10 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R is arginine, and K is lysine.

32. The method of claim 3 1 wherein said peptide comprises at least 10 amino acids of the sequence LRSRTKIIRIRH (SEQ ID NO: 1 ).

33. The method of claim 31 wherein said virus is a herpesvirus.

34. The method of claim 33 wherein said herpesvirus is an a- herpesvirus.

35. The method of claim 34 wherein said a-herpesvirus is HSV- 1 .

36. A method for blocking the binding of a virus to a target cell, said method comprising the step of contacting said target cell with a peptide comprising at least 10 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4) wherein X designates any amino acid, R designates the amino acid arginine, and K designates the amino acid lysine.

37. The method of claim 36 wherein said peptide comprises at least 10 amino acids of the sequence MPRRRRI RRRQK (SEQ ID NO: 3).

38. The method of claim 36 wherein said virus is a herpesvirus.

39. The method of claim 38 wherein said herpesvirus is selected from the group consisting of an ot-herpesvirus, a β-herpesvirus, and a γ-herpesvirus.

40. The method of claim 39 wherein said a-herpesvirus is HSV- 1 .

41 . The method of claim 39 wherein said β-herpesvirus is cytomegalovirus (CMV).

42. The method of claim 39 wherein said γ-herpesvirus is human herpesvirus-8 (HHV-8).

43. A method for the treatment of a patient who is susceptible to a viral infection, said method comprising the step of administering to said patient a peptide comprising at least 10 consecutive amino acids of the sequence XRXRXKXXRXRX (SEQ ID NO: 2) wherein X is any amino acid, R is arginine, and K is lysine.

44. A method for the treatment of a patient who is susceptible to a viral infection, said method comprising the step of administering to said patient a peptide comprising at least 10 consecutive amino acids of the sequence XXRRRRXRRRXK (SEQ ID NO: 4) wherein X is any amino acid, R arginine, and K is lysine.

45. A peptide-therapeutic compound conjugate, comprising:

(a) a peptide of any one of claims 1 -27 and

(b) a therapeutic compound;

wherein said peptide is coupled to said therapeutic compound to generate said conjugate.

46. The peptide-therapeutic compound conjugate of claim 45 wherein said therapeutic compound is selected from the group consisting of a nucleoside analog, a small molecule, and an oligosaccharide.

47. The peptide-therapeutic compound conjugate of claim 46 wherein said nucleoside analog is a guanosine analog.

48. The peptide-therapeutic compound conjugate of claim 47 wherein said guanosine analog is acyclovir or valacyclovir. .

49. The peptide-therapeutic compound conjugate of claim 48 wherein said guanosine analog is acyclovir as depicted in Formula 1:

Formula I

The peptide-therapeutic compound conjugate of claim 46 wherein said oligosaccharide is capable of binding to one or more of HSV-1 glycoproteins gB, gC, and gD.

51. The peptide-therapeutic compound conjugate of claim 46 wherein said oligosaccharide is selected from the group consisting of a tetrasaccharide, a hexasaccharide, an octasaccharide, and a decasaccharide.

52. The peptide-therapeutic compound conjugate of claim 51 wherein said oligosaccharide is HS octasaccharide 1 having the structure of Formula II: Formula II

53. The peptide-therapeutic compound conjugate of claim 46 wherein said therapeutic compound is a small molecule.

54. The peptide-therapeutic compound conjugate of claim 46 wherein said small molecule is Bis-2-methyl-4-amino-quinolyl-6-carbamide (Surfen) having the structure of Formula III: