WO2012037498A2 - Peptides anti-héparane sulfate bloquant l'infection au virus herpès simplex in vivo - Google Patents

Peptides anti-héparane sulfate bloquant l'infection au virus herpès simplex in vivo Download PDF

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WO2012037498A2
WO2012037498A2 PCT/US2011/052002 US2011052002W WO2012037498A2 WO 2012037498 A2 WO2012037498 A2 WO 2012037498A2 US 2011052002 W US2011052002 W US 2011052002W WO 2012037498 A2 WO2012037498 A2 WO 2012037498A2
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peptide
herpesvirus
amino acids
seq
peptides
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PCT/US2011/052002
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WO2012037498A3 (fr
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Deepak Shukla
Vaibhav Tiwari
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University Of Illinois
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Priority to EP11826047.0A priority Critical patent/EP2616478A4/fr
Publication of WO2012037498A2 publication Critical patent/WO2012037498A2/fr
Publication of WO2012037498A3 publication Critical patent/WO2012037498A3/fr
Priority to US13/842,193 priority patent/US20130189784A1/en
Priority to US14/555,000 priority patent/US9464113B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D473/00—Heterocyclic compounds containing purine ring systems
    • C07D473/02—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6
    • C07D473/18—Heterocyclic compounds containing purine ring systems with oxygen, sulphur, or nitrogen atoms directly attached in positions 2 and 6 one oxygen and one nitrogen atom, e.g. guanine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
    • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
    • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D215/38—Nitrogen atoms
    • C07D215/42—Nitrogen atoms attached in position 4
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C08B37/0081—Reaction with amino acids, peptides, or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16111—Cytomegalovirus, e.g. human herpesvirus 5
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16411—Rhadinovirus, e.g. human herpesvirus 8
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/16011—Herpesviridae
    • C12N2710/16611—Simplexvirus, e.g. human herpesvirus 1, 2

Definitions

  • 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.
  • 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.
  • HSV herpes simplex virus
  • 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.
  • HSV- 1 and HSV-2 herpes simplex virus type-1 and -2
  • 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.
  • 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).
  • 3-OS HS 3-O-sulfortransferases
  • HSV envelope glycoproteins B and C bind HS and mediate virus attachment to cells. Shieh et al, J. Cell Biol. ⁇ 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.
  • 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
  • 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).
  • 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.
  • HAV human immunodeficiency virus
  • 30S HS also plays a role in hepatitis B virus replication (Zhang et al, Virology 406:280-285
  • HS-binding peptides or compounds can be used to prevent genital HPV, HIV, and cytomegalovirus infections.
  • peptides including anti-HS peptides and anti-3-OS HS peptides that significantly inhibit viral infection and/or receptor-mediated cell-to-cell fusion.
  • 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.
  • 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.
  • these peptides are 10 or 12 amino acids in length.
  • each X may be independently selected from the group consisting of leucine (L), serine (S), threonine (T), isoleucine (I), and histidine (H).
  • 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.
  • 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).
  • 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.
  • these peptides are 10 or 12 amino acids in length.
  • X may be independently selected from the group consisting of methionine (M), proline (P), isoleucine (I), and glutamine (Q).
  • 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.
  • 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).
  • 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 ⁇ -herpesvirus, and a ⁇ -herpesvirus.
  • the a- herpesvirus is HSV- 1.
  • the ⁇ -herpesvirus is cytomegalovirus (CMV).
  • the ⁇ -herpesvirus is human herpesvirus-8 (HHV-8).
  • 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.
  • 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).
  • 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).
  • Still further embodiments of the present disclosure provides methods for blocking the binding of a virus to a target cell
  • 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.
  • 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).
  • Yet further embodiments of the present disclosure provides methods for blocking the binding of a virus to a target cell
  • 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.
  • 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).
  • 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 ⁇ -herpesvirus, and a ⁇ -herpesvirus.
  • the a-herpesvirus is HSV-1.
  • the ⁇ -herpesvirus is cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • the ⁇ -herpesvirus is human herpesvirus-8 (HHV-8).
  • 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.
  • 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).
  • kits for the treatment of a patient who is susceptible to a viral infection comprising 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.
  • 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).
  • 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.
  • a therapeutic compound such as an antiviral compound selected from a nucleoside analog, an oligosaccharide, and a small molecule.
  • 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.
  • 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.
  • oligosaccharide can be an HS octasaccharide 1 having the structure of Formula II:
  • 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
  • Figure 1 demonstrates that the - inhibition of HSV-1 entry by 12-mer synthetic peptides is not specific to any particular gD receptor.
  • Figure 2 demonstrates that G l and G2 peptides block HSV-1 entry into human target cells.
  • Figure 3 demonstrates that HSV- 1 entry blocking activity of G2 peptide is not HSV-1 strain specific.
  • 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.
  • FIG. 5 demonstrates that G2 blocks cellular entry by representative members of beta and gamma herpesvirus subfamilies (CMV and HHV-8).
  • Figure 6 demonstrates that G2 functions by preventing HSV- 1 attachment to cells, which results in loss of binding and viral replication.
  • Figure 7 demonstrates the activity of G2 against HSV-1 glycoprotein induced cell-to-cell fusion and spread.
  • Figure 8 demonstrates that G l or G2 effectively block infection by HSV- 1 in a mouse model of corneal keratitis.
  • 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.
  • a virus such as a herpes simplex virus ⁇ e.g., HSV-1
  • 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.
  • 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 ⁇ -herpesvirus-, a ⁇ -herpesvirus-, and/or a ⁇ -herpesvirus-mediated disease.
  • 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.
  • 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).
  • 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.
  • HS moieties are frequently up-regulated during pathological conditions and may contribute to inflammation. Esko and Lindahl, J. Clin. Invest.
  • the presently disclosed peptides may also find utility in blocking the pathological effects of HS and in regulating inflammation.
  • 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.
  • 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.
  • 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).
  • Group 1 peptides of the present disclosure comprise at least 10 or at least
  • X is any amino acid, R is arginine, and is lysine.
  • X may be independently selected from the group consisting of leucine, serine, threonine, isoleucine, and histidine.
  • 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.
  • Group 2 peptides of the present disclosure comprise at least 10 or at least
  • X is any amino acid
  • R is arginine
  • K is lysine.
  • X may be independently selected from the group consisting of methionine, proline, isoleucine, and glutamine.
  • 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.
  • 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, ⁇ -herpesviruses, and ⁇ -herpesviruses.
  • the ct-herpesvirus is HSV-1 .
  • the ⁇ - herpesvirus is cytomegalovirus (CMV).
  • the ⁇ -herpesvirus is human herpesvirus-8 (HHV-8).
  • 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.
  • 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.
  • 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).
  • 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.
  • arginine has been found important for charge-charge interaction with HS. Knappe et al , J. Biol. Chem. 282:27913-22 (2007).
  • 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).
  • 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).
  • 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.
  • a 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.
  • “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.
  • 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.
  • 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.
  • 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.
  • physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological salt buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • 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.
  • 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).
  • disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • sterile saline optionally about 5% v/v dimethylsulfoxide (“DMSO”) for solubility
  • benzalkonium chloride optionally about 5% v/v dimethylsulfoxide (“DMSO”) for solubility
  • sulfuric acid to adjust pH.
  • 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.
  • acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
  • aqueous solutions up to about 10% v/v DMSO or Trappsol can be used to maintain solubility of some peptides.
  • sterile, fixed oils may be conventionally employed as a solvent or suspending medium such as, for example, synthetic mono- or di-glycerides.
  • fatty acids such as oleic acid or neutral fatty acids
  • Pluronic block copolymers can be formulated with lipids for time release from solid form over a period of weeks or months.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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).
  • HSV-1 herpes simplex virus type I
  • HSV-2 herpes simplex virus type II
  • VZV varicella zoster virus
  • Acyclovir is less active against Epstein-Barr virus (EBC) and cytomegalovirus (CMV).
  • conjugates that comprise one or more oligosaccharide such as a herpesvirus HS oligosaccharide.
  • oligosaccharide such as a herpesvirus HS oligosaccharide.
  • 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.
  • 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.
  • 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.
  • nESl nanoelectrospray ionization
  • 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:
  • 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.
  • the enzymatic steps can be varied.
  • the Cs-epi can be removed and 2-0 sulfotransferase R189A mutant can be used for step c of the synthetic pathway shown above.
  • 2-OST R189A specifically sulfates the GlcUA, not IdoUA. Bethea et al., Proc.
  • 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 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).
  • 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
  • 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 ).
  • 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 ).
  • 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
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • 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).
  • a linker may be employed between the HS and 3-OS HS binding peptide and the therapeutic compound.
  • 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.
  • 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).
  • 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).
  • 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 ⁇ - herpesvirus, and/or a ⁇ -herpesvirus).
  • a herpesvirus e.g., an a-herpesvirus, a ⁇ - herpesvirus, and/or a ⁇ -herpesvirus.
  • the ct-herpesvirus is HSV- 1 .
  • the ⁇ -herpesvirus is cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • the ⁇ -herpesvirus is human herpesvirus-8 (HHV-8).
  • 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.
  • G 1 and/or G2 a peptide(s) prevents HSV-1 spread in the cornea.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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 ⁇ g/kg a day, more typically from 1 to 10,000 ⁇ g kg or from 1 to 100 ⁇ g kg of body weight or from 1 to 10 ⁇ g 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.
  • a phage display library (PhD,TM-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).
  • 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 10 ⁇ g/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 NaHC0 3 (pH 8.6) buffer.
  • BSA bovine serum albumin
  • 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 ⁇ 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 ⁇ 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.
  • PEG polyethylene glycol
  • 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.
  • the eluted phages were plaque purified and titered on soft-agar plates.
  • 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.
  • PBS phosphate buffer saline
  • the presently described examples employed a variety of cell types, including wild-type Chinese hamster ovarian (CHO-K 1 ), mutant CHO-745, and CHO- ⁇ 8 cells.
  • CHO-K 1 wild-type Chinese hamster ovarian
  • mutant CHO-745 mutant CHO-745
  • CHO- ⁇ 8 cells CHO- ⁇ 8 cells.
  • primary cultures of human corneal fibroblasts (CF), retinal pigment epithelial (RPE), human conjunctival (HCjE), Vero, and HeLa cells were also used.
  • CHO cell lines were grown in Ham's F- 12 medium (Gibco BRL, Carlsbad,
  • 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.
  • DMEM Dulbecco's Modified Eagle Medium
  • 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).
  • HSV-1 strains including the ⁇ -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).
  • HSV-1 entry was assayed as described in Shukla et al , Cell 99: 13-22
  • 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).
  • pBG38 gD receptors nectin-1
  • HVEM pBec l O
  • 3-OST-3 isoform pDS43
  • pCDNA3.1 empty vector
  • LipofectAMINE Gibco/BRL
  • HSV- 1 HSV- 1 (KOS) gL86
  • PFUs plaque forming units
  • ⁇ -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).
  • ONPG o-nitrophenyl-p-D-galactopyranoside
  • X-gal Sigma
  • the enzymatic activity was measured at 410 nm using a micro-plate reader (Spectra Max 190, Molecular Devices, Sunnyvale, CA).
  • the cells were then washed and permeabilized with 2 mM MgCl 2 , 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).
  • Natural target RPE cells were incubated with G l and G2 peptides for 60 min at room temperature before the cells were infected with ⁇ -galactosidase expressing CMV (ATCC) for 8 h.
  • ⁇ -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).
  • 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).
  • GFP green fluorescent protein
  • 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
  • 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.
  • KOS HSV- 1 glycoproteins
  • 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 was examined using reporter lysis assay (Promega) at 24 hr post mixing.
  • HSV-1 binding to human CF Monolayers of approximately 5 ⁇ 10 6 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).
  • 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).
  • DIC differential interference contrast
  • 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 ⁇ phosphate saline buffer for 60 min at room temperature.
  • HSV-1 ⁇ -galactosidase-expressing recombinant virus HSV-1 ( OS) HSV-1 gL86 (25 pfu/cell) virus was used for infection.
  • OS ⁇ -galactosidase-expressing recombinant virus HSV-1
  • HSV-1 gL86 25 pfu/cell virus
  • ONPG substrate 3.0 mg/ml
  • 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).
  • 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.
  • 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.
  • Mock-treated cells served as a control.
  • Pretreated cells were infected with a ⁇ 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 ⁇ -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 ⁇ objective of Zeiss Axiovert 100.
  • the Peptide Inhibitors are also Active against a Variety of HSV-1 Strains
  • 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 ⁇ - 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.
  • G2 Represents a Class of Broad Spectrum Anti-HS Peptides with Activity against Multiple Herpesviruses
  • 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 ).
  • HCV-8 human conjunctival epithelial cells
  • 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.
  • This Example demonstrates that G2 peptide prevents target cell infection by herpesviruses by blocking viral HS binding sites and, hence, viral attachment.
  • 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).
  • SD standard deviation
  • This Example demonstrates, via flow cytometry detection, a significant reduction of GFP reporter virus binding to cells pretreated with G2.
  • 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.
  • 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.
  • Figure 7 shows "effector" CHO- 1 cells expressing HSV- 1 glycoproteins
  • 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).
  • 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 ).
  • 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 ⁇ 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 10 6 PFU. At 4 or 7 days post infection, immunohistochemistry was performed using anti-HSV- 1 gD polyclonal antibody.

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Abstract

Cette invention concerne des peptides anti-héparane sulfate et des méthodes utilisant ces peptides pour prévenir ou traiter les infections virales, notamment les infections à Herpès simplex de type α, β et γ, par exemple les infections à HSV-1, à CMV et à HHV-8 respectivement. Les peptides comprennent au moins 10 acides aminés de séquence XRXRXKXXRXRX et/ou XXRRRRXRRRXK, X représentant n'importe quel acide aminé, par exemple les peptides comprenant au moins 10 acides aminés de séquence LRSRTKIIRIRH et/ou au moins 10 acides aminés de séquence MPRRRRIRRRQK. Les peptides peuvent être couplés à un ou à plusieurs composés thérapeutiques pour générer des conjugués peptides-composé thérapeutique, le composé thérapeutique pouvant être un ou plusieurs des éléments suivants : analogue de nucléoside, oligosaccharide et petite molécule.
PCT/US2011/052002 2010-09-16 2011-09-16 Peptides anti-héparane sulfate bloquant l'infection au virus herpès simplex in vivo WO2012037498A2 (fr)

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EP11826047.0A EP2616478A4 (fr) 2010-09-16 2011-09-16 Peptides anti-héparane sulfate bloquant l'infection au virus herpès simplex in vivo
US13/842,193 US20130189784A1 (en) 2010-09-16 2013-03-15 Anti-heparan sulfate peptides that block herpes simplex virus infection in vivo
US14/555,000 US9464113B2 (en) 2010-09-16 2014-11-26 Anti-heparan sulfate peptides that block herpes simplex virus infection in vivo

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Cited By (5)

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CN103788232A (zh) * 2013-12-23 2014-05-14 深圳市海普瑞药业股份有限公司 一种硫酸乙酰肝素十糖及其制备方法和应用
WO2015102738A1 (fr) * 2013-10-18 2015-07-09 The Charles Stark Draper Laboratory, Inc. Compositions antivirales multivalentes, méthodes de préparation et utilisations de celles-ci
US9683017B2 (en) 2014-07-17 2017-06-20 University Tennessee Research Foundation Inhibitory peptides of viral infection
US20170209524A1 (en) * 2015-07-28 2017-07-27 Carnegie Mellon University Methods and Compounds to Suppress Viral Genome Release and Packaging
US10046050B2 (en) 2014-08-26 2018-08-14 University Of Tenessee Research Foundation Targeting immunotherapy for amyloidosis

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US5166173A (en) * 1991-01-29 1992-11-24 Genelabs Incorporated Method of treating herpes simplex virus infection
WO2000010591A1 (fr) * 1998-08-18 2000-03-02 The Trustees Of The University Of Pennsylvania Peptides permettant d'inhiber l'entree de l'herpes simplex
US20060051368A1 (en) * 2004-07-23 2006-03-09 Northwestern University Herpes simplex virus mutations
WO2009006618A2 (fr) * 2007-07-05 2009-01-08 University Of Kansas Protéine mutante icp0 du virus de l'herpès simplex.

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
See also references of EP2616478A4
TIWARI ET AL., J. GEN. VIROL., vol. 88, 2007, pages 1075 - 1079

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015102738A1 (fr) * 2013-10-18 2015-07-09 The Charles Stark Draper Laboratory, Inc. Compositions antivirales multivalentes, méthodes de préparation et utilisations de celles-ci
CN103788232A (zh) * 2013-12-23 2014-05-14 深圳市海普瑞药业股份有限公司 一种硫酸乙酰肝素十糖及其制备方法和应用
US9683017B2 (en) 2014-07-17 2017-06-20 University Tennessee Research Foundation Inhibitory peptides of viral infection
US10308685B2 (en) 2014-07-17 2019-06-04 University Of Tennessee Research Foundation Inhibitory peptides of viral infection
USRE47838E1 (en) 2014-07-17 2020-02-04 University Of Tennessee Research Foundation Inhibitory peptides of viral infection
US10046050B2 (en) 2014-08-26 2018-08-14 University Of Tenessee Research Foundation Targeting immunotherapy for amyloidosis
US10213506B2 (en) 2014-08-26 2019-02-26 University Of Tennessee Research Foundation Targeting immunotherapy for amyloidosis
US10646568B2 (en) 2014-08-26 2020-05-12 University Of Tenessee Research Foundation Targeting immunotherapy for amyloidosis
US20170209524A1 (en) * 2015-07-28 2017-07-27 Carnegie Mellon University Methods and Compounds to Suppress Viral Genome Release and Packaging
US10702572B2 (en) * 2015-07-28 2020-07-07 Carnegie Mellon University Methods and compounds to suppress viral genome release and packaging

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