WO2003051835A2 - Methodes et compositions d'inhibition de la production virale - Google Patents

Methodes et compositions d'inhibition de la production virale Download PDF

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Publication number
WO2003051835A2
WO2003051835A2 PCT/US2002/041028 US0241028W WO03051835A2 WO 2003051835 A2 WO2003051835 A2 WO 2003051835A2 US 0241028 W US0241028 W US 0241028W WO 03051835 A2 WO03051835 A2 WO 03051835A2
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group
peptidomimetic
peptide
protein
ncr
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PCT/US2002/041028
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WO2003051835A3 (fr
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Yuval Reiss
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Proteologics, Inc.
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Priority to AU2002359792A priority Critical patent/AU2002359792A1/en
Publication of WO2003051835A2 publication Critical patent/WO2003051835A2/fr
Publication of WO2003051835A3 publication Critical patent/WO2003051835A3/fr

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    • 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
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1016—Tetrapeptides with the first amino acid being neutral and aromatic or cycloaliphatic
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1024—Tetrapeptides with the first amino acid being heterocyclic
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • Gag late (L) domain is essential for efficient virus budding, but it is not sufficient.
  • the primary functional motifs of Gag L domains may be either a PPxY sequence (RSV, murine leukemia virus and Mason Pfizer monkey virus), P(T/S)AP (HTV-rl) or YxxL (equine anemia virus-, EIAV), both P(T/S)AP and PPxY (Rabies virus, VSV, filoviruses) or two overlapping elements PTAPPEY (Ebola virus).
  • the tetrapeptide PPxY is recognized by a family of protein interaction modules called WW domains.
  • the YxxL sequence is recognized by the AP-2 adaptor protein and is essential for clathrin-mediated endocytosis in the plasma membrane.
  • P(T/S)AP is recognized by TsglOl.
  • Each of the above domains may be recognized by more than one type of protein.
  • Ubiquitination is essential for viral assembly, and host E3 ubiquitin Hgases, such as Nedd4 (a WW domain protein), have been implicated in viral maturation. It is thought that the L-domains promote formation of one or more protein complexes involved hi ubiquitination and membrane reorganization steps of viral maturation. Complex assembly may be further aided by formation of particular membrane domains referred to as lipid rafts and barges. These structures may form around Gag oligomers and participate in the recruitment of other proteins involved in maturation.
  • the invention relates to methods and compositions for inhibiting viral reproduction in a cell, optionally by inhibiting an aspect of viral reproduction such as assembly, budding and/or release.
  • the invention relates to methods and compositions for treating viral infections in a subject.
  • Certain methods of the invention comprise administering to a subject a pharmaceutically effective amount of an L-Domain (LD) therapeutic, which inhibits virus reproduction by inhibiting the association of a virus L-domain containing protein (such as, for example, Gag or a functional equivalent of Gag) with one or more host proteins.
  • LD L-Domain
  • the LD therapeutic has the potential to inhibit the association of Gag with at least two different host proteins.
  • the LD therapeutic comprises a peptide, p ⁇ ptidomimetic or a small molecule, natural or synthetic, or other compound that mimics an L-domain and thereby competes with virus L-domain containing proteins for interaction with proteins, optionally host proteins, that bind to the L-domain.
  • an LD therapeutic comprises an LD peptide, such as a peptide comprising an amino acid motif contained in a viral L-domain, such as YxxL, the HIN1 L-domain motif PTAP, the Ebola L-domain motif PTAPPEY, Hie expanded HIN1 L-domain PTAPPEE or any peptide having one or more.
  • a viral L-domain such as YxxL, the HIN1 L-domain motif PTAP, the Ebola L-domain motif PTAPPEY, Hie expanded HIN1 L-domain PTAPPEE or any peptide having one or more.
  • PPxY, YxxL and/or P(T/S)AP motifs in any order and with any spacing, such that the peptide will act as a potent binding site for a . host protein of a retro virus, rhabdovirus and/or filoviruses, and • particularly HIN1, Ebola virus, Rabies virus and NSN.
  • the LD therapeutic comprises a mixture of two or more of the above LD peptides, such as, for example, a mixture comprising a peptide comprising a PPxY domain and a peptide comprising a P(T/S)AP domain.
  • an LD therapeutic comprises a peptide that contains a combination of PPxY, P(T/S)AP and/or YxxL and acts as a potent inhibitor of a virus such as a retrovirus, a rhabdovirus and/or a filovirus.
  • the LD therapeutic comprises a polypeptide including at least two of PPxY, P(T/S)AP and YxxL, optionally overlapping.
  • an LD peptide may comprise a hydrophobic modification, such as, for example, a lipid (eg. a fatty acid, a sterol, a triglyceride, an isoprenoid) or hydrophobic amino acid, etc.
  • a hydrophobic modification improves the effectiveness offheLD peptide, optionally by concentrating the therapeutic agent at a site of virus assembly, such as, for example, the plasma membrane or a vesicular membrane, or a specialized membrane structure such as a lipid raft or barge.
  • Hydrophobic modifications may be connected to subject peptides in essentially any way known to one of skill in the art, including attachment to an N-terminus, C-terminus or a side chain, and peptides may comprise more than one such modification.
  • An exemplary LD therapeutic comprises a polypeptide having an N-terminal myristoyl or palmitoyl group.
  • an LD therapeutic may comprise a polypeptide including an amino acid sequence that directs modification in vivo, such as, for example, an N-te ⁇ s ⁇ al myristylation or palmitylation signal sequence.
  • LD therapeutics may also comprise peptides that contain either HIV1 PTAP with an N-terminal myristylation or palmitylation signal, a peptide with the Ebola L-domain motif PTAPPEY with an N- terminal myristylation or palmitylation signal or any peptide expressing PPxY or P(T/S)AP or combinations thereof at any order and with any spacing containing an N- terminal myristylation or palmitylation signal that inhibit retrovirus, rhabdovirus and or filovirus reproduction and/or infectivity.
  • LD peptides inhibit BTN1, Ebola virus, Rabies virus and or NSN.
  • an LD peptide includes a modified amino acid.
  • a polypeptide of an LD therapeutic may be modified with a lipid moiety, a saccharide, a phosphate, etc.
  • the LD therapeutic comprises a PTAPxxY, PTAPPEY, PTAPPxxY peptide or peptidomimetic thereof, optionally including an ⁇ -terminal fatty acid modification, and optionally including a phosphorylated residue.
  • an LD therapeutic may comprise a fusion protein wherein a first portion of the fusion protein comprises an LD peptide.
  • a second portion of the fusion protein may comprise any of a variety of peptides that confer some desirable property, such as, for example, increased stability (a "stabilizing peptide"), targeting or entry of the desired cells (a "carrier peptide"), a peptide that facilitates production by facilitating, for example, expression or purification (a "production peptide”), etc.
  • Exemplary targeting peptides include viral targeting proteins such as the Tat profe ⁇ a from HIN or an antibody (eg. IgG, Fab, single chain antibody, etc.).
  • a viral targeting protein is selected to target the fusion protein to a cell type that is normally infected by the virus type that the LD therapeutic is intended to inhibit.
  • the invention provides nucleic acids comprising a coding sequence for an LD peptide or for a single chain antibody that binds to an L domain peptide sequence.
  • nucleic acids may be used, for example, to produce LD peptides, including fusion proteins.
  • nucleic acids may be administered to a subject so as to cause production of the LD peptide in
  • the LD therapeutic comprises a peptido imetic of an LD peptide described herein.
  • a peptidomimetic of an LD peptide described herein.
  • LD peptides, peptidomimetics and small molecules are able to compete with the native L domain peptide sequence for binding to a cell or molecule that has a binding site for the native L domain peptide sequence.
  • the compounds of the present invention are represented by formula 1:
  • Ri is -OH, -CH(OH)CH 3 , -CH 2 OH, -(CH 2 ) complicatOH, or -CHOH(CH 2 ) ⁇ CH 3 ;
  • R 2 is H, -CH 3) -CH 2 R b , or -SH;
  • Yi, Y 2 , Y , and Y 4 are trivalent radicals selected independently from the group consisting of -NR CRbC(O)-, -(O)CCR b NR b -, -CRbNRbC(O>, -C(O)NR b CR b -, - NCR b C(O)R b , R (O)CCR b N-, -NCH 2 C(O)-, -(O)CCH 2 N-, -NRbCR b N b -,-.
  • n is an integer in the range 1 to 6 inclusive;
  • R b is independently selected for each occurrence from the group consisting of a bond, H, halide, alkyl, alkenyl, alkynyl, hydroxyl, alkoxyl, silyloxy, amino, ⁇ itro, sulfhydryl, alkylthio, imino, amido, phosphoryl, phosphonate, phosphine, phosphonamide, carboxyl, carboxamide, keto, silyl, thioalkyl, alkylsulfonyl, arylsulfonyl, selenoalkyl, formyl, ester, heteroalkyl, cyano, guanidine, amidine, acetal, ketai, amine oxide, aryl, heteroaryl, aralkyl, hetero aralkyl, azide, aziridine, carbamate, epoxide, hydroxamic acid, imido, oxime, sulfonamine, sulfon
  • R 8 o represents an aryl, cycloalkyl, cycloalkenyl, heterocycly ⁇ , or polycyclyl group; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention have formula 1, wherein at least one of Yj, Y 2 , Y 3 , or Y 4 is a peptoid.
  • the compounds of the present invention have formula 1, wherein at least one of Yi, Y 2; Y 3 , or Y 4 is a benzodiazepine.
  • the compounds of the present invention have formula 1, wherein at least one of Yi, Y 2 , Y 3 , or Y is a gamma lactam ring.
  • the compounds of the present invention have formula 1, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a keto methylene group.
  • the compounds of the present invention have formula 1, ' wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is abeta amino alcohol group.
  • the compounds of the present invention have formula 1, wherein at least one of Yi, Y 2 , Y , or Y is a diamino ketone group.
  • the compounds of the present invention have formula 1,
  • the compounds of the present invention have formula 1, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a vinylogous peptide.
  • vinylogous peptide is a viylogous sulfonamino ' peptide.
  • the vinylogous peptide is a trans vinyiogous_peptide.
  • the compounds of the present invention have fo ⁇ mi ⁇ a 1, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a fluoroalkene group.
  • the compounds of the present invention have formula 1, wherein at least one of Yj, Y 2 , Y 3 , or Y is a phosphonamide.
  • the compounds of the present invention have formula 1, wherein at least one of Y ⁇ , Y 2 , Y 3 , or Y is a retro-inverso peptide analog.
  • R 3 may be - CH 2 CH 2 CH 2 - wherein R 3 and the nitrogen atom are part of a five membered ring .
  • Yi and Y 3 are trivalent radicals selected independently from the group consisting of -NR b CR b C(O)-, -(O)CCR b NR b -, -CR b NR b C(O)-, -C(O)NR b CR b -, R b (O)CCR b N-, -NCH 2 C(O)-, -(O)CCH 2 N-, -N feC bN *-, -C(O NR b CR b C(O)(C(R b ) 2 ) n C(O)-, -C(O)(C(R b ) 2 ) n C(O)CR b NR b -, - C(O)C(R b ) 2 CR b NR b -, -(O)C(R b ) 2 CR b -, -NR b -, -(O
  • Y 4 is a divalent radical selected from the group consisting of -NR b CR b C(O)Rb, - (O)CCR b N(R b ) 2 , -CR b NR b C(O)R b , -C(O)NR b C(R b ) 2 -, - NCH 2 C(O)R b , -(O)CCH 2 NR b , -NRbCR N Rb 2, ⁇ -C(O)CRbC(O)R h ,
  • n is an integer in the range 1 to 6 inclusive;
  • R b is independently selected for each occurrence from the group consisting of a bond, H, halide, alkyl, alkenyl, alkynyl, hydroxyl, alkoxyl, silyloxy, amino, nitro, sulfhydryl, alkylthio, imino, amido, phosphoryL phosphonate, phosphine, phosphonamide, carboxyl, carboxamide, keto, silyl, thioalkyl, alkylsulfonyl, arylsul&nyl, selenoalkyl, formyl, ester, heteroalkyl, cyano, guanidine, amidine, acetal, ketai, amine oxide, aryl, heteroaryl, aralkyl, hetero aralkyl, azide, aziridine, carbamate, epoxide, hydroxamic acid, imido, oxime, sulfonamine, sulfonamide,
  • Rso represents an aryl, cycloalkyl, cycloalkenyi, heterocyclyl, or p ⁇ lyeycT l group; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention have formula 2, wherein at least one of Y Y 2 , Y 3 , or Y 4 is a peptoid. In certain embodiments the compounds of the present invention have formula 2, wherein at least one of Y ⁇ , Y 2 , Y 3 , or Y 4 is a benzodiazepine.
  • the compounds of the present invention have formula 2, wherein at least one of Yj, Y 2 , Y 3 , or Y 4 is a gamma lactam ring.
  • the compounds of the present invention have formula 2, wherein at least one of Yi, Y 2 , Y 3 , or Y is a keto methylene group.
  • the compounds of the present invention have formula 2, wherein at least one of Yj, Y 2 , Y 3 , or Y 4 is a beta amino alcohol group.
  • the compounds of the present invention have formula 2, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a diamino ketone group.
  • the compounds of the present invention have formula 2, wherein at least one of Yi, Y 2 , Y 3 , or Y is a methyleneamino group.
  • the compounds of the present invention have formula 2, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a vinylogous peptide.
  • the vinylogous peptide is a viylogous sulfonamino peptide.
  • the vinylogous peptide is a trans vinylogous peptide.
  • the compounds of the present invention have formula 2, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a fluoroalkene group.
  • the compounds of the present invention have formula 2, wherein at least one of Yi, Y 2 , Y 3 , or Y is a phosp onamide.
  • the compounds of the present invention have formula 2, wherein at least one of Y l3 Y 2 , Y 3 , or Y 4 is a sulfonamide. In certain embodiments the compounds of the present invention have formula 2, wherein at least one of Y l5 Y 2 , Y 3 , or Y 4 is a retro-inyerso peptide analog.
  • the compounds of the present invention are represented by formula 3:
  • R 3 is H, CH 3 , CH(CH 3 ) 2 , CH 2 CH(CH 3 ) 2) CH(CH 3 )CH 2 CH 3 , CH 2 CH 2 SCH 3 , CH 2 (C 6 H 5 ), CH 2 OH, CH(OH)CH 3 , CH 2 SH, CH 2 (C 6 H 4 OH), CH 2 C(O)NH 2 , CH 2 CH 2 C(O)NH 2 , CH 2 C(O)OH; CH 2 CH 2 C(O)OH, CH 2 CH 2 CH 2 CH 2 NH 2 ,
  • R may be -CH 2 CH 2 CH 2 - wherein R and the nitrogen atom are part of a five membered ring;
  • R is a branched or unbranched C to C 6 alkyl
  • Y ⁇ is a divalent radical selected from the group consisting of N(R b ) 2 C b C(O)-, R b (0)CCR b NR b -, -C(R b ) 2 NR b C(0)-, R b C(O)NR b CR b -,
  • Y 2 and Y 3 are each trivalent radicals selected from the group consisting of - NR b CR b C(O)-, -(O)CCR NR b -, -CR b NR b C(O)-, -C(O)NR b CR b -, -NCR b QO ⁇ , R b (O)CCR b N-, -NCH 2 C(O)-, -(O)CCH 2 N-, -NRbCR b NR b -, -C(O)CRbC(OK - NR b CR b C(0)(C(R b ) 2 ) n C(O)-, -C(0)(C(R b ) 2 ) n C(O)CR b NR b -, -N bCRbCOW GK - C(O)C(R h ) 2 CR b NR b -, -(C
  • Y 4 is a divalent radical selected from the group consisting of -NR b CR b C(O)R b , - (0)CCR b N(R b ) 2 , -CR b NR b C(O)R b , -C(O)NR b C(R b ) 2 -, -NCRbC(O)Rb, Rb(0)CCR b NR b -, - NCH 2 C(O)R b , -(O)CCH 2 NR b , -NR b CR b N(R b ) 2 , -C(O)CR b C(O)R b ,
  • n is an integer in the range 1 to 6 inclusive;
  • R b is independently selected for each occurrence from the group consisting of a bond, H, halide, alkyl, alkenyl, alkynyl, hydroxyl, alkoxyl, silyloxy, amino, nitro, sulfhydryl, alkylthio, i ino, amido, phosphoryl, phosphonate, phosp ne, phosphonamide, carboxyl, carboxamide, keto, silyl, thioalkyl, alkylsulfonyl, arylsulfonyl, selenoalkyl, formyl, ester, heteroalkyl, cyano, guanidine, amidine, acetal, ketai, amine oxide, aryl, heteroaryl, aralkyl, heteroaralkyl, azide, aziridine, carbamate, epoxide, hydroxamic acid, imido, oxime, sulfonamine, sulfonamide
  • R 8 o represents an aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl group; and m is an integer in the range 0 to 8 inclusive.
  • the compounds of the present invention have formula 3,
  • Yj, Y 2 , Y3, or Y 4 is a peptoid.
  • the compounds of the present invention have formula 3, wherein at least one of Y s Y 2 , Y 3 , or Y 4 is a benzodiazepine.
  • the compounds of the present invention have formula 3, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a gamma lactam ring.
  • the compounds of the present invention have formula 3, wherein at least one of Y ls Y 2 , Y 3 , or Y is a eto methylene group.
  • the compounds of the present invention have formula 3, wherein at least one of Y Y 2 , Y 3 , or Y is a beta amino alcohol group.
  • the compounds of the present invention have formula 3, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a diamino ketone group.
  • the compounds of the present invention have formula 3, wherein at least one of Y l ⁇ Y 2 , Y 3 , or Y is a methyleneam ⁇ no group.
  • the compounds of the present invention have formula 3, wherein at least one of Yi, Y 2 , Y 3 , or Y is a vinylogous peptide.
  • the vinylogous peptide is a viylogous srdfonamino peptide.
  • the vinylogous peptide is a trans vinylogous peptide.
  • the compounds of the present invention have formula 3, wherein at least one of Yj, Y 2 , Y 3 , or Y 4 is a fluoroalkene group.
  • the compounds of the present invention have formula 3, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a phosphonamide.
  • the compounds of the present invention have formula 3, wherein at least one of Yi, Y 2 , Y 3 , or Y 4 is a sulfonamide.
  • the compounds of the present invention have formula 3, wherein at least one of Y l5 Y 2 , Y 3 , or Y 4 is a retro-inverso peptide analog.
  • an LD therapeutic comprises a peptide, peptidomimetic or small molecule that mimics an L-domain recognition site of a host protein and can compete for binding to a virus L-domain.
  • Figure 1 Release of virus-like particles (VLP) from cells. Phospho-images of SDS- PAGE gels of immunoprecipitated 35S- labeled gag proteins from cell (upper panel) and viral (lower panel) lysates from transfected HeLa cells. Cells were transfected with one of the following plasmids: HTV-expressing plasmids - PTAPPEE, ATAAPEE, PTAPPEY and ATAPPEY. Cells that were transfected with PTAPPEE and PTAPPEY were also either untreated or treated with TSG101 RNAi (50nM for 48 hours). The chase time is presented from left to right for each image.
  • VLP virus-like particles
  • Figure 2 Release of virus-like particles from cells. Graphs of the results of the pulse chase experiments, as quantified by image analysis, are presented as the percentage of p24 released from the cells (VLP) versus the total amount of Gag inside and outside the cells over time.
  • amino acid motif is a sequence of amino acids, optionally a generic set of conserved amino acids, associated with a particular functional activity.
  • binding refers to a direct association between two molecules, due to, for example, covalent, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions, and including interactions such as salt bridges and water bridges.
  • Cells “host cells” or “recombinant host cells” are terms used interchangeably herein. It is understood that such terms refer not only to the particular subject cell but to the progeny. or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.
  • a "chimeric protein” or “fusion protein” is a fusion of a first amino acid sequence
  • a chimeric protein may present a foreign domain which is found (albeit in a different protein) in an organism which also expresses the first protein, or it may be an "interspecies", “intergenic”, etc. fusion of protein structures expressed by different kinds of organisms.
  • compound used herein interchangeably and are meant to include, but are not limited to, peptides, nucleic acids, carbohydrates, small organic molecules, natural product extract libraries, and any other molecules (including, but not limited to, chemicals, metals and organometallic compounds).
  • amino acids refers to grouping of amino acids on the basis of certain common properties.
  • a functional way to define common properties between individual amino acids is to analyze the normalized frequencies of amino acid changes between corresponding proteins of homologous organisms (Schulz, G. E. and R. H. Schirmer., Principles of Protein Structure, Springer- Verlag). According to such analyses, groups of amino acids may be defined where amino acids within a group exchange preferentially with each other, and therefore resemble each other most in their impact on the overall protein structure (Schulz, G. E. and R. BL Schirmer.,
  • a small-residue group consisting of Ser, Thr, Asp, Asn, Gly, Ala, Glu, Gin and
  • each amino acid residue may form its own group, and the group formed by an individual amino acid may be referred to simply by the one and/or three letter abbreviation for that amino acid commonly used in the art.
  • a “conserved residue” is an amino acid that is relatively invariant across a range of similar proteins. Often conserved residues will vary only by being replaced with a similar amino acid, as described above for “conservative amino acid substitution”.
  • the term "consisting essentially of as used in reference to a peptide including one or more designated amino acid sequences indicates that no more than' 20 to 30 amino acids are added to the designated amino acid sequence(s), and furthermore that these additional amino acids do not substantially alter the function of the designated amino acid sequence(s).
  • the term “consisting essentially of 1 as used in reference to a peptidomimetic indicates that no more than 20-30 amino acid mimetic units are added to the designated sequence, and that these added units do not substantially alter the function of the designated sequence.
  • an "effective amount" of, e.g., an LD peptide or peptidomimetic, with respect to the subject methods of treatment refers to an amount of active ingredient in a preparation which, when applied as part of a desired dosage regimen brings about, e.g., a change in the course of viral infection, including changes in rate of viral reproduction, changes in the viral load, changes in the infectivity of the virus, etc.
  • Homology or “identity” or “similarity” refers to sequence similarity between two peptides or between two nucleic acid molecules. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an equivalent position in the compared sequences is occupied by the same base or amino acid, then the molecules are identical at that position; when the equivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences.
  • a sequence which is "unrelated” or “non-homologous” shares less than 40% identity, though preferably less than 25% identity with a sequence of the present invention.
  • the absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases the identity and homology/similarity.
  • the term "homology” describes a mathematically based comparison of sequence similarities which is used to identify genes or proteins with similar functions or motifs.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters of the respective programs e.g., XBLAST and BLAST
  • hydrophobic refers to the tendency of chemical moieties with nonpolar atoms to interact with each other rather than water or other polar atoms.
  • Materials that are “hydrophobic” are, for the most part, insoluble in water.
  • Natural products with hydrophobic properties include lipids, fatty acids, phospholipids, sphingolipids, acylglycerols, waxes, sterols, steroids, terpenes, prostaglandins, thromboxanes, leukotrienes, isoprenoids, retenoids, biotin, and hydrophobic amino acids such as tryptophan, phenylalanine, isoleucine, leucine, valine, methionine, alanine, proline, and tyrosine.
  • a chemical moiety is also hydrophobic or has hyckophobic properties if its physical properties are determined by the presence of nonpolar atoms.
  • the term includes lipophilic groups.
  • identity means the percentage of identical nucleotide or amino acid residues at corresponding positions in ' two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford Umversity Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)).
  • the BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM H Bethesda, Md. 20894; Altschul, S., et al., J. Mol. Biol. 215: 403-410 (1990).
  • the well known Smith Waterman algorithm may also be used to determine identity.
  • Inhibit or “inhibition” as used herein with respect to binding between a host protein and a late domain peptide is intended to indicate any reproducibly detectable decrease in the binding interaction. Generally, inhibition is measured using a fixed. concentration of the host protein and late domain peptide and varying thexoncentration of the inhibitor to create an inhibition curve. Inhibition may be as little as 5% or greater, optionally 10%, 20%, 30%, 50%, 75%, 95% or more, although the exact level of inhibition will depend on the concentration of reagents used.
  • inhibition of VLP production may be as little as 5% or greater, optionally 10%, 20%, 30%, 50%, 75%, 95% or more, although the exact level of inhibition will depend on the concentration of reagents used.
  • isolated when applied to polypeptides means a polypeptide or a portion thereof which, by virtue of its origin or manipulation: (i) is present in a host cell as the expression product of a portion of an expression vector; or (ii) is linked to a protein or other chemical moiety other than that to which it is linked in nature; or (iii) does not occur in nature, for example, a protein that is chemically manipulated by appending, or adding at least one hydrophobic moiety to the protein so that the protein is in a form not found in nature.
  • isolated it is further meant a protein that is : (i) synthesized chemically; or (ii) expressed in a host cell and purified away from associated and contaminating proteins.
  • the term generally means a polypeptide that has been separated from other proteins and nucleic acids with which it naturally occurs.
  • the polypeptide is also separated from substances such as antibodies or gel matrices (polyacrylamide) which are used to purify it
  • a "lipid” includes any of a group of organic compounds, including the fats, oils, waxes, sterols, and triglycerides, that are insoluble in water but soluble in nonpolar organic solvents, are oily to the touch, and together with carbohydrates and proteins constitute the principal structural material of living cells.
  • lipophilic group in the context of being attached to a-polypept ⁇ de, refers to a group having high hydrocarbon content thereby giving the group high affinity to lipid phases.
  • a hpophilic group can be, for example, a relatively long chain alkyl or cycloalkyl (preferably n-alkyl) group having approximately 7 to 30 carbons.
  • the afl yl group may terminate with a hydroxy or primary amine "tail".
  • hpophilic molecules include naturally-occurring and synthetic aromatic and non-aromatic moieties such as fatty acids, esters and alcohols, other lipid molecules, cage structures such as adamantane and buckminsterfullerenes, and aromatic hydrocarbons such as benzene, perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, and naphthacene.
  • a "patient” or “subject” to be treated by the subject method can mean either a human or non-human animal.
  • a cell to be contacted with subject LD therapeutics includes a cell in culture, a cell that is part of an organized assemblage, such as an organ or tissue, or a cell that is part of an organism.
  • peptide refers to unmodified amino acid chains, and also include minor modifications, such as phosphorylations, glycosylations and lipid modifications.
  • peptide and “peptidomimetic” are not mutually exclusive and include substantial overlap.
  • An "LD peptide” is a peptide comprising an amino acid motif of a late domain, preferably selected from PPxY, P(T/S)AP and YxxL.
  • a “peptidomimetic” includes any modified form of an amino acid chain, such as a phosphorylation, capping, fatty acid modification and including unnatural backbone and/or side chain structures. As described below, a peptidomimetic comprises the structural continuum between an amino acid chain and a non-peptide small molecule. Peptidomimetics generally retain a recognizable peptide-like polymer unit struc ⁇ ure.
  • An "LD peptidomimetic” is a peptidomimetic designed to mimic an LD peptide, retaining certain structural elements of the LD peptide.
  • Small molecule as used herein, is meant to refer to a composition, which has a molecular weight of less than about 5 kD and most preferably less than about 2.5 kD.
  • Small molecules can be nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic (carbon containing) or inorganic molecules.
  • Many pharmaceutical companies have extensive libraries of chemical and/or biological mixtures comprising arrays of small molecules, often fungal, bacterial, or algal extracts, which can be screened with any of the assays of the invention.
  • an "x" in an amino acid sequence may be replaced by a mimic of the amino acid present in the target sequence, or the amino acid may be replaced by a spacer of essentially any form that does not interfere with the activity of the peptidomimetic.
  • compositions and methods of the invention relate to late (L) domain function.
  • the late (L) domain often part of a Gag polypeptide, is typically involved in viral budding and assembly.
  • the primary functional motifs of Gag L domains may be either a PPxY sequence (RSV, murine leukemia virus and Mason Pfizer monkey virus), P(T/S)AP (HIV-1) or YxxL (equine anemia virus, EIAV), both PCT ⁇ JP and PPxY (Rabies virus, VSV, filoviruses) or two overlapping elements PTAPPEY (Ebola virus).
  • Each of these L domain sequences is thought to recruit, either through direct or indirect interactions, different types of host proteins.
  • PPxY recruits a family of protein interaction modules called WW domains. WW domains are present in proteins such as the Nedd4 and Nedd4-like proteins.
  • YxxL recruits, for example, the AP-2 adaptor protein.
  • AP-2 is essential for clathrin-mediated endocytosis in the plasma membrane. Indeed, AP-2 adaptor protein co-localizes with budding structures in EIAV, supporting the hypothesis that it may be part of the budding machinery.
  • P(T/S)AP is thought to recruit the TsglOl protein.
  • TsglOl is implicated in sorting of proteins into lysosomes and recruits the HIV 1 -Gag via the L-domain PTAP motif.
  • Another protein implicated in lysosome sorting, Vps4, is required for the release of both HIN1 and MLN.
  • the invention relates to LD therapeutics designed to act as mimics or decoys of an LD peptide comprising an amino acid motif selected from the group: YxxL, P(T/S)AP and PPxY. While not wishing to be bound to mechanism, it is expected that such peptides may affect a viral life cycle by disrupting L-domain function.
  • Such therapeutics may, for example, comprise LD peptides, peptidomimetics,. small molecules based on the amino acid sequence of an L domain.
  • An LD therapeutic may also comprise an antibody that binds to an appropriate L domain, hi certain embodiments, an LD therapeutic decreases the infectivity of viral particles, by, for example, causing a cell to produce defective virus particles, whether or not the rate of production is affected.
  • the invention relates to the observation that multiple L domain recruitment systems may operate in the process of viral assembly and l ⁇ udding.
  • both the P(T/S)AP and PPxY systems may operate in H1N.
  • the HIV-1 p6Gag protein contains the sequence PTAPPEE (p ⁇ wt) and thus we observed that this domain has the potential, if mutated to change a single amino acid, to comprise a tandem arrangement of two L-domain motifs, PTAP and PPxY.
  • PTAPPEY p ⁇ Ebola
  • a therapeutic for HIV comprises elements designed to interfere with both P(T/S)AP and PPxY.
  • the invention relates to methods and compositions for decreasing viral replication by contacting an infected cell or organism with an LD therapeutic composition that mimics two or more of the L domain tetrapeptides selected from the group consisting of: P(T/S)AP, PPxY and YxxL.
  • an LD therapeutic of this nature may limit the ability of the target virus to mutate and avoid inhibition by, for example, creating an alternative L domain tetrapeptide.
  • the invention relates to peptides, peptidomimetics, and small molecules designed to mimic, or act as decoys for, sequences comprising sequences including but not limited to: PPxY, P(T/S)AP, YxxL, and combinations thereof, such as, for example, sequences comprising PPxYx n P(T/S)AP, PPxYx n YxxL; P(T/S)APx n PPxY, P(T/S)APx n YxxL, YxxLx n PPxY, YxxLx n P(T/S)AP, PPxYxxL and P(T/S)APPxY, where x n indicates anywhere from 0 to many intervening amino acids.
  • P(T/S)APPEE sequence An additional subject sequence is the P(T/S)APPEE sequence.
  • Such peptides, peptidomimetics and small molecules may be used singly or in combination. Combinations may be generated by mixing the compounds prior to contacting with infected cells or organisms, or combinations may be achieved by applying the subject mimics separately to infected cells
  • a model for virus release is that the L-domain recruits the lysosome sorting machinery. To do so, either Gag or an ancillary protein is ubiquitinated. This is facilitated by the recruitment of a ubiquitin-protein ligase to Gag. Once this occurs, the lysosome-sorting complex is recruited to the sites of virus assembly and budding to facilitate the release of the budding virions by an unknown mechanism. 3. Hydrophobic modifications
  • the invention relates to LD peptides, peptidomimetics and small molecules comprising a Hpophilic moiety.
  • targeted therapeutics may have improved efficacy by tending to partition to membrane domains at which viral assembly and/or budding is occuring.
  • certain viruses such as HIV1
  • lipid rafts are detergent insoluble membrane microenvironments composed primarily of cholesterol and sphingolipids and sequester GPI-linked proteins. Lipid rafts appear to participate in the process of signal transduction and virus assembly. It has been demonstrated that HIN1 particles produced by human T-cells lines acquire GPI (glycosylphosphatidyl inositol)-linked proteins Thy-1 and CD59 as well as the
  • Gag ganglioside GM1 which are known to partition preferentially into lipid rafts. HIV Gag associates with lipid rafts and cholesterol depletion greatly abrogates virus release. Finally, Gag oligomers assemble in "Barges". Barges have higher density than rafts probably due to the presence of the Gag-Gag ohgomers. The association of Gag with barges is mediated by an oligomerization domain within the nucleocapsid ( ⁇ C) and by the ⁇ -terminus of the protein. Furthermore, ⁇ -myristoylation of Gag is necessary for barge association. It is suggested that multimerization of myristoyiated Gag drives association of the Gag oligomers into rafts .
  • the ubiquitin protein llgas ⁇ s ⁇ edd4 and CM also partition into lipid rafts in response to IgE-triggered cell signaling.
  • hpophilic moieties with which LD therapeutics may be derivatived.
  • a hpophilic group can be, for example, a relatively long chain alkyl or cycloalkyl (preferably n-alkyl) group having approximately 7 to 30 carbons.
  • the alkyl group may terminate with a hydroxy or primary amine "tail".
  • hpophilic molecules include alicyclic hydrocarbons, saturated and unsaturated fatty acids and other lipid and phospholipid moieties, waxes, cholesterol, isoprenoids, terpenes and polyalicyclic hydrocarbons including adamantane and buckminsterfullerenes, vitamins, polyethylene glycol or oligoethylene glycol, (Cl-C18)-alkyl phosphate diesters, -O-CH2- CH(OH)-O-(C12-C18)-alkyl, conjugates with pyrene derivatives, esters and alcohols, other lipid molecules, cage structures such as adamantane and buckminsterfullerenes, and aromatic hydrocarbons such as benzene, perylene, phenanthrene, anthracene, naphthalene, pyrene, chrysene, and naphthacene.
  • the hpophilic moiety can be a hpophilic dye suitable for use in the invention include, but are not limited to, diphenylhexatriene, Nile Red, N-phenyl-1-naphthylamine, Prodan, Laurodan, Pyrene, Perylene, rhodamine, rhodamine B, tetramethykhodamine, Texas Red, sulforhodamine, l, -didodecyl-3,3,3',3'tetramethylindocarbocyanine perchlorate, octadecyl rhodamine B and the BODIPY dyes available from Molecular Probes Inc.
  • exemplary hpophilic moietites include aliphatic carbonyl radical groups such as decanoyl, dodecanoyl, dodecenoyl, tetradecadienoyl, decynoyl or dodecynoyl.
  • the N-terminal amine of a protein can be modified preferentially relative to other amines in a protein because its lower pKa results in higher amounts of the reactive unprotonated form at neutral or acidic pH.
  • Aryl halides, aldehydes and ketones, acid anhydrides, isocyanates, isothiocyanates, imidoesters, acid halides, " N- hydroxysuccinimidyl (e.g., sulfo-NHS-acetate), nitrophenyl esters, acylimidazoles, and other activated esters and thioesters are among those known to react with amine functions.
  • Active thioesters include acylated derivatives of thiols, wherein the thiol has a molecular weight less than 500 a u and a pKa lower than that of ethanethiol, preferably aromatic or heteroaromatic thiols, even more preferably acylated thiophenols bearing one, or preferably two or more electron withdrawing groups.
  • Suitable electron- withdrawing substituents include nitro, carbonyl (e.g., ester, aldehyde, ketone, amide, carboxyl, or thioester groups), cyano, halogen (preferably chloro or fluoro), sulfonyl (e.g., sulfonamide, sulfonate, sulfonic acid, or sulfone groups), or phosphonyl (e.g., phosphate, phosonate, etc.) groups, preferably nitro, carbonyl, cyano, or halogen groups.
  • the active thioesters comprise derivatives of fatty acids having from 2 to 24 carbon atoms.
  • the active thioesters are derivatives of natural or unnatural amino acids, preferably tripeptides or smaller.
  • the active thioesters include compounds having two or more active thioester moieties, in order to link two or more polypeptides together.
  • Heterobifunctional cross-linkers provide the ability to design more specific coupling methods for conjugating to proteins, thereby reducing the occurrences of unwanted side reactions such as homo-protein polymers.
  • a wide variety of heterobifunctional cross-linkers are known in the art. These include: succinimidyl 4- (N-maleimidomethyl) cyclohexane- 1-carboxylate (SMCC), m-Maleimidobenzoyl-N- hydroxysuccinimide ester (MBS); N-succinimidyl (4-iodoacetyl) aminobenzoate (SLAB), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride (EDC); 4-succimmidyloxycarbonyl- a-methyl-a-(2-pyridyldithio)-tolune (SMPT), N-
  • cross-linking agents having disulfide bridges within the linking chain can be synthesized instead as the alkyl derivatives so as to reduce the amount of linker cleavage in vivo.
  • DSS Disuccinimidyl suberate
  • BMH bismaleimidohexane
  • DMP dimemylpimelimidate.2 HC1
  • BASED bis-[ ⁇ -(4- azidosaiicylamido)ethyl]disulfide
  • BASED bis-[ ⁇ -(4- azidosaiicylamido)ethyl]disulfide
  • SANPAH N-succiniraidyl-6(4'-az ⁇ do-2'- nitrophenyl- afnino)hexanoate
  • heterobifunctional cross-linkers contain the primary amine reactive group, N-hydroxysuccinimide (NHS), or its water soluble analog N-hydroxysulfosuccinimide (sulfo-NHS).
  • NHS N-hydroxysuccinimide
  • sulfo-NHS water soluble analog N-hydroxysulfosuccinimide
  • thiol reactive group Another reactive group useful as part of a heterobifunctional cross-linker is athiol reactive group.
  • Common thiol reactive groups include maleimides, halogens, and pyridyl disulfides. Maleimides react specifically with free sulfhydryls (cysteine residues) in minutes, under slightly acidic to neutral (pH 6.5-7.5) conditions. Halogens (iodoacetyl functions) react with -SH groups at physiological pH's. Both of these reactive groups result in the formation of stable thioether bonds.
  • the third component of the heterobifunctional cross-linker is the spacer aim. or bridge.
  • the bridge is . the structure that connects the two reactive ends.
  • the most apparent attribute of the bridge is its effect on steric hindrance.
  • a longer bridge can more easily span the distance necessary to link two complex biomolecules.
  • SMPB has a span of 14.5 angstroms.
  • Preparing protein-moiety conjugates using heterobifunctional reagents is a two- step process involving the amine reaction and the sulfhydryl reaction.
  • the protein chosen should contain a primary amine. This can be Iysine epsilon amines or a primary alpha amine found at the N-terminus of most proteins.
  • the protein should not contain free sulfhydryl groups. In cases where both proteins to be conjugated contain free sulfhydryl groups, one protein can be modified so that all sulfhydryls are blocked using for instance, N-ethylmaleimide (see Partis et al. (1983) J. Pro. Chem.
  • Ellman's Reagent can be used to calculate the quantity of sulfhydryls in a particular protein (see for example Ellman et al. (1958) Arch. Biochem. Biophys. 74:443 and Riddles et al. (1979) Anal. Biochem. 94:75, incorporated by reference herein).
  • the reaction buffer should be free of extraneous amines and sulfhydryls.
  • the pH of the reaction buffer should be 7.0-7.5. This pH range prevents maleimide groups from reacting with amines, preserving the maleimide group for the second reaction with sulfhydryls.
  • the NHS-ester containing cross-linkers have limited water solubility. They should be dissolved in a minimal amount of organic solvent (DMF or DMSO) before introducing the cross-linker into the reaction mixture.
  • the cross-liriker/solvent forms an emulsion which will allow the reaction to occur.
  • the sulfo-NHS ester analogs are more water soluble, and can be added directly to the reaction buffer. Buffers of high ionic strength should be avoided, as they have a tendency to "salt out" the sulfo-NHS esters. To avoid loss of reactivity due to hydrolysis, the cross-linker is added to the reaction mixture immediately after dissolving the protein solution.
  • the reactions can be more efficient in concentrated protein solutions.
  • the rate of hydrolysis of the NHS and sulfo-NHS esters will also increase with increasing pH. Higher temperatures will increase the reaction rates for both hydrolysis and acylation.
  • the protein is now activated with a sulfhydryl reactive moiety.
  • the activated protein may be isolated from the reaction mixture by simple gel filtration or dialysis.
  • the sulfhydryl reaction the Hpophilic group chosen for reaction with maleimides, activated halogens, or pyridyl disulfides must contain a free sulfhydryl.
  • a primary amine may be modified to add a sulfhydryl
  • the buffer should be degassed to prevent oxidation of sulfliydryl groups.
  • EDTA may be added to chelate any oxidizing metals that may be present in the buffer.
  • Buffers should be free of any sulfhydryl containing compounds.
  • the hpophilic moiety employed is a lipid moiety.
  • a "lipid” is a member of a heterogenous class of hydrophobic substances characterized by a variable solubility in organic solvents and insolubility, for the most part, in water.
  • the principal class of lip ids that are encompassed within this invention are fatty acids and sterols (e.g., cholesterol).
  • Derivatized proteins ' of the invention contain fatty acids which are cyclic, acyclic (i.e., straight chain), saturated or unsaturated, mono- carboxylic acids.
  • Exemplary saturated fatty acids have the generic formula: CH3 (CH2)n COOH.
  • CH3 (CH2)n COOH The following table lists examples of some fatty acids that can be derivatized conveniently using conventional chemical methods.
  • lauric acid 12 myristic acid 14 palmitic acid 16 stearic acid 18 arachidic acid 20 behenic acid 22 lignoceric acid.
  • lipids that can be attached include branched-chain fatty acids and those of the phospholipid group such as the phosphatidylinositols (i.e., phosphatidylinos ⁇ tol 4- monophosphate and phosphatidylinositol 4,5-biphosphate), phosphatidycholine, phosphatidylethanolamine, phosphatidylserine, and isoprenoids such as famesyl or geranyl groups.
  • phosphatidylinositols i.e., phosphatidylinos ⁇ tol 4- monophosphate and phosphatidylinositol 4,5-biphosphate
  • phosphatidycholine phosphatidylethanolamine
  • phosphatidylserine phosphatidylserine
  • isoprenoids such as famesyl or geranyl groups.
  • lipid modification on proteins starts with the formation of a thioester intermediate and the lipid moiety is then transferred to the alpha-amine of the N-terminus through the formation of a cyclic intermediate.
  • the reactive lipid moiety can be in the form of thioesters of saturated or unsaturated carboxylic acids such as a- Coenzyme A thioesters.
  • Such materials and their derivatives may include, for example, commercially available Coenzyme A derivatives such as palmitoleoyl Coenzyme A, arachidoyl Coenzyrne A, arachidonoyl Coenzyme A, lauroyl Coenzyme A and the like. These materials are readily.
  • LD peptidomimetics having a hpophilic moiety
  • modifications to the procedures that may be necessary to accommodate any unusual chemistry of the peptidomimetic form Often, however, a peptidomimetic will have a natural primary amine at the N-terminus or on a side chain, and this it will generally be possible to modify such an amine according to the methods described herein.
  • lipids can be attached to the same or other sites using enzymically catalyzed reactions. Palmitoylation of proteins in vivo is catalyzed by a class of enzymes known as palmitoyl-CoA:protein S-palmitoyltransferases. Using purified enzymes, in vitro acylation of protein substrates has been demonstrated.
  • myristoylation of the amino terminus may be carried out using an N-myristoyl transferase (NMT), a number of which have been well characterized in both mammals and in yeast. Both of these strategies would require the use of fatty acyl- coenzyme A derivatives as substrates.
  • NMT N-myristoyl transferase
  • a protein with aa engineered recognition sequence may be myristoylated or palmitoylated in vivo.
  • Another method of modifying a peptide or peptidomimetic with a hydrophobic moiety is to create a recognition site for the addition of an isoprenoid group at the C-terminus of the protein.
  • LD peptides include peptides that comprise a structural motif contained in a viral L-domain with an amino acid sequence such as any of those described above.
  • the peptide will act as a potent binding site for a host protein involved in retrovirus, rhabdovirus and/or filo virus infection, and particularly HIV1, Ebola virus, Rabies virus and VSV.
  • two or more of the above domains overlap, as in the PTAPPEY motif of Ebola virus.
  • LD peptides also include peptides that comprise or mimic an L-domain binding site of a host protein (a WW domain, for example) and thereby competes with the host L-domain binding protein for interaction with viral proteins containing the L-domain.
  • a host protein a WW domain, for example
  • a subject LD therapeutic comprises a peptidomimetic of an LD peptide (an LD peptidomimetic).
  • Peptidomimetics are compounds based on, or derived from, peptides and proteins.
  • the LD peptidomimetics of the present invention typically can be obtained by structural modification of a known LD peptide sequence using one or more unnatural amino acids, conformational restraints, isosteric replacements, and the like.
  • the subject peptidomimetics constitute the coatirium of structural space between peptides and non-peptide synthetic structures; LD peptidomimetics may be useful, therefore, in delineating pharmacophores and in helping to translate peptides into nonpeptide compounds with the activity of the parent LD peptides.
  • LD peptidomimetics can have such attributes as being non-hydrolyzable (e.g., increased stability against proteases or other physiological conditions which degrade the corresponding peptide), increased specificity and/or potency for inhibition of interaction between L-domains and L-domain binding proteins, increased cell permeability for intracellular localization of the peptidomimetic, and increased tendency to partition into lipid assemblies such as rafts and/or barges.
  • peptide analogs of the present invention can be generated using, for example, benzodiazepines (e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G.R.
  • the present invention specifically contemplates the use of confbrmationally restrained mimics of peptide secondary structure.
  • Numerous surrogates have been developed for the amide bond of peptides. Frequently exploited surrogates for the amide bond include the following groups (i) trans-olefins, (ii) fiuoroalkene, (iii) methyleneamino, (iv) phosphonamides, and (v) sulfonamides.
  • peptidomimietics based on more substantial modifications of the backbone of the LD peptide can be used.
  • Peptidomimetics which fall in this category include (i) retro-inverso analogs, and (ii) N-alkyl glycine analogs (so-called peptoids, see eg. Simon et al. (1992) Proc. Nati. Acad. Sci. USA 89: 9367-71) and others mentioned above.
  • retro-inverso analogs can be made according to the methods known in the art, such as that described by the Sisto et al. U.S. Patent 4,522,752.
  • a retro- inverso analog can be generated as follows. The first step is to form a geminal diamine analog of an amino acid.
  • the geminal diamine corresponding to the N-terminal amino acid for example an N-terminal proline is synthesized by treating an N-protected proline analog with ammonia under HOBT-DCC coupling conditions to yield an unsubstituted amide, and then effecting a Hofmann-type rearrangement with LI-bis- (trifluoroacetoxy)iodobenzene (TLB), as described in Radhakrishna et al. (1979) J. Org. Chem. 44:1746, effectively removing the carbonyl moiety from the unsubstituted amide.
  • TLB LI-bis- (trifluoroacetoxy)iodobenzene
  • the product amine is then coupled to a side-chain protected (e.g., as the benzyl ester) second amino acid, such as N-Fmoc D-thr residue under standard conditions to yield the pseudodipeptide.
  • a side-chain protected (e.g., as the benzyl ester) second amino acid such as N-Fmoc D-thr residue under standard conditions to yield the pseudodipeptide.
  • the Fmoc (flu ⁇ renylmethoxycarbonyl) group is removed with piperidine in dimethylformamide, and the resulting amine is trimethylsilylated with bistrimethylsilylacetamide (BSA) before condensation with suitably alkylated, side-chain protected derivative of Meldrum's acid, as described in U.S. Patent 5,061,811 to Pinori et al, to yield a retro-inverso tripeptide.
  • BSA bistrimethylsilylacetamide
  • Meldrum's acid is a cyclic malonate analog where the R substitution on the fifth carbon (the carbon between the carbonyl groups) determines the amino acid moiety added. For instance, if the R group in Meldrum's acid is a methyl group, then an alanine moiety is added and the retro-inverso tripeptide has the formula PTA. The remaining ester group from the ring opening reaction with Meldrum's acid is further coupled with an amino acid analog under standard conditions to give the protected tetrapeptide analog. The protecting groups are removed to release the product, and the steps repeated to elongate the tetrapeptide to the full length peptidomimetic. It will be understood that a mixed peptide, e.g.
  • Retro-enantio analogs such as this can be synthesized commercially available D- amino acids (or analogs thereof) and standard solid- or solution-phase peptide-synthesis techniques.
  • a suitably amino- protected (t-butyloxycarbonyl, Boc) residue eg. D-proline
  • a solid support such as chloromethyl resin.
  • the resin is washed with dichloromethane (DCM), and the BOC protecting group removed by treatment with TFA in DCM.
  • the resin is washed and neutralized, and the next Boc-protected D-amino acid (eg. D-thr) is introduced by coupling with diisopropylcarbodiimide.
  • the resin is again washed, and the cycle repeated for each of the remaining amino acids in turn (D-ala, D-pro, etc).
  • the protecting groups are removed and the peptide cleaved from the solid support by treatment with hydrofluoric acid/anisole/dimethyl sulfide/thioanisole.
  • the final product is purified by HPLC to yield the pure retro-enantio analog.
  • trans olefin analog of a LD peptide can be synthesized according to the method of Y.K. Shue et al. (1987) Tetrahedron Letters 28:3225.
  • Other pseudodipeptides can be made by the method set forth above merely by substitution of the appropriate starting Boc amino acid and Wittig reagent. Variations in the procedure may be necessary according to the nature of the reagents used, but any such variations will be purely routine and will be apparent to one of skill in the art.
  • pseudodipeptides synthesized by the above method to other pseudodipeptides, to make peptide analogs with several olefinic functionalities in place of amide functionalities.
  • pseudodipeptides corresponding to Pro-Pro or Glu-Tyr, etc. could be made and then coupled together by standard techniques to yield an analog of the LD peptide which has alternating olefinic bonds between residues.
  • the LD peptidomimetic may incorporate the l-azabicyclo[4.3.0]nonane surrogate (see Kim et al. (1997) J. Org. Chem. 62:2847), or an N-acyl piperazic acid (see Xi et al. (1998) J. Am. Chem. Soc. 120:80), or a 2-substituted piperazine moiety as a constrained amino acid analogue (see Williams et al. (1996) J. Med. Chem. 39:1345-1348).
  • the LD peptidomimetic may incorporate the l-azabicyclo[4.3.0]nonane surrogate (see Kim et al. (1997) J. Org. Chem. 62:2847), or an N-acyl piperazic acid (see Xi et al. (1998) J. Am. Chem. Soc. 120:80), or a 2-substituted piperazine moiety as a constrained amino acid an
  • certain amino acid residues can be replaced with aryl and bi-aryl moieties, e.g., monocyclic or bicyclic aromatic or heteroaromatic nucleus, or a biaromatic, aromatic-heteroaromatic, or bihetero aromatic nucleus.
  • the subject LD peptidomimetic is capped at either the N- terminus (or other end structure at the normal N-terminus such as a carbonyl) or the C- terminus (or other end structure at the normal C-terminus, such as an amine) or both.
  • an LD therapeutic may comprise a polypeptide comprising one or more L domain sequences where the polypeptide has either an acetyl cap at the N-terminus of an amide cap at the C-terminus or both.
  • Exemplary peptidomimetics of this type include: Ac-Pro-Thr-Ala-Pro-Am, Ac-Pro-Thr-Ala-Pro-Pro-Glu-Tyr-Am, etc.
  • the subject LD peptidomimetics can be optimized by, e.g., combinatorial synthesis techniques combined with such high throughput screening as described herein.
  • peptidomimetics include, but are not limited to, protein-based compounds, carbohydrate-based compounds, lipid-based compounds, nucleic acid-based compounds, natural organic compounds, synthetically derived organic compounds, anti-idiotypic antibodies and/or catalytic antibodies, or fragments thereof.
  • a peptidomimetic can be obtained by, for example, screening libraries of natural and synthetic compounds for compounds capable of inhibiting the interaction between an L- domain and a host protein.
  • a peptidomimetic can also be obtained, for example, from libraries of natural and synthetic compounds, in particular, chemical or combinatorial libraries (i.e., libraries of compounds that differ in sequence or size but that have the same building blocks).
  • Another aspect of the invention pertains to an antibody specifically reactive with an LD peptide.
  • an LD peptide by using an LD peptide, anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., an LD peptide or an antigenic fragment which is capable of eliciting an antibody response, or a fusion protein as described above).
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An LD peptide can be administered in the presence of adjuvant.
  • the progress of immunization can be monitored by detection of antibody titers in plasma or serum.
  • Standard ELISA or other immunoassays can be used with the immunogen as antigen to assess the levels of antibodies.
  • antibodies are specific to an LD sequence selected from the group: PPxY, P(T/S)AP and YxxL.
  • an antibody is able to bind to two or more of such sequences or to combinations of such sequences, such as, for example, PTAPPEY.
  • anti-LD antisera can be obtained and, if desired, polyclonal anti-LD antibodies isolated from the serum.
  • antibody-producing cells lymphocytes
  • immortalizing cells such as myeloma cells to yield hybridoma cells.
  • Hybridoma cells can be screened immtimochemically for production of antibodies specifically reactive with an LD peptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells.
  • antibody as used herein is intended to include fragments thereof which are also specifically reactive with one of the subject LD peptides.
  • Antibodies can be fragmented using conventional techniques and the fragments screened for utility in the same manner as described above for whole antibodies. For example, F(ab)2 fragments
  • the resulting F(ab)2 fragment can be generated by treating antibody with pepsin.
  • the resulting F(ab)2 fragment can be generated by treating antibody with pepsin.
  • the antibody of the present invention is further intended to include bispecific, single-chain, and chimeric and humanized molecules having affinity for an LD peptide conferred by at least one CDR region of the antibody.
  • the antibodies, the antibody further comprises a label attached thereto and able to be detected, (e.g., the label can be a radioisotope, fluorescent compound, enzyme or enzyme co- factor).
  • Anti-LD antibodies can be introduced into cells as nucleic acids encoding- single chain antibodies to be expressed in infected cells (see below).
  • subject LD therapeutic molecules such as peptidomimetics and small molecules
  • subject LD therapeutic molecules may be obtained by rational design.
  • the three-dimensional structure of a target peptide of the present invention can be analyzed by, for example, - nuclear magnetic resonance (NMR) or x-ray crystallography.
  • the three-dimensional structure can then be used to predict-structures of potential LD therapeutics by, for example, computer modeling.
  • the predicted LD therapeutic structures can then be produced by, for example, chemical synthesis, recombinant DNA technology, or by isolating a molecule from a natural source (e.g., plants, animals, bacteria and fungi).
  • a natural source e.g., plants, animals, bacteria and fungi
  • the p6 domain of HIV1 Gag is known to be disordered in crystal and solution structures.
  • an LD therapeutic may be modeled on an unconstrained polypeptide chain of one of the above-mentioned sequences.
  • the desired structure may be modeled without resort to NMR or X-ray crystallography, and any of a number of programs for modeling short peptide chains may be employed.
  • the structure of the appropriate late domain sequence may be analyzed (eg. by NMR or X-ray crystallography) in a complex with a binding partner, such as a WW protein, TsglOl or AP2, and an LD therapeutic may be modeled based on the structure of late domain sequence adopted during binding.
  • LD peptides, peptidomimetics and small molecules are able to compete with the native L domain peptide sequence for binding to a cell or molecule that has a binding site for the native L domain peptide sequence.
  • an LD peptide, peptidomimetic or small molecule comprising a portion based on the tetrapeptide P(T/S)AP will compete for binding to a cell or protem having a P(T/S)AP binding site, such as, for example, a TsglOl polypeptide.
  • an LD peptide, peptidomimetic or small molecule comprising a portion based on the tetrapeptide PPxY will compete for binding to a cell or protein having a PPxY binding site, such as, for example, a Nedd4, Nedd4-like or other WW domain polypeptide.
  • an LD peptide, peptidomimetic or small molecule comprising a portion based on the tetrapeptide YxxL will compete for binding to a cell or protein haying a YxxL binding site, such as, for example, an AP2 polypeptide.
  • peptides, peptidomimetics and small molecules have a K D that is no more than ten times greater than the K D of the L domain on which they are based, and optionally have a roughly equivalent K D or a K D ten times lower or less.
  • Peptides, peptidomimetics and small molecules comprising portions based on two or more L domains may compete for binding to two or more appropriate host proteins.
  • an LD peptide, peptidomimetics or small molecule may inhibit viral particle production in a Virus-like Particle assay (VLP).
  • VLP Virus-like Particle assay
  • An exemplary embodiment of a VLP assay comprises the use of a HeLa cell line transfected with an HLV-encoding plasmid, where the encoded HIN is rendered uninfections by a deletion in the env region.
  • the production of VLPs may be monitored in a number of ways, such as, for example, by measuring the level of a viral protein in a VLP particle fraction versus the level of a viral protein in the total fraction. High levels of viral protein in the NLP fraction correlate with high levels of VLP production.
  • LD peptides, peptidomimetics and antibodies may be incorporated into fusion proteins.
  • the fusion protein provides a second functional portion, such as, for example, a carrier protein, a production protein or a stabilizing protein.
  • Useful carrier proteins include, for example, bacterial hemolysins or "blending agents", such as alamethicin or sulfhydryl activated lysins.
  • Other carrier moieties which. may be used include cell entry components of bacterial toxins, such as Pseu ⁇ monas exotoxin, tetanus toxin, ricin toxin, and diphtheria toxin.
  • mehttin from bee venom.
  • Other useful carrier proteins include proteins which are viral receptors, cell receptors or cell ligands for specific receptors that are internalized, i.e., those which cross mammalian cell membranes via specific interaction with cell surface receptors, recognized and taken into the cell by cell surface receptors.
  • Such cell ligands include, for example, epidermal growth factor, fibroblast growth factor, transferrin and platelet- derived growth factor.
  • the ligand may be a non-peptide, such as mannose- 6-phosphate, which permits internalization by the mannose-6-phosphate receptor.
  • the transport moiety may also be selected from bacterial immunogens, parasitic immunogens, viral immunogens, immunoglobuhns or fragments thereof that bind to target molecules, cytokines, growth factors, colony stimulating factors and hormones.
  • a transport moiety may also be derived from the tat protein of HLV-1.
  • Such carrier peptides may also be considered targeting peptides when they confer some specificity of cell type to which the LD therapeutic is delivered.
  • Stability proteins include proteins that increase the stability of the LD peptide, peptidornimetic or antibody.
  • Production proteins include proteins that assist in production of the LD peptide. It is widely appreciated that fusion proteins can facilitate the expression of proteins, and accordingly, can be used in the expression of the LD peptides of the present invention.
  • Exemplary production peptides include GST, polyhistidine, cellulose binding protein, chitin binding protein, etc.
  • LD peptides can be generated as glutathione-S-transferase (GST-fusion) proteins.
  • Such GST-fusion proteins can enable easy purification of LD peptides, as for example by the use of glutathione-derivatized matrices (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. (N.Y.: John Wiley & Sons, 1991)).
  • a fusion gene coding for a purification leader sequence such as a poly-(His)/enterokiriase cleavage site sequence, can be placed at the N-terminus of the LD peptide in order to permit purification of the poly(His)-LD peptide protein by affinity chromatography using a Ni2+ metal resin.
  • the purification leader sequence can, if desired, be subsequently removed by treatment with enterokinase (e.g., see Hochuli et al. (1987) J. Chromatography 411:177; and Janknecht et al. PNAS 88:8972).
  • enterokinase e.g., see Hochuli et al. (1987) J. Chromatography 411:177; and Janknecht et al. PNAS 88:8972.
  • fusion genes are known to those skilled in the art. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt- ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkafine phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • Nucleic acids encoding fusion proteins may be operatively linked to regulatory sequences and introduced into appropriate expression systems using conventional recombinant DNA procedures.
  • LD therapeutics may be administered directly to cells infected with a virus such as a retrovirus, rhabdovirus and or filo virus. Direct delivery of such LD therapeutics may be facilitated by formulation of the compound in any pharmaceutically acceptable dosage form, e.g., for delivery orally, intratumorally, peritumorally, interlesionally, intravenously, intramuscularly, subcutaneously, periolesionally, or (preferably) topical routes, to exert local therapeutic effects.
  • a virus such as a retrovirus, rhabdovirus and or filo virus.
  • Direct delivery of such LD therapeutics may be facilitated by formulation of the compound in any pharmaceutically acceptable dosage form, e.g., for delivery orally, intratumorally, peritumorally, interlesionally, intravenously, intramuscularly, subcutaneously, periolesionally, or (preferably) topical routes, to exert local therapeutic effects.
  • Topical administration of the therapeutic is advantageous since it allows localized concentration at the site of administration with minimal systemic adsorption. This simplifies the delivery strategy of the agent to the disease site and reduces the extent of toxicological characterization. Furthermore, the amount of material to be applied is far less than that required for other administration routes. Effective delivery requires the agent to diffuse into the affected cells.
  • Successful intracellular delivery of agents not naturally taken up by cells has been achieved by exploiting the natural process of intracellular membrane fusion, or by direct access of the cell's natural transport mechanisms which include endocytosis and pinocytosis (Duzgunes (1985) Subcellular Biochemistry 11:195-286). Such processes are also useful in the direct delivery and uptake of the subject LD peptides and peptidomimetics by infected cells.
  • the membrane barrier can be overcome by associating the LD therapeutic protein in a carrier such as a hpid formulation closely resembling the lipid composition of natural cell membranes.
  • a carrier such as a hpid formulation closely resembling the lipid composition of natural cell membranes.
  • the subject LD peptidomi ⁇ etics are encapsulated in liposomes to form pharmaceutical preparations suitable for administration to living cells and, in particular, suitable for topical administration to human skin.
  • the Yarosh U.S. Patent 5,190,762 demonstrates that proteins can be delivered across the outer skin layer and into living cells, without receptor binding, by liposome encapsulation.
  • carrier lipids are able to fuse with the cell membranes on contact, and in the process, the associated LD peptidomimetic is delivered intracellularly.
  • Lipid complexes can not only facilitate intracellular transfers by fusing with cell membranes but also by overcoming charge repulsions between the cell membrane and the molecule to be inserted.
  • the carrier lipids of the formulations comprise an amphipathic lipid, such as the phospholipids of cell membranes, and form hollow lipid vesicles, or liposomes, in aqueous systems. This property can be used to entrap the LD peptidomimetic within the liposomes.
  • Liposomes offer several advantages as carriers: They are non-toxic and biodegradable in composition; they display long circulation half-lives; and recognition molecules can be readily attached to their surface for targeting to tissues. Finally, cost effective manufacture of liposome-based pharmaceuticals, either in a liquid suspension or lyophilized product, has demonstrated the viability of this technology as an acceptable drug delivery system.
  • Liposomes have been described in the art as in vivo carriers.
  • the structure of various types of lipid aggregates varies, depending on composition and method of forming the aggregate.
  • Such aggregates include Hposomes, unilamellar vesicles, multilameller vesicles, micelles and the like, having particle sizes in the nanometer to micrometer range.
  • Methods of making lipid aggregates are by now well-known in the art.
  • the liposomes may be made from natural and synthetic phospholipids, glycolipids, and other lipids and lipid congeners; cholesterol, cholesterol derivatives and other cholesterol congeners; charged species which impart a net charge to the membrane; reactive species which can react after liposome formation to link additional molecules to the liposome membrane; and other lipid soluble compounds which have chemical or biological activity.
  • pH sensitive liposomes are a preferred type of liposome for use as a carrier with the present invention.
  • One pathway for the entry of liposomes into cellular cytoplasm is by endocytosis into lysozymes of low pH. Accordingly, liposomes which are stable at neutral pH but release their contents at acidic pH can be used to deliver enzymes into the lysozymes of the cytoplasm, whereupon the contents are released.
  • Liposomes can be made sensitive to the low pH of the lysozymes by the hpid composition.
  • pH sensitive liposomes can be prepared by using phospholipids which form lipid bilayers when charged but fail to stack in an ordered fashion when neutralized.
  • An example of such a phospholipid is phosphatidylethanolamine, which is negatively charged above pH 9.
  • the net charge of a phospholipid can be maintained at a pH which would otherwise neutrafize the head groups by including charged molecules in the lipid bilayer which themselves can become neutralized. Examples of these charged molecules are oleic acid and choleste yl hemisuccinate, which are negatively charged at neutral pH but become neutralized at pH 5.
  • the effect of combining these together in a lipid bilayer is that at pH 9 all molecules are charged; at pH 7 the net negative charge of the oleic acid and cholesteryl hemisuccinate maintains the stability of the phosphatidylethanolamine, and at pH 5 all components are protonated and the lipid membrane is destabilized. Additional neutral molecules, such as phosphatidylcholine, can be added to the liposomes as long as they do not interfere with stabilization of the pH sensitive phospholipid by the charged molecules.
  • the LD peptidomimetic is formulated with a positively charged synthetic (cationic) lipid carrier N-[l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA), in the form of liposomes, or small vesicles which can fuse with the negatively charged lipids .o the Gel! membranes of mammalia ⁇ cells, resulting in uptake of the contents of the liposome (see, for example, Feigner et at (1987) PNAS 84:7413-7417; and U.S. Pat. No. 4,897,355 to Eppstein, D. et al.).
  • DOTMA l-(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride
  • DOTMA l,2-bis(oleoyloxy)-3-(trimethyl-ammonio)pro ⁇ ane
  • LipofectinTM (Bethesda Research Laboratories, Gaithersburg, Md.) and/or LipofectAM NETM, commercially available reagents, can be used as carriers to deliver the LD peptidomimetic directly into cells. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and can efficiently deliver functional LD peptidomimetic into, for example, keratinocytes.
  • Sells et al. (1995) Biotechniques 19:72-76 describe a procedure for delivery of purified proteins into a variety of cells using such polycationic lipid preparations.
  • lipids vesicles for direct delivery of the LD peptidomimetic include vesicles containing a quaternary ammonium surfactant (Ballas et al. (1988) Biochim. Biophys Acta 939:8-18); Hpophilic derivatives of spermine (Behr et al. (1989) PNAS 86:6982-6986).
  • the lipid formulations of the subject LD peptidomimetic can be used in pharmaceutical formulations to deliver the LD peptidomimetic by various routes and to various sites in the animal body to achieve the desired therapeutic effect.
  • Local or systemic delivery of the therapeutic agent can be achieved by administration comprising application or insertion of the formulation into body cavities, inhalation or insufflation of an aerosol, or by parenteral introduction, comprising intramuscular, intravenous, intradermal, peritoneal, subcutaneous and topical administration.
  • Topical formulations are those advantageously applied to the skin or mucosa.
  • Target mucosa can be that of the vaginal, cervical, vulvar, penal or anorectal mucosa, or target mucosa can be that of the gastrointestinal tract, comprising the mouth, larynx, esophagous and stomach.
  • Lipids present in topical formulations can act to facilitate introduction of therapeutic LD peptidomimetic into the target tissue, such as the stratum or corneum of the skin, by perturbing the barrier properties of the protective membrane, or by introducing perturbing agents or penetration enhancers such as DMSO, AzoneTM or by promoting the activity of these penetration enhancers.
  • compositions comprising the cationic lipids of the invention are topical preparations containing an anesthetic or cytostatic agent, immunomodulators, bioactive peptides or oligonucleotides, sunscreens or cosmetics.
  • Preparations for topical use are conveniently prepared with hydrophilic and hydrophobic bases in the form of creams, lotions, ointments or gels; alternatively, the preparation may be in the form of a liquid that is sprayed on the skin.
  • the effect of the cationic lipids is to facilitate the penetration of the active antiviral agent through the stratum corneum of the dermis.
  • composition and form of pharmaceutical preparations comprising the liposome, in combination with the LD peptidomimetic, can vary according to the intended route of administration.
  • the liposomes can be made to bind to specific sub-populations of cells.
  • the therapeutic LD peptidomimetic can be delivered by way of a carrier that is an artificial viral envelope (ANE).
  • a number of viral envelopes are available which exploit molecular recognition of cell surface receptors by viral surface proteins as a means for selective intracellular delivery of macromolecules, including proteins.
  • ANE artificial viral envelope
  • the ANEs be generated as viral mimetics of a number of human viruses including human immunodeficiency virus type 1 and 2 (HIN- 1/2 ).
  • direct delivery of an LD peptidomimetic may be facilitated by chemical modification of the polypeptide itself.
  • One such modification involves increasing the lipophilicity of the LD peptidomimetic in order to increase binding to the cell surface, in turn, stimulating non-specific endocytosis of the protein.
  • Lipophilicity may be increased by adding a lipophilic moiety (e.g., one or more fatty acid molecules) to the LD peptidomimetic.
  • a lipophilic moiety e.g., one or more fatty acid molecules
  • the protein may be palmitoylated or myristoylated.
  • Such lipid modifications may assist in targeting the therapeutic to lipid assemblies where viral maturation is thought to occur, in part.
  • a lipopeptide may be produced by fusion or cross-linking, to permit the LD peptidomimetic to resemble the natural lipopeptide from E.coli, tripalmitoyl-S-glycerylcysteil-seryl-serine, at its amino terminus.
  • This lipopeptide has been shown to increase the uptake of fused peptides (P. Hoffinann et al., (1988) Immunobiol. 177:158-70). Lipophilicity may also be increased by esterification of the protein at tyrosine residues or other amino acid residues.
  • uptake of the LD peptidomimetic may be increased by addition of a basic polymer such as polyarginine or polylysine (Shen et al. (1978) PNAS 75:1872-76).
  • Direct delivery of LD peptidomimetics may also be effected by the use of carriers that are transport moieties, such as protein carriers tnown to cross cell membranes.
  • an LD peptide may be fused to a carrier protein, preferably by a genetic fusion which may be expressed in a system such as E.coli, baculovirus, CHO cell, Cos cell or yeast.
  • the amino terminus of an LD peptide or peptidomimetic may be fused to the carboxy terminus of a transport moiety using standard techniques.
  • an LD therapeutic may be administered serially or in combination with other therapeutics used in the treatment of viral infections.
  • therapeutics include protease inhibitors, such as, for example, indinavir and saquinavir, etc., and reverse transcriptase inhibitors such as, for example, azidothymidine (AZT), dideoxyinosine, dideoxycytosine, nevirapine, etc.
  • AZA azidothymidine
  • combination therapies may advantageously utilize less than conventional dosages of those agents, or involve less radical regimens, thus avoiding any potential toxicity or risks associated with those therapies.
  • any of the above enumerated delivery methods may be augmented, where topical apphcation is being carried out, by the use of ultrasound or iontophoretic delivery devises which facilitate transdermal delivery of proteins. See, for example, Banga et al. (1993) Pharm Res 10:697-702; and Mitragotri et al. (1995) Science 269:850-853.
  • the present invention relates to constructs containing a nucleic acid encoding, for example in an exemplary method, a LD peptide of the present invention, operably linked to at least one transcriptional regulatory sequence for introduction into and expression in a virus-infected cell.
  • the gene constructs of the present invention are formulated to be used as a part of, for example, a gene therapy protocol to deliver the subject therapeutic protein to an animal to be treated.
  • any of the methods known to the art for the insertion of DNA fragments into a vector may be used to construct expression vectors consisting of appropriate transcriptional/ translational control signals and the desired nucleic acid sequence, such as an LD peptide-encoding nucleotide sequence or a nucleic acid encoding a single chain antibody that binds to an L domain sequence.
  • the desired nucleic acid sequence such as an LD peptide-encoding nucleotide sequence or a nucleic acid encoding a single chain antibody that binds to an L domain sequence.
  • Expression of a subject nucleic acid may be regulated by a second nucleic acid sequence so that the encoded polypeptide is expressed in a host infected or transfected with the recombinant DNA molecule.
  • expression of aLD peptide may be controlled by any promoter/enhancer element known in the art.
  • the promoter activation may be tissue specific or inducible by a metabolic product or aclministered substance. .
  • Promoters/enhancers which may be used to control the expression of the LD peptide in vivo include, but are not limited to, a promoter used by the target virus, the cytomegalovirus (CMN) promoter/enhancer (Karasuyama et al, 1989, J. Exp. Med., 169:13), the human b-actin promoter (Gunning et al. (1987) P ⁇ AS 84:4831-4835), the glucocorticoid-inducible promoter present in the mouse mammary tumor virus long terminal repeat (MMTN LTR) (Klessig et al. (1984) Mol. Cell Biol.
  • CTN LTR mouse mammary tumor virus long terminal repeat
  • Rous sarcoma virus (RSV) (Yamamoto et al., 1980, Cell, 22:787-797), the herpes simplex virus (HSV) thymidine kinase promoter/enhancer (Wagner et al. (1981) PNAS 82:3567 ⁇ 71), and the herpes simplex virus LAT promoter (Wolfe et al. (1992) Nature Genetics, 1:379-384), and Keratin gene promoters, such as Keratin 14.
  • Expression constructs of the subject LD therapeutic polypeptides may be administered in any biologically effective carrier, e.g.
  • Approaches include insertion of the LD peptide coding sequence in viral vectors including recombinant retroviruses, adenovirus, adeno-associated virus, and herpes simplex virus-1, or recombinant eukaryotic plasmids.
  • Viral vectors transfect cells directly; plasmidDNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g. antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPO4 precipitation carried out in vivo.
  • lipofectin cationic liposomes
  • derivatized e.g. antibody conjugated
  • polylysine conjugates e.g. antibody conjugated
  • gramacidin S e.g. antibody conjugated
  • a preferred approach for in vivo introduction of nucleic acid into a cell is by use of a viral vector containing nucleic acid encoding the particular LD peptide desired.
  • Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid.
  • molecules encoded within the viral vector e.g., the recombinant LD peptide, are expressed efficiently in cells which have taken up viral vector nucleic acid.
  • non-viral methods can also be employed to cause expression of a LD peptide in the tissue of an animal.
  • Most nonviral methods of gene transfer rely on normal mechanisms used ' by mammalian cells for the uptake and intracellular transport of macromolecules.
  • non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of the LD peptide-encoding gene by the targeted cell.
  • Exemplary gene delivery systems of this type include liposomal derived systems, poly- lysine conjugates, and artificial viral envelopes.
  • the gene delivery systems for the therapeutic LD peptide coding sequence can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g. by intravenous injection, and specific transduction of the protein in the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlhng expression of the receptor gene, or a combination thereof.
  • initial delivery of the recombinant gene is more limited with introduction into the animal being quite locahzed.
  • the gene delivery vehicle can be introduced by catheter (see U.S.
  • the gene therapy construct of the present invention is applied topically to an infected or transformed cells of the skin or mucosal tisue.
  • An LD peptide gene construct can, in one embodiment, be delivered in a gene therapy construct by electroporation using techniqu.es described, for example, by Dev et al. ((1994) Cancer Treat Rev 20:105-115).
  • the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.

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Abstract

La présente invention porte sur des motifs d'acides aminés de domaine L, ainsi que sur des méthodes et des compositions permettant de réduire la reproduction virale et/ou l'infectivité virale.
PCT/US2002/041028 2001-12-18 2002-12-18 Methodes et compositions d'inhibition de la production virale WO2003051835A2 (fr)

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US9180180B2 (en) 2003-07-11 2015-11-10 Novavax, Inc. Functional influenza virus-like particles (VLPs)
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US9474799B2 (en) 2003-07-11 2016-10-25 Novavax, Inc. Functional influenza virus-like particles (VLPS)
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US6960431B2 (en) 2001-08-22 2005-11-01 Myriad Genetics, Inc. Therapeutic compositions and methods for treating viral infection
US9951317B2 (en) 2003-07-11 2018-04-24 Novavax, Inc. Highly efficient influenza matrix (M1) proteins
US9956280B2 (en) 2003-07-11 2018-05-01 Novavax, Inc. Functional influenza virus-like particles (VLPs)
US9474799B2 (en) 2003-07-11 2016-10-25 Novavax, Inc. Functional influenza virus-like particles (VLPS)
US9623104B2 (en) 2003-07-11 2017-04-18 Novavax, Inc. Functional influenza virus-like particles (VLPS)
US9694066B2 (en) 2003-07-11 2017-07-04 Novavax, Inc. Functional influenza virus like particles (VLPs)
US10729760B2 (en) 2003-07-11 2020-08-04 Novavax, Inc. Functional influenza virus like particles (VLPs)
US9464276B2 (en) 2003-07-11 2016-10-11 Novavax, Inc. Highly efficient influenza matrix (M1) proteins
US9180180B2 (en) 2003-07-11 2015-11-10 Novavax, Inc. Functional influenza virus-like particles (VLPs)
US9937253B2 (en) 2003-07-11 2018-04-10 Novavax, Inc. Functional influenza virus-like particles (VLPS)
US10548968B2 (en) 2003-07-11 2020-02-04 Novavax, Inc. Functional influenza virus-like particles (VLPS)
US10188723B2 (en) 2003-07-11 2019-01-29 Novavax, Inc. Functional influenza virus like particles (VLPs)
US10544399B2 (en) 2003-07-11 2020-01-28 Novavax, Inc. Highly efficient influenza matrix (M1) proteins
US10160756B2 (en) 2014-03-31 2018-12-25 The Trustees Of The University Of Pennsylvania Antiviral compounds and methods using same
WO2017156146A1 (fr) * 2016-03-08 2017-09-14 University Of Vermont And State Agricultural College Arénavirus modifié
US11260090B2 (en) 2016-03-08 2022-03-01 University Of Vermont And State Agricultural College Modified arenavirus

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