WO2010089129A1 - Inhibiteurs de fusion virale et leurs utilisations - Google Patents

Inhibiteurs de fusion virale et leurs utilisations Download PDF

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WO2010089129A1
WO2010089129A1 PCT/EP2010/000723 EP2010000723W WO2010089129A1 WO 2010089129 A1 WO2010089129 A1 WO 2010089129A1 EP 2010000723 W EP2010000723 W EP 2010000723W WO 2010089129 A1 WO2010089129 A1 WO 2010089129A1
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amino acid
peptide
virus
inhibitor
seq
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PCT/EP2010/000723
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Riccardo Cortese
Antonello Pessi
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Cormus Srl
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/554—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound the modifying agent being a steroid plant sterol, glycyrrhetic acid, enoxolone or bile acid
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Definitions

  • the present invention relates to novel inhibitors of viral entry into cells and their use for the prophylaxis and treatment of viral diseases.
  • Enveloped viruses such as orthomyxoviruses, paramyxoviruses, retroviruses, flaviviruses, rhabdoviruses and alphaviruses, are surrounded by a lipid bilayer originating from the host plasma membrane (Ono and Freed, Adv. Virus Res., 2005, 273, 5419-5442).
  • This envelope contains glycoproteins that mediate receptor binding and fusion between viral and host cell membranes.
  • Cholesterol and sphingolipids such as sphingomyelin are often enriched in these viral lipid bilayers, particularly in lipid-rich rafts in their plasma membrane (Aloia et al, PNAS, 1988, 85, 900-4 and 1993, 90, 5181-5).
  • a number of transmembrane proteins and receptors including CD4 which is the primary receptor for HIV envelope gpl20, are particularly enriched in lipid rafts.
  • gp41 To accomplish the fusion and mixing of cellular and viral contents, gp41 must undergo a complex series of conformational changes apparently triggered by the attachment of gpl20 to the CD4 primary receptor and the CCR5 or CXCR4 coreceptors of the target cell. The completion of co-receptor binding leads to the fusion-active conformation of the viral transmembrane fusion protein gp41.
  • the ectodomain of gp41 contains two heptad repeat regimes: HRl (proximal to the N terminus) and HR2 (proximal to the C terminus).
  • the hydrophobic fusion peptide region inserts into the host cell membrane, whereas the HRl region of gp41 forms a trimeric coiled coil structure. HR2 regions then fold back within the hydrophobic grooves of the HRl coiled coil, forming a hairpin structure containing a thermodynamically stable six-helix bundle that draws the viral and cellular membranes together for fusion (Matthews et al, Nature Reviews Drug Discovery, 2004, 3, 215-225). It has been shown that interference with HRl HR2 interaction inhibits viral entry and hence viral replication (WO 2005/067960 Al). A number of HRl binding peptides have been described in the prior art and enfuvirtide has been approved for the treatment of HIV. However, the doses required to interfere with viral entry are still substantial and there is a need in the art to improve the effectiveness of such HRl binding compounds.
  • RSV respiratory syncytial virus
  • LRTI viral lower respiratory tract illness
  • the enveloped viruses designated as human parainfluenza viruses types 1, 2, and 3
  • HPIVl, HPIV2 and HPIV3, respectively are also important respiratory pathogens in infancy and early childhood: -25% of individuals in this age group will develop clinically significant
  • HPIVs were recovered from 18% of outpatients with upper respiratory tract illness (URTI), 22% with LRTI, and 64% with croup (3).
  • URTI upper respiratory tract illness
  • HPIV-I and HPIV-2 are the principal causes of croup, which occurs primarily in children 6—48 months of age.
  • HPIV3 causes bronchiolitis and pneumonia predominantly in children ⁇ 12 months of age.
  • HPIVs are second only to RSV as important causes of viral LRTI in young children (1).
  • RSV Like RSV,
  • HPIV3 can cause severe LRTI in immunocompromised patients.
  • HPIV3 also infects children early in life: -60% and ⁇ 80% will have been infected before the ages of 2 and 4 years, respectively. Infection with HPIVl and HPIV2 occurs when children are slightly older, but, by 5 years of age, most children have been infected with these viruses at least once. RSV epidemics occur during the winter and early spring in temperate climates and during the rainy season in some, but not all, tropical climates.
  • HPIVl epidemics occur in the fall of odd-numbered years
  • HPIV2 epidemics occur biennially or annually in the fall
  • HPIV3 epidemics occur annually in the spring and summer.
  • RSV and HPIVs but also many other enveloped viruses especially of the paramyxovirus Paramyxoviridae family of the Mononegavirales order can reinfect individuals throughout life, many of which will cause upper respiratory tract infections (URTI).
  • URTI upper respiratory tract infections
  • Primary infection with RSV or HPIV3 does not always elicit immune responses that will protect the lower respiratory tract, because RSV- and HPIV3-associated LRTI can occur in young children experiencing second infections.
  • Licensed vaccines for RSV and HPIVs and enveloped viruses are not currently available. Furthermore, it is frequently observed that primary infection with an enveloped virus such as a paramyxovirus generates merely a suboptimal immune response especially in young infants.
  • the present inventors have identified novel and improved inhibitors of viral fusion based on fusogenic proteins of enveloped virus, which are surprisingly effective against multiple different enveloped viruses.
  • These inhibitors comprise: (a) a polypeptide comprising a peptide capable of binding to a HRl domain of a Type 1 viral fusogenic protein of an enveloped virus (HRl domain) selected from the group consisting of HRl domains with an amino acid sequence according to SEQ ID NO: 1 to 17 and 105, wherein the peptide has a length of at least ten contiguous amino acids and (b) a membrane integrating lipid selected from the group consisting of cholesterol, a sphingolipid, a glycolipid, a glycerophospholipid and membrane integrating derivatives thereof, which is attached to the C-terminal region of the polypeptide; or a pharmaceutically acceptable salt thereof.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the inhibitor of the present invention or a pharmaceutically acceptable salt thereof or a combination of the present invention and a pharmaceutically acceptable excipient.
  • the present invention relates to an inhibitor of the present invention or a pharmaceutically acceptable salt thereof or a combination of the present invention for the treatment or prevention of infection by an enveloped virus.
  • the invention provides a method for making a broad-spectrum inhibitor of viral fusion effective against at least three different enveloped viruses, wherein the method comprises the steps of:
  • said enveloped viruses are paramyxoviruses.
  • the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", Leuenberger, H.G.W, Nagel, B. and Klbl, H. eds.
  • the inhibitor molecules of the invention comprise amino acids which are designated following the standard one- or three-letter code according to WIPO standard ST.25 unless otherwise indicated.
  • Viral fusogenic proteins mediate penetration into the host cell.
  • the fusogenic proteins also called penetrenes of enveloped animal viruses can be divided on the basis of common structural motifs into at least three classes.
  • Orthomyxoviruses, retroviruses, paramyxoviruses, arenaviruses, and coronaviruses for example encode type I penetrenes which are also known as type I viral fusion proteins or ⁇ -penetrenes.
  • Type I viral fusogenic proteins are well known in the art and comprise one or more of the following: a "fusion peptide” which is a cluster of hydrophobic and aromatic amino acids located at or near the amino terminus, an amino terminal helix (N-helix, HRl), a carboxyl terminal helix (C-helix, HR2), usually an aromatic amino acid (aa) rich pre-membrane domain and a carboxyl terminal anchor (see e.g. Garry et al., "Proteomics computational analyses suggest that baculovirus GP64 superfamily proteins are class III penetrenes", Virology Journal 2008, 5:28 and see also: Harrison S., "Viral membrane fusion", Nat Struct MoI Biol.
  • the present inventors have identified novel inhibitors of viral fusion based on fusogenic proteins of enveloped virus. These inhibitors are based on peptides that bind to a HRl domain of a Type 1 viral fusogenic protein since it is known that the interaction of the HRl domain with the respective cellular receptor facilitates fusion with the cell membrane. Accordingly, it has been observed that the provision of HRl domain binding peptides interferes with this process.
  • the inhibitors of viral fusion provided herein comprise, essentially consist or consist of:
  • a polypeptide comprising, essentially consisting or consisting of a peptide capable of binding to a HRl domain selected from the group consisting of HRl domains with an amino acid sequence according to SEQ ID NO: 1 to 17 and 105, wherein the peptide has a length of at least ten contiguous amino acids
  • a membrane integrating lipid selected from the group consisting of cholesterol, a sphingolipid, a glycolipid, a glycerophospholipid and membrane integrating derivatives thereof, which is attached to the C-terminal region of the polypeptide; and (c) optionally a linker connecting the membrane integrating lipid preferably cholesterol or derivative thereof with said polypeptide or a pharmaceutically acceptable salt thereof.
  • HRl domain binding peptides that are part of the polypeptide comprised in the inhibitors of viral fusion and entry of the present invention can be identified with art known high throughput assay system, preferably phage display, wherein bacteria are transformed by phage each expressing a different peptide in fusion to a phage capsid protein. These fusion proteins will "display" the respective peptide on the surface of the bacterial cell, which then can be tested for interaction with the HRl domain of interest, i.e. one of the HRl domains having a sequence according to SEQ ID NO: 1 to 17 and 105.
  • HR 1 domain binding peptides may be designed using a rational peptide design approach.
  • HR2 domain of a Type 1 viral fusogenic protein of an enveloped virus (HR2 domain) from which the respective HRl domain originates.
  • HR2 domain is mutated at one or multiple positions and then assayed for binding activity to the HRl domain.
  • Suitable assays include any art known protein-protein interaction assay including without limitation in vitro interaction assays, preferably GST or antibody-pull-down assays wherein one of the two binding partners is expressed as a GST fusion protein or as a fusion with an antibody tag and the other binding partner is labelled, and in vivo interaction assays, preferable two-hybrid assays, as well as functional assays, wherein the respective derivative is tested for its ability to inhibit replication or entry of the respective virus.
  • Such assays have been described in the art and have also been employed in the present invention to assess the suitability of a particular HR2 domain derivative to be used in the inhibition of viral fusion.
  • the HR2 domain that is tested for binding to the respective HRl domain is the HR2 domain that is naturally present in the glycoprotein from which the peptide according to SEQ ID NO: 1 to 17 and 105 is derived or on which it is based.
  • the inhibitor of the present invention has a long half life in blood serum. It has been shown that the ability of synthetic peptides or synthetic protein fragments to survive the degradative action of aminopeptidases and serum proteolytic enzymes can be remarkably enhanced by modifications at their N-terminal alpha-amino group.
  • the polypeptide in (a) comprises at its N- terminus a chemical modification selected from the group consisting of one or more D-amino acids, an acyl group, beta-alanine, 9H-fIuoren-9-ylmethoxycarbonyl (Fmoc), Benzyloxy- carbonyl and (t)ert-(B)ut(o)xy(c)arbonyl (Boc).
  • peptide inhibitors of the invention may in one embodiment comprise or consist of L- and/or D-amino acids.
  • membrane integrating lipid can be any lipid as specified as long as it has the capability to insert into a cell membrane or an equivalent artificial lipid bilayer.
  • a “membrane integrating lipid” and membrane integrating derivatives thereof are capable of forming rafts as described in Xu, J. Biol. Chem. 276, (2001) 33540-33546 and Wang, Biochemistry 43, (2004) 1010-8.
  • the membrane integrating lipid is a glycolipid selected from the group consisting of a ganglioside, a cerebroside, a globoside and a sulfatide.
  • the ganglioside may be e.g. selected from the group consisting of GDIa, GDIb, GMl, GD3, GM2, GM3, GQIa and GQIb.
  • the membrane integrating lipid is sphingomyelin or ceramide. If the membrane integrating lipid is a glycerophospholipid then in a preferred embodiment it is selected from the group of glycerophospholipids consisting of phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine.
  • the present invention also comprises membrane integrating derivatives of cholesterol.
  • Such derivatives are structurally related to cholesterol in that they have the same steroid basic structure, i.e. (8R,9S,10R,13S,14S)-10,13-dimethyl- 1,2,6,7,8,9,11, 12,14,15, lojy-dodecahydrocyclopentafajphenanthren and have a comparable ability to insert into a lipid bilayer with the lipid composition as human cells as cholesterol.
  • Preferred integrating derivatives of cholesterol include ergosterol, 7-dihydrocholosterol and stigmasterol.
  • the ability to insert into a lipid bilayer can be tested by art known methods using, e.g. fluorescently labelled cholesterol and structural derivatives thereof on an artificial lipid bilayer.
  • an integrating derivative of a membrane integrating lipid useful in the invention has the ability to integrate into a lipid raft comprised in a cell membrane.
  • a membrane integrating lipid that can integrate into a lipid raft can generally also form one. Thus, if a lipid can integrate into and thus form a lipid raft can be tested e.g. as described in Xu, J. Biol. Chem. 276, (2001) 33540-33546, Wang, Biochemistry 43, (2004) 1010-8.
  • the inhibitor of the present invention has to be in a certain orientation. This orientation is achieved, if the membrane integrating lipid, e.g. cholesterol is attached at (e.g. covalently linked to) the C- terminal region of the polypeptide.
  • the term "C-terminal region" is used to refer to the 5 most C- terminally located amino acids, preferably the four most C-terminally located amino acids, preferably the three most C-terminally located amino acids, the two most C-terminally located amino acids or the C-terminal amino acid.
  • the C-terminal amino acid is generally that amino acid, which has a free carboxy group.
  • the membrane integrating lipid e.g.
  • cholesterol or a functional derivative thereof is attached to the polypeptide through the C-terminal amino acid of the polypeptide.
  • the free carboxy group is modified to increase stability and/or biological half life. In such cases it is preferred that the attachment is through a side chain of the amino acid rather than through the carboxy group.
  • the skilled person for the purpose of the determining the "C-terminal region" or the "C-terminal amino acid” is still capable of this determination by assessing the orientation of the peptide bonds between the amino acids preceding the modified C-terminal amino acid.
  • the polypeptide that forms part of the viral inhibitor of the present invention in addition to the peptide may comprise linker amino acids, preferably at its C-terminus.
  • the C-terminus to which the membrane integrating lipid, e.g. cholesterol or its derivative will be linked will also comprise such linker amino acids.
  • linking refers to a covalent bond between an amino acid in the C- terminal region of the polypeptide and the membrane integrating lipid, e.g. cholesterol or to a linker as described herein that is located between the membrane integrating lipid, e.g. cholesterol and an amino acid in the C-terminal region.
  • the viral inhibitor consists of (i) a polypeptide comprising, essentially consisting or consisting of a HRl domain binding peptide as set out above and one or more linker amino acids and a membrane integrating lipid selected from the group consisting of cholesterol, a sphingolipid, a glycolipid, a glycerophospholipid and membrane integrating derivatives thereof or of (ii) a polypeptide comprising, essentially consisting or consisting of a HRl domain binding peptide as set out above and one or more linker amino acids, a linker and said membrane integrating lipid, preferably cholesterol, or membrane integrating derivatives thereof.
  • the membrane integrating lipid preferably cholesterol or linker is attached to a side chain of one of the amino acids in the C-terminal region of the polypeptide.
  • the linker or membrane integrating lipid is covalently attached to the C-terminus of the polypeptide of the inhibitor of the invention.
  • said "C-terminal region” consists of the C-terminal 5 amino acids of the polypeptide of the inhibitor of the invention.
  • said membrane integrating lipid is selected from a sphingolipid, a glycolipid, a glycerophospholipid and membrane integrating derivatives thereof, wherein the membrane integrating lipid is covalently attached via a free -OH, -NH 3 or -COOH group of the lipid to the C-terminal region of said polypeptide as described above.
  • said membrane integrating lipid is attached covalently to a linker described herein over a free -OH, -NH 3 or -COOH group of the lipid and wherein the linker is attached to the C-terminus or C-terminal region of said polypeptide of the inhibitor.
  • the membrane integrating lipid is a sphingolipid having a structure according to formula IX:
  • pharmaceutically acceptable salt refers to a salt of the viral inhibitor of the present invention.
  • Suitable pharmaceutically acceptable salts of the viral inhibitor of the present invention include acid addition salts which may, for example, be formed by mixing a solution of the viral inhibitor of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.
  • suitable pharmaceutically acceptable salts thereof may include alkali metal salts (e.g., sodium or potassium salts); alkaline earth metal salts (e.g., calcium or magnesium salts); and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate).
  • alkali metal salts e.g., sodium or potassium salts
  • alkaline earth metal salts e.g., calcium or magnesium salts
  • suitable organic ligands e.g., ammonium, quaternary ammonium and amine cations formed using counteranions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sul
  • Illustrative examples of pharmaceutically acceptable salts include but are not limited to: acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate
  • Certain viral inhibitor of the present invention contain both basic and acidic functionalities, e.g. GIu, Asp, GIn, Asn, Lys, or Argm that allow the compounds to be converted into either base or acid addition salts.
  • an inhibitor according to the invention which comprises said membrane integrating lipid has the surprising property of not only significantly enhancing the inhibitory activity of the polypeptide but also to extend the inhibitory activity of the polypeptide inhibitors to enveloped viruses that are not inhibited when the membrane integrating lipid is absent.
  • the inhibitor of the invention inhibits the fusion of at least two and preferably at least three different types of viruses selected from the group consisting of Influenza virus, Parainfluenza virus, Sendai virus, Measles virus, Newcastle disease virus, Mumps virus, respiratory syncytical virus, Hendra virus, Nipah virus, Ebola virus, and Marburg virus, with a host cell.
  • the inhibitor of the invention is capable of interfering with the viral fusion with the host cell of at least the viruses HPIV3, NiV, RSV and/or SV5.
  • Figures 2-5 show preferred peptide sequences that may be comprised in a broad-spectrum antiviral agent of the invention.
  • said at least ten contiguous amino acids of the peptide are at least ten contiguous amino acids of SEQ ID NO: 99 or of a derivative thereof, wherein the derivative consists of the following amino acids: amino acid 1 is selected from VaI, Leu, and Tyr; amino acid 2 is selected from Ala, Ser, Asp, Tyr, and Phe; amino acid 3 is selected from Leu, He, Pro, and Thr; amino acid 4 is selected from Asp and Leu; amino acid 5 is selected from Pro, VaI, and Lys; amino acid 6 is selected from He, Leu, Phe, and VaI; amino acid 7 is selected from Asp and GIu; amino acid 8 is selected from He and Phe; amino acid 9 is selected from Ser and Asp; amino acid 10 is selected from He, GIn, Ala, and Ser; amino acid 11 is selected from GIu, As
  • an inhibitor of the invention wherein the derivative of SEQ ID NO: 99 mentioned above consists of the following amino acids:
  • Amino acid 1 can be VaI or Tyr; Amino acid 2 can be Ala, Ser, VaI, or Phe; Amino acid 3 is Leu; Amino acid 4 can be Asp or Leu; Amino acid 5 can be Pro, VaI, or Lys; Amino acid 6 can be He or Phe; Amino acid 7 is Asp; Amino acid 8 can be He or Phe; Amino acid 9 can be Ser or
  • Amino acid 10 can be He, Ala, or Ser; Amino acid 11 can be GIu or GIn; Amino acid 12 is
  • Amino acid 1 can be VaI or Tyr; Amino acid 2 can be Ala, Ser, VaI, or Phe; Amino acid 3 is Leu; Amino acid 4 can be Asp or Leu; Amino acid 5 can be Pro, VaI, or Lys; Amino acid 6 can be He or Phe; Amino acid 7 is Asp; Amino acid 8 can be He or Phe; Amino acid 9 can be Ser or Asp; Amino acid 10 can be He, Ala, or Ser; Amino acid 11 can be GIu or GIn; Amino acid 12 is Leu; Amino acid 13 can be Asn or Ser; Amino acid 14 can be Lys, GIn, or Ser; Amino acid 15 can be Ala, VaI, or He; Amino acid 16 can be Lys or Asn; Amino acid 17 can be Ser, Lys, GIu, or
  • Amino acid 1 can be VaI or Tyr; Amino acid 2 can be Ala, Ser, VaI, or Phe; Amino acid 3 is Leu; Amino acid 4 can be Asp or Leu; Amino acid 5 can be Pro, VaI, or Lys; Amino acid 6 can be He or Phe; Amino acid 7 is Asp; Amino acid 8 is He; Amino acid 9 can be Ser or Asp; Amino acid 10 can be He, Ala, or Ser; Amino acid 11 can be GIu, GIn, or VaI; Amino acid 12 is Leu; Amino acid 13 can be Asn or Ser; Amino acid 14 can be Lys, GIn, or Ser; Amino acid 15 can be Ala, VaI, or He; Amino acid 16 can be Lys or Asn; Amino acid 17 can be Ser, Lys, GIu, or
  • the inventors have shown that substituting two amino acids (QK) of a wildtype HPIV3 polypeptide according to SEQ ID NO: 98 with two amino acids (KI) from
  • Hendravirus produced a polypeptide which effectively inhibits the viral fusion of four different paramyxoviruses, when said hybrid polypeptide was coupled to a membrane integrating lipid
  • amino acid 31 of the derivative is Lys and amino acid 32 of the derivative is He.
  • amino acid 31 of the derivative is GIy and amino acid 32 of the derivative is Lys.
  • SEQ ID NOs: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 and 119 specify preferred amino acid substitutions and preferred conserved amino acids as derived from multiple sequence alignments as shown e.g. in figures 2-5.
  • said at least ten contiguous amino acids of the peptide are at least ten contiguous amino acids of an amino acid sequence selected from the group consisting of
  • said peptide of the inhibitor of the invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 and 119; wherein each X recited in the sequences specified by said SEQ ID NOs is individually selected from any amino acid with the proviso that the peptide has at least 70% sequence identity with SEQ HD NO: 98.
  • the peptide of the inhibitor according to the invention has the amino acid sequence V 1 XXDXXDISXXL 12 XXXK I6 XXLXXS 22 XXXI 26 XXS 29 XKILXXI 36 (SEQ ID NO: 110) or a derivative thereof, wherein the derivative comprises at least one of the following amino acids substitutions:
  • Vi may be substituted with L, A or I;
  • S 22 may be substituted with A
  • I 26 may be substituted with L or V;
  • S 29 may be substituted with A; and/or
  • I 36 may be substituted with V or L; wherein each X is individually selected from any amino acid with the proviso that the peptide has at least 70% sequence identity with SEQ ID NO: 98.
  • said at least ten contiguous amino acids of the peptide are at least ten contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 and 119; wherein each X recited in the sequences specified by said SEQ ID NOs is individually selected from any amino acid with the proviso that said amino acid sequence has at least 80% sequence identity with SEQ ID NO: 98.
  • said peptide of the inhibitor of the invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 and 119; wherein each X recited in the sequences specified by said SEQ ID NOs is individually selected from any amino acid with the proviso that the peptide has at least 80% sequence identity with SEQ ID NO: 98.
  • the peptide of the inhibitor according to the invention has the amino acid sequence V I XXDXXDISXXL 12 XXXK 16 XXLXXS 22 XXXI 26 XXS 29 XKILXXI 36 (SEQ ID NO: 110) or a derivative thereof, wherein the derivative comprises at least one of the following amino acids substitutions:
  • Vi may be substituted with L, A or I;
  • S 22 may be substituted with A
  • I 26 may be substituted with L or V;
  • S 29 may be substituted with A; and/or
  • I 36 may be substituted with V or L; wherein each X is individually selected from any amino acid with the proviso that the peptide has at least 80% sequence identity with SEQ ID NO: 98.
  • said at least ten contiguous amino acids of the peptide are at least ten contiguous amino acids of an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 and 119; wherein each X recited in the sequences specified by said SEQ ID NOs is individually selected from any amino acid with the proviso that said amino acid sequence has at least 87% sequence identity with SEQ ID NO: 98.
  • said peptide of the inhibitor of the invention has an amino acid sequence selected from the group consisting of SEQ ID NOs: 106, 107, 108, 109,
  • each X recited in the sequences specified by said SEQ ID NOs is individually selected from any amino acid with the proviso that the peptide has at least 87% sequence identity with SEQ ID NO: 98.
  • the peptide of the inhibitor according to the invention has the amino acid sequence
  • K 16 may be substituted with N or H
  • S 22 may be substituted with A
  • I 26 may be substituted with L or V;
  • S 29 may be substituted with A; and/or I 36 may be substituted with V or L; wherein each X is individually selected from any amino acid with the proviso that the peptide has at least 90% sequence identity with SEQ ID NO: 98.
  • the inhibitors of viral fusion outlined above have a length of between 10 and 50 amino acids more preferably between 32 and 50 amino acids and most preferably between 36 and 50 amino acids.
  • the peptide is derived from or consists of a HR2 domain since HR2 domains are known to be capable of binding to HRl domains.
  • HR2 domains are known to be capable of binding to HRl domains.
  • Preferred naturally occurring or synthetic HR2 domains are outlined in Table 1 below as SEQ ID NO: 18-104 and SEQ ID NO: 106-119. Table 1
  • the peptide of the inhibitor of the invention is a peptide that has at least 80% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NO: 18 to 104. Most preferably, the peptide of the inhibitor of the invention is a peptide that has at least 90% or 100% amino acid sequence identity with a sequence selected from the group consisting of SEQ ID NO: 96 to 104. As mentioned, the peptide must be capable of binding to an HRl domain with an amino acid sequence according to any of SEQ ID NO: 1 to 17 and 105. Thus, it is preferred that the peptide has at least 70% of the binding activity that a peptide has, which is 100% identical to the respective SEQ ID NO: 96 to 104.
  • the term "identity" or “identical” in the context of polynucleotide, peptide or protein sequences refers to the number of residues in the two sequences (a reference sequence as indicated herein as SEQ ID NO and a second sequence) that are identical when aligned over the entire length of the reference sequence for maximum correspondence as is well known in the art. Specifically, the percent sequence identity of the two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches (nucleotides or amino acids, respectively) between the reference sequence and the aligned second sequence divided by the length of the reference sequence and multiplied by 100.
  • Alignment tools that can be used to align two sequences are well known to the person skilled in the art and can, for example, be obtained on the World Wide Web, e.g., ClustalW (www.ebi.ac.uk/clustalw) or Align (http://www.ebi.ac.uk/emboss/align/index.html).
  • Matrix Blosum62 for protein sequences and "DNAfull" for nucleic acid sequences
  • Gap Open 10.0
  • Gap Extend 0.5.
  • the term "the peptide is derived" has one of the following meanings:
  • the peptide (i) consists of 10 or more contiguous amino acids from above indicated peptides with an amino acid sequence according to SEQ ID NO: 18 to 104, i.e.
  • the peptide is a fragment of one of those peptides, or (ii) the peptide consists of 10 or more contiguous amino acids of above peptides having the amino acid sequences set out in SEQ ID NO: 18 to 104 or has the amino acid sequence as set out in SEQ ID NO: 18 to 104 and comprises one, two or three amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 104, without substantially altering the HRl domain binding activity of the peptide on which it is based.
  • the HRl domain binding activity is not substantially altered, if the binding is at least 50%, preferably at least 60%, preferably at least 70%, preferably at least 80%, preferably at least 90% or at least 100% or more of the binding observed for the HR2 domain peptide on which the derivative having one, two or three mutations is based.
  • HRl binding activity may be assayed as set out above using in vitro and in vivo binding assays as well as functional assays.
  • the HRl domain that is preferably tested for binding to the respective HR2 domain or peptide derivative is the HR-I domain that is naturally present in the glycoprotein from which the peptide according to SEQ ID NO: 18 to 104 is derived or on which it is based, i.e.
  • the HRl domain used in in vitro or in vivo binding assays may be from any of the viruses that the HR2 domain was derived from. In some of above cases the HR2 peptide is similar or identically present in two viruses. In those cases the HRl domain may be derived from either virus or either virus may be used in a functional assay testing the HRl binding activity of the respective derivative.
  • Preferred HRl domains that can be used have the amino acid sequences as set out in SEQ ID NO: 1 to 17 and 105.
  • the peptide of the inhibitor of the invention has a length of not more than 50 amino acids. In a preferred embodiment the peptide has a length of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 94 and 96-104.
  • the peptide has a length of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 94 and 96-104 and comprises one amino acid change with respect to the amino acid sequences according to SEQ ID NO: 18 to 94 and 96-104.
  • the peptide has a length of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 94 and 96-104 and comprises two amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 94 and 96-104.
  • the peptide has a length of at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 94 and 96-104 and comprises three amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 94 and 96-104.
  • the peptide has a length of at least 24 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 31 and 34 to 94 and 96-104.
  • the peptide has a length of at least 24 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 31 and 34 to 94 and 96-104 and comprises one amino acid change with respect to the amino acid sequences according to SEQ ID NO: 18 to 31 and 34 to 94 and 96-104.
  • the peptide has a length of at least 24 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 31 and 34 to 94 and 96-104 and comprises two amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 31 and 34 to 94 and 96-104.
  • the peptide has a length of at least 24 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 31 and 34 to 94 and 96-104 and comprises three amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 31 and 34 to 94 and 96-104.
  • the peptide has a length of at least 25 or at least 26 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 30 and 35 to 94 and 96-104.
  • the peptide has a length of at least 25 or at least 26 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 30 and 32 to 94 and 96-104 and comprises one amino acid change with respect to the amino acid sequences according to SEQ ID NO: 18 to 30, 34 to 49 and 53 to 94 and 96-104.
  • the peptide has a length of at least 25 or at least 26 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 30, 34 to 49 and 53 to 94 and 96-104 and comprises two amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 30, 34 to 49 and 53 to 94 and 96-104.
  • the peptide has a length of at least 25 or at least 26 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 30, 34 to 49 and 53 to 94 and 96-104 and comprises three amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 30, 34 to 49 and 53 to 94 and 96-104.
  • the peptide has a length of at least 27, at least 28 or at least 29 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 30, 35 to 49 and 53 to 83 and 96-104.
  • the peptide has a length of at least 27, at least 28 or at least 29 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 30, 35 to 49 and 53 to 83 and 96-104 and comprises one amino acid change with respect to the amino acid sequences according to SEQ ID NO: 18 to 30, 35 to 49 and 53 to 83 and 96-104.
  • the peptide has a length of at least 27, at least 28 or at least 29 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 30, 35 to 49 and 53 to 83 and 96-104 and comprises two amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 30, 35 to 49 and 53 to 83 and 96-104.
  • the peptide has a length of at least 27, at least 28 or at least 29 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 30, 35 to 49 and 53 to 83 and 96-104 and comprises three amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 30, 35 to 49 and 53 to 83 and 96-104.
  • the peptide has a length of at least 30 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 53 to 83 and 96- 104.
  • the peptide has a length of at least 30 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 53 to 83 and 96-104 and comprises one amino acid change with respect to the amino acid sequences according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 53 to 83 and 96-104.
  • the peptide has a length of at least 30 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 53 to 83 and 96-104 and comprises two amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 53 to 83 and 96- 104.
  • the peptide has a length of at least 30 contiguous amino acids of above peptides according to 18 to 21, 27 to 30, 35 to 49 and 53 to 83 and 96-104 and comprises three amino acid changes with respect to the amino acid sequences according to 18 to 21, 27 to 30, 35 to 49 and 53 to 83 and 96-104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 54 to 82 and 96- 104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 54 to 82 and 96-104 and comprises one amino acid change with respect to the amino acid sequences according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 54 to 82 and 96-104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 54 to 82 and 96-104 and comprises two amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 49 and 54 to 82 and 96- 104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to 18 to 21, 27 to 30, 35 to 49 and 54 to 82 and 96-104 and comprises three amino acid changes with respect to the amino acid sequences according to 18 to 21, 27 to 30, 35 to 49 and 54 to 82 and 96-104.
  • the peptide has a length of at least 32 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, 40 to 49 and 54 to 82 and 96-104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, 40 to 49 and 54 to 82 and 96-104 and comprises one amino acid change with respect to the amino acid sequences according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, 40 to 49 and 54 to 82 and 96-104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, 40 to 49 and 54 to 82 and 96-104 and comprises two amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, 40 to 49 and 54 to 82 and 96-104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to 18 to 21, 27 to 30, 35 to 38, 40 to 49 and 54 to 82 and 96-104 and comprises three amino acid changes with respect to the amino acid sequences according to 18 to 21, 27 to 30, 35 to 38, 40 to 49 and 54 to 82 and 96-104.
  • the peptide has a length of at least 33 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, and 40 to 49 and 96- 104.
  • the peptide has a length of at least 32 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, and 40 to 49 and 96-104 and comprises one amino acid change with respect to the amino acid sequences according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, and 40 to 49 and 96-104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, and 40 to 49 and 96-104 and comprises two amino acid changes with respect to the amino acid sequences according to SEQ ID NO: 18 to 21, 27 to 30, 35 to 38, and 40 to 49 and 96-104.
  • the peptide has a length of at least 31 contiguous amino acids of above peptides according to 18 18 to 21, 27 to 30, 35 to 38, and 40 to 49 and 96-104 and comprises three amino acid changes with respect to the amino acid sequences according to 18 to 21, 27 to 30, 35 to 38, and 40 to 49 and 96-104.
  • the peptide has an amino acid sequence according to SEQ ID NO: 18 to 104 or is a HRl domain binding derivative thereof, which comprises one, two or three amino acid changes with respect to the amino acid sequence according to SEQ ID NO: 18 to 104.
  • the C-terminus of the polypeptide is modified, preferably by amidation.
  • the N-terminus is modified by reacting the free amino group with a mono or dicarboxylic organic acid, preferably acetic acid or succinic acid.
  • both the N-terminus and the C-terminus of the polypeptide are modified.
  • polypeptides comprised in the inhibitor of the present invention comprise an amino acid sequence as set out in SEQ ID NO: 18 to 104 and 106-119 and derivatives of the peptides according to SEQ ID NO: 106-119 as defined above and have an acetylated or succinilated N-terminus and an amidated C — terminus.
  • the polypeptide comprised in the inhibitor of the present invention may comprise additional amino acids at its N- and/or C-terminus.
  • the number of additional amino acids at the N-terminus and/or C-terminus preferably does not exceed 15 amino acids to avoid interference with the interaction with the HRl domains on the surface of the enveloped virus.
  • the C- and/or N-terminal amino acids are linker amino acids.
  • linker amino acid is used to refer to small amino acids that preferably form unstructured domains, i.e. that do not adopt alpha-helical or beta-sheet structure and are, thus, suitable to provide structural flexibility between the HRl binding peptide comprised in the polypeptide and the membrane integrating lipid, preferably cholesterol or membrane inserting derivative thereof.
  • Preferred examples of such amino acids comprise Ala, GIy, Ser and Pro.
  • the one or more linker amino acids comprises a cysteine at its C-terminus.
  • linker amino acids are the following: (Gly) m+ i,
  • the polypeptide consists of a HRl binding peptide, preferably a HRl binding peptide selected from the peptides having an amino acid sequence as set out in SEQ ID NO: 18 to 104, and 106- 119 and derivatives of the peptides according to SEQ ID NO: 106-119 as defined above and one of the above indicated preferred linkers, in particular GlySerGlySerGly or GlySerGlySerGlyCys.
  • said membrane integrating lipid e.g. cholesterol or membrane inserting derivative thereof is attached to the C-terminal region of the polypeptide via a linker.
  • linker preferably refers to an organic molecule that adopts a linear conformation.
  • linkers comprise, consist essentially of or consist of a moiety selected from the group consisting of Y, -(CH 2 CH 2 X) n -, -(CH 2 CH 2 CH 2 X) n -, -Y-(CH 2 CH 2 X) n -, -Y-(CH 2 CH 2 CH 2 X) n -, -Y-(CH 2 CH 2 X) n -Z, -Y-(CH 2 CH 2 CH 2 X) n -Z, -Y-(CH 2 CH 2 X) n -CH 2 -Z, -Y-(CH 2 CH 2 CH 2 X) n -CH 2 -Z, -Y-(CH 2 CH 2 CH 2 X) n -CH 2 -Z, -Y-(CH 2 CH 2 X) n -CH 2 -Z, -Y-(CH 2 CH 2 X) n -CH 2 -Z, -Y-(CH 2 CH 2
  • X is -O- or -NH-
  • the membrane integrating lipid is cholesterol or a membrane integrating derivative thereof, then it is preferred that the cholesterol or membrane integrating derivative thereof is attached directly or via the linker to the polypeptide through the oxygen moiety at the 3 position of the cholesterol or membrane integrating derivative thereof.
  • the C-terminal amino acid is Cys and, accordingly, in a preferred embodiment the membrane integrating lipid, preferably cholesterol or the linker is attached to the sulphur moiety of the amino acid linker or to the Cys residue that may naturally occur in a HR2 domain.
  • X in each instance is independently selected from -NH- and -O-;
  • R designates the bond to the polypeptide, preferably a sulphur moiety, preferably of Cys, and n is an integer of 1 to 15, i.e. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • the most preferred inhibitors of the present invention are those wherein the membrane integrating lipid, preferably cholesterol is linked through a linker as set out above in formulas (I) to (VIII) and the polypeptide consists of a peptide derived from SEQ ID NO: 18 to 104 as outlined above, in particular one of the peptides having an amino acid sequence as set out in
  • the paramyxovirus fusion proteins as well as the hemagglutinin HA2 protein of influenza virus share some common features including that both proteins are formed by proteolytic cleavage of a large precursor and mediate virus-target membrane fusion and in both proteins, the fusion peptide is located at the N-terminus and the transmembrane domain at the C-terminus of the ectodomain.
  • the site that the fusion occurs In particular, HA2 is internalized by endocytosis and mediates fusion of the viral and endosome membranes only when inside the cell and only in the presence of an acidic pH, while the paramyxovirus fusion process occurs at the surface of the target cell at neutral pH.
  • the inhibitor of the present invention comprises
  • a polypeptide comprising a peptide capable of binding to a HRl domain of a Type 1 viral fusogenic protein of an orthomyxovirus (HRl domain) with an amino acid sequence according to SEQ ID NO: 105, wherein the peptide has a length of at least ten, preferably at least 20 and most preferably at least 31 contiguous amino acids and
  • a membrane integrating lipid selected from the group consisting of cholesterol, a sphingolipid, a glycolipid, a glycerophospholipid and membrane integrating derivatives thereof, which is attached to the C-terminal region of the polypeptide; or a pharmaceutically acceptable salt thereof.
  • the peptide is derived from a HR2 domain of a
  • HR2 domains with an amino acid sequence according to SEQ ID NO: 83 to 95.
  • the invention provides a method for making a broad-spectrum inhibitor of viral fusion effective against at least three different paramyxoviruses, wherein the method comprises the steps of:
  • generating a polypeptide comprising a hybrid peptide capable of binding to a HRl domain of a Type 1 viral fusogenic protein of an enveloped virus (HRl domain) selected from the group consisting of HRl domains with an amino acid sequence according to SEQ ID NO: 1 to 17 and 105, wherein the hybrid peptide comprises amino acids from HR2 domains of at least two different enveloped viruses; and
  • a membrane integrating lipid selected from the group consisting of cholesterol, a sphingolipid, a glycolipid, a glycerophospholipid and membrane integrating derivatives thereof to the C-terminal region of the polypeptide.
  • a further aspect of the invention is an inhibitor producible according to the method of the invention.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the inhibitor of the present invention or a pharmaceutically acceptable salt thereof or a combination of the present invention as set out above and a pharmaceutically acceptable excipient.
  • pharmaceutically acceptable carriers can be either solid or liquid. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid excipient can be one or more substances, which may also act as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material.
  • the excipient is preferably a finely divided solid, which is in a mixture with the finely divided inhibitor of the present invention.
  • the inhibitor of the present invention is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • the powders and tablets preferably contain from 5% to 80%, more preferably from 20% to 70% of the active compound.
  • Suitable excipients are magnesium carbonate, magnesium stearate, talc, sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose, a low melting wax, cocoa butter, and the like.
  • the term "preparation" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with it. Similarly, cachets and lozenges are included.
  • Tablets, powders, capsules, pills, cachets, and lozenges can be used as solid dosage forms suitable for oral administration.
  • any oral administration form retards the release of the inhibitors of the present invention to the lower intestinal tract, wherein protease activity is reduced.
  • a low melting wax such as a mixture of fatty acid glycerides or cocoa butter
  • the active component is dispersed homogeneously therein, as by stirring.
  • the molten homogeneous mixture is then poured into convenient sized molds, allowed to cool, and thereby to solidify.
  • Preferred administration forms are liquid form preparations include solutions, suspensions, and emulsions, for example, water or water/propylene glycol solutions.
  • liquid preparations can be formulated in solution in, e.g. aqueous polyethylene glycol solution.
  • the pharmaceutical preparation is preferably in unit dosage form.
  • the preparation may be subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, an injection vial, a tablet, a cachet, or a lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • the inhibitors of the present invention or a pharmaceutically acceptable salt thereof or a combination of the present invention is for the treatment or prevention of infection by an enveloped virus, such as a paramyxovirus.
  • Preferred enveloped viruses treatable are selected from the group consisting of Influenza virus, preferably human Influenza virus, Parainfluenza virus, preferably human Parainfluenza virus, e.g. type 1 to 4 (HPIVl, HPIV2, HPIV3 or HPIV4), Sendai virus, Measles virus, Newcastle disease virus, Mumps virus, respiratory syncytical virus (RSV), preferably human RSV, Hendra virus (HeV), Nipah virus, Ebola virus (EBOV), and Marburg virus.
  • Influenza virus preferably human Influenza virus
  • Parainfluenza virus preferably human Parainfluenza virus, e.g. type 1 to 4 (HPIVl, HPIV2, HPIV3 or HPIV4)
  • Sendai virus Sendai virus
  • Measles virus Newcastle disease virus
  • Mumps virus respiratory syncytical virus
  • RSV respiratory syncytical virus
  • HeV Hendra virus
  • Nipah virus Nipah virus
  • the present invention relates to the use inhibitors of the present invention or a pharmaceutically acceptable salt thereof or a combination of the present invention for the production of a medicament for treating or preventing infection by an enveloped virus, such as a paramyxovirus.
  • enveloped viruses treatable are selected from the group consisting of Influenza virus, preferably human Influenza virus, Parainfluenza virus, preferably human Parainfluenza virus, e.g.
  • HPIVl type 1 to 4 (HPIVl, HPIV2, HPIV3 or HPIV4), Sendai virus, Measles virus, Newcastle disease virus, Mumps virus, respiratory syncytical virus (RSV), preferably human RSV, Hendra virus (HeV), Nipah virus, Ebola virus (EBOV), and Marburg virus.
  • RSV respiratory syncytical virus
  • HeV Hendra virus
  • EBOV Ebola virus
  • Marburg virus Marburg virus
  • Cholesterol-derivatized inhibitors are active against influenza. Cholesterol- derivatized inhibitors no. 1, 3 and 5 of table 3 inhibit influenza A/H3N2 in a plaque reduction assay. Non-cholesterol-containing peptides (compounds no. 2, 4 and 6 of table 3) are inactive.
  • Fig. 2-5 Preferred peptide sequences that may be comprised in a broad-spectrum antiviral agent of the invention.
  • " — " may be any three amino acids, preferably PSD or no amino acids. Amino acids that can be used to substitute the respective amino acids of the reference sequence "VALDPIPSDDISIELNKAKSDLEESKEWIRRSNGKLDSI” or "VALDPIDI SIELNKAKSDLEESKEWIRRSNGKLDSI (if “—” means no amino acids at the indicated position) are indicated below the reference sequence.
  • the amino acid at position 1 may be any amino acid selected from the group consisting of VaI, Leu and Tyr.
  • the amino acid at position 2 may be any amino acid selected from the group consisting of Ala, Ser, Asp, Tyr and Phe.
  • Peptide Synthesis Methods of making peptides comprising naturally and non-naturally occurring amino acids are well known in the art. Synthetic or microbiological methods can be used, hi The following a more specific description is provided of methods to prepare peptides derivatized with cholesterol.
  • the required cholesterol derivatives bearing a bromoacetyl group can be made as described in the Examples, or by analogy, thereto, by using commercially available compounds or by well known methods from commercially available compounds.
  • Derivatives of cholesterol are commercially available or can be made from commercially available materials by well known methods
  • Trifluoroacetic acid (2 mL, 26 mmol) was added to a solution of 1 (1.48 g, 2 mmol) in 10 ml of CH 2 Cl 2 and the mixture was stirred at room temperature for 3h. AU the volatiles were removed under vacuo and the crude was lyophilized to obtain an incolor oil that was dissolved in 60 mL of CH 2 Cl 2 .
  • Bromoacetic anhydride (0.62 g, 2.4 mmol) was added followed by NJV- diisopropylethylamine (0.65 mL, 3.7 mmol) and the mixture was stirred at room temperature for 3h.
  • Amine-dPEG ⁇ TM acid (1.65g, 2.7 mmol, Product N° 10287, Quanta BioDesign, Ltd.) was dissolved in 15 mL of dichloromethane and Boc-anhydride (0.7g, 3.2 mmol) was added followed by triethyl amine (0.75ml, 5.4 mmol). The mixture was stirred at room temperature for 2h and then the solvent was evaporated under reduced pressure.
  • Trifluoroacetic acid (1.7 mL, 22 mmol) was added to a solution of 3 (1.57 g, 1.5 mmol) in 8.5 ml of CH 2 Cl 2 and the mixture was stirred at room temperature for 3h until disappearance of starting material. All the volatiles were removed under vacuum and the crude was lyophilized to obtain an incolor oil that was dissolved in 45 mL of CH 2 Cl 2 .
  • Bromoacetic anhydride (0.48 g,
  • Amino acids were activated with equimolar amounts of HBTU (2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate) and a 2-fold molar excess of DIEA (N,N-diisopropyl-ethylamine).
  • the side chain protecting groups were: tert-butyl for Asp, GIu, and Ser; trityl for Asn and Cys; tert- butoxy-carbonyl for Lys and Trp; and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl for Arg.
  • the dry peptide-resin was treated with 82.5% TFA, 5% phenol, 5% water, 5% thioanisole, 2.5% ethanedithiol for 1.5h at room temperature.
  • the resin was filtered and the solution was evaporated and the peptide pellet treated several times with diethylether to remove the organic scavengers.
  • the final pellet was dried, resuspended in 1 :1 (v/v) H 2 O: acetonitrile and lyophilized.
  • the crude peptide was analyzed by liquid chromatography-mass spectrometry using a Waters-Micromass LCZ Platform with a Phenomenex, Jupiter C 4 column (150 x 4.6 mm, 5 ⁇ m) using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and the following linear gradient: 30%(B)-60%(B) in 20'- 80%(B) in 3'-80%(B) for 3', flow 1 ml/min.
  • the crude peptide was dissolved at 1 mg/ml in 70% eluent A/30% eluent B.
  • the HPLC trace of crude PEP2667 is shown in Figure 4.
  • the crude peptide was purified by reverse-phase HPLC with a XBridge Cl 8 semi- preparative column (50 x 150 mm, 5 ⁇ m, 130 A), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and an isocratic step at 25%(B) for 5' followed by the linear gradient: 25%(B)-40%(B) in 20'-80%(B) in 2'-80%(B) for 3', flow 30 ml/min.
  • the purified peptide was characterized by HPLC/MS on a Waters-Micromass LCZ platform as described above (theoretical M. W. 4543.1 Da, found 4542.5 Da).
  • reaction was monitored by liquid chromatography-mass spectrometry using a Waters-Micromass LCZ Platform with a Phenomenex, Jupiter C 4 column (150 ⁇ 4.6 mm, 5 ⁇ m, ) using a linear gradient of eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, flow rate 1 ml/min.
  • the cholesterol-peptide product was purified by reverse-phase HPLC with semi-preparative Waters RCM Delta-PakTM C 4 cartridges (25 x 200 mm, 15 ⁇ m), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and an isocratic step at 35%(B) for 5' followed by the linear gradient: 35%(B)- 55%(B) in 25'-80%(B) in 2'-80%(B) for 3', flow 30 ml/min.
  • the purified peptide was characterized by HPLC/MS on a Waters-Micromass LCZ platform as described above (theoretical M. W. 4970 Da, found 4969.3 Da).
  • the peptide was prepared by conjugation between bromoacetyl-oxa ⁇ cholesterol and the peptide prepared in 6. above (SEQ ID NO. 49). 2.26 ⁇ mol of the peptide (SEQ ID No. 49) were dissolved in 600 ⁇ L of DMSO and 2.49 ⁇ mol (1.1 eq) of bromoacetyl-oxa ⁇ cholesterol dissolved in 188 ⁇ L of THF, were added. Then 8 ⁇ L of DIEA (N,N-diisopropyl-ethylamine) were added to the mixture which was left stirring at room temperature.
  • DIEA N,N-diisopropyl-ethylamine
  • reaction was monitored by liquid chromatography-mass spectrometry using a Waters-Micromass LCZ Platform with a Phenomenex, Jupiter C 4 column (150 x 4.6 mm, 5 ⁇ m) using a linear gradient of eluents (A) 0.1% TFA in water and (B) 0.1 % TFA in acetonitrile, flow rate 1 ml/min.
  • the cholesterol-peptide product was purified by reverse-phase HPLC with semi-preparative Waters RCM Delta-PakTM C 4 cartridges (25 x 200 mm, 15 ⁇ m), using as eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, and an isocratic step at 45%(B) for 5' followed by the linear gradient: 45%(B)- 65%(B) in 30'-80%(B) in 2'-80%(B) for 3', flow 30 ml/min.
  • the purified peptide was characterized by HPLC/MS on a Waters-Micromass LCZ platform as described above (theoretical M. W. 5216 Da, found 5215.2 Da).
  • the peptide was prepared by conjugation between bromoacetyl-oxa ⁇ -cholesterol and the peptide prepared in 6. above (SEQ ID NO. 49). 2.26 ⁇ mol of the peptide (SEQ ID No. 49) were dissolved in 600 ⁇ L of DMSO and 2.49 ⁇ mol (1.1 eq) of bromoacetyl-oxa ⁇ -cholesterol dissolved in 188 ⁇ L of THF, were added. Then 8 ⁇ L of DIEA (N,N-diisopropyl-ethylamine) were added to the mixture which was left stirring at room temperature.
  • DIEA N,N-diisopropyl-ethylamine
  • reaction was monitored by liquid chromatography-mass spectrometry using a Waters-Micromass LCZ Platform with a Phenomenex, Jupiter C 4 column (150 x 4.6 mm, 5 ⁇ m) using a linear gradient of eluents (A) 0.1% TFA in water and (B) 0.1% TFA in acetonitrile, flow rate 1 ml/min.
  • the purified peptide was characterized by HPLC/MS on a Waters-Micromass LCZ platform as described above (theoretical M.W. 5569 Da, found 5268.5 Da).
  • the peptide was prepared by standard Solid-phase Peptide synthesis, using Fmoc chemistry on an APEX396 synthesizer (Advanced Chemtech) using AM-Polysryrene LL resin (100-200 mesh, Novabiochem) derivatized with a modified Rink linker p-[(R,S)- ⁇ -[9H-Fluoren- 9-yl-methoxyformamido]-2,4-dimethoxybenzyl]-phenoxyacetic acid.
  • the following side chain protecting groups were used: OtBu for Asp and GIu; tBu for Ser; Boc for Lys and Trp; Trt for Asn and Cys.
  • the dry peptide-resin was treated with the cleavage mixture, 82.5% TFA, 5% phenol, 5% Tioanisole, 5% water, 2.5% EDT for 1.5 h at room temperature.
  • the resin was filtered and the solution was added to cold methyl-t-butyl ether in order to precipitate the peptide.
  • the peptide pellets were washed with fresh cold methyl-t-butyl ether to remove the organic scavengers. The process was repeated twice. Final pellets were dried, resuspended in 30% acetonitrile and lyophilized
  • the purified peptide was lyophilized and structure and purity was confirmed by analytical UPLC and electrospray mass spectrometry on a SQ Detector Waters platform.
  • the peptide was synthesized by chemoselective thioether conjugation between the peptide prepared in 10. above (SEQ ID No. 101) and bromoacetyl-oxa 4 -cholesterol. 21.5 mg of bromoacetyl-oxa ⁇ cholesterol (28.4 umol, 1.2 eq) were dissolved in 1.1 mL of anhydrous THF (cone. 20 mg/mL) and added to 110 mg of purified SEQ ID No. 101 (23.7 umol) dissolved in 3.66 mL of DMSO (cone. 30 mg/mL). Then 145 uL (3% by volume) of DIPEA (N,N- diisopropyl-ethylamine) were added to the mixture which was left stirring at room temperature.
  • DIPEA N,N- diisopropyl-ethylamine
  • the resulting cholesterol-peptide product was purified by reverse-phase HPLC; in order to have final compound as acetate salt we used water and acetonitrile with 0.2% acetic acid as eluents for the purification.
  • the antiviral activity of the compounds indicated in Table 2 were tested against HPIV3 and HeV.
  • the compounds in each case comprised a polypeptide with the respectively indicated amino acid sequence (see SEQ ID NOs: 35 to 49) that was acetylated at its N-terminus and amidated at its C-terminus.
  • SEQ ID NO: 49 was linked to cholesterol either directly or via two linkers of different length.
  • NA not active.
  • ND not done.
  • Figure 1 shows that the inhibitors of table 3 with the cholesterol group are effective against influenza virus, while the peptides lacking cholesterol are inactive.
  • the inhibition was determined using a plaque reduction assay as described in Porotto, M., et al. (2007) J. Virol. 81:10567-74; Porotto, M., et al. (2007) J. Virol. 81:3216-3228; Porotto, M., et al. (2009) J Virol 83:5148-5155.
  • inhibitory peptides with their respective lipid modification that are effective against different enveloped viruses are shown in table Table 5.
  • Peptides used in experiments 1-3 were derived from the HR2 domain of respiratory syncytial virus (RSV).
  • the peptide used in experiment no. 1 is inactive against RSV, human parainfluenza virus type 3 (HPIV3), and
  • Simian Virus 5 (S V5) (IC 50 >10 ⁇ M).
  • the peptide used in experiment no. 2 has the same sequence as in experiment no. 1, plus the spacer sequence Gly-Ser-Gly-Ser-Gly and a cysteine alkylated with iodoacetamide. It is the control for the peptide used in experiment no. 3, in which the cysteine is derivatized with cholesterol.
  • the peptides used in experiments no. 4, 5, and 6, are all derived from the HR2 domain of HP rV3.
  • Peptide used in experiment no. 5 has the same sequence as peptide used in experiment no. 4, plus the spacer sequence Gly-Ser-Gly-Ser-Gly and a cysteine alkylated with iodoacetamide. It is the control for the peptide used in experiment no. 6, in which the cysteine is derivatized with cholesterol. As shown, the peptide used in experiment no.
  • the peptide used in experiment no. 5 is inactive (IC 50 >10 ⁇ M) against two other viruses, RSV and SV5. However, when cholesterol is attached to this sequence, producing peptide used in used in experiment no.
  • Table 4 shows the alignment of the sequences of five paramyxoviruses (Human parainfluenza virus type 3 (HPIV3), Simian parainfluenza virus 5 (VS5), human respiratory syncytial virus (RSV), Hendra virus (HeV), and Nipah Virus (NiV)), taking into account that RSV binds to the hydrophobic pocket of its inner coiled-coil with two Phe residues (Phe488, Phe493) which are separated by an extra helical turn with respect to the other paramyxoviruses.
  • HPIV3 Human parainfluenza virus type 3
  • Simian parainfluenza virus 5 VS5
  • RSV human respiratory syncytial virus
  • HeV Hendra virus
  • Nipah Virus Nipah Virus
  • composition of a hybrid peptide, active on multiple paramyxoviruses can be designed as outlined in Figures 2-5, indicating the identity of the residue at each position of the peptide.
  • the parent sequence is taken from the HR2 domain of HPIV3.
  • similar consensus can be derived by taking as parent sequence the
  • the peptide used in experiment no. 4 also includes the substitution GIn 31 GIy (i.e. the 31 st amino acid of the peptide is substituted as indicated). In contrast to the peptide used in experiment no. 4, the peptide used in experiment no.
  • the peptides used in experiments no. 9-10 combine the substitutions of the peptides used in experiments no. 7 and no. 8. (AIa 15 IIe, GIn 31 LyS, LyS 32 IIe, i.e. the 15 th , 31 st and 32 nd amino acid of the peptide is substituted as indicated).
  • the peptide used in experiment no. 10 has the same sequence as the peptide used in experiment no. 9, plus the spacer sequence Gly-Ser-Gly- Ser-Gly and a cysteine alkylated with iodoacetamide. It is the control for Peptide used in experiment no. 11, in which the cysteine is derivatized with cholesterol.

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Abstract

La présente invention concerne de nouveaux inhibiteurs de l'entrée de virus dans des cellules et leur utilisation pour la prophylaxie et le traitement de maladies virales.
PCT/EP2010/000723 2009-02-06 2010-02-05 Inhibiteurs de fusion virale et leurs utilisations WO2010089129A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130216613A1 (en) * 2010-10-15 2013-08-22 Guy Jean Marie Fernand Pierre Baudoux Cytomegalovirus gb antigen
CN104774161B (zh) * 2014-01-13 2017-08-25 成都福瑞康生物科技有限公司 多肽、蛋白质peg修饰剂合成方法
WO2019223644A1 (fr) * 2018-05-23 2019-11-28 中国人民解放军军事科学院军事医学研究院 Polypeptide, composition pharmaceutique et utilisation associées
WO2023159113A1 (fr) * 2022-02-16 2023-08-24 Greene Warner C Inhibiteurs de fusion de peptides présentant une activité inhibitrice de pan-coronavirus

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20130216613A1 (en) * 2010-10-15 2013-08-22 Guy Jean Marie Fernand Pierre Baudoux Cytomegalovirus gb antigen
CN104774161B (zh) * 2014-01-13 2017-08-25 成都福瑞康生物科技有限公司 多肽、蛋白质peg修饰剂合成方法
WO2019223644A1 (fr) * 2018-05-23 2019-11-28 中国人民解放军军事科学院军事医学研究院 Polypeptide, composition pharmaceutique et utilisation associées
WO2023159113A1 (fr) * 2022-02-16 2023-08-24 Greene Warner C Inhibiteurs de fusion de peptides présentant une activité inhibitrice de pan-coronavirus

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