METHOD TO BLOCK THE INFECTION BY FLAVIVIRUSES, MOLECULES AND USES

• US 20110212105A1
• Filed: 04/26/2007
• Published: 09/01/2011
• Est. Priority Date: 04/28/2006
• Status: Active Grant

• Current Assignee: Centro De Ingenieria Genetica Y Biotecnologia
• Original Assignee: Centro De Ingenieria Genetica Y Biotecnologia
• Inventors: Guirola Cruz, Osmany, Fleitas Salazar, Noralvis, Chinea Santiago, Glay, Cabrales Rico, Ania, Sarría Núñez, Mónica, González López, Luis Javier, Padrón Palomares, Gabriel Ramón, Besada Pérez, Vladimir Armando, Toledo Mayora, Patricia Gabriela, Garay Pérez, Hilda Elisa, Martín Dunn, Alejandro Miguel, Sánchez Puente, Aniel, Musacchio Lasa, Alexis, Huerta Galindo, Vivian, Reyes Acosta, Osvaldo
• Granted Patent: US 8,420,311 B2
• Time in Patent Office: Days
• US Class Current: 424/159.1
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/70596 Molecules with a "CD"-designation not provided for elsewhere
• CPC Class Codes: C07K 14/8107 Endopeptidase (E.C. 3.4.21-99) inhibitors
• CPC Class Codes: C12N 2770/24122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: Y02A 50/30 Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The present invention is related to a method for blocking the infection of cells by dengue virus, based on interfering the direct interaction of the viral envelope protein with a cellular receptor or its indirect interaction with said cellular receptor through a carrier protein, as well as related uses; wherein said cellular receptor is the alpha-2 macroglobulin receptor, also known as the low density receptor-related protein or as CD91, and said carrier protein is human alpha-2 macroglobulin.

Claims

1. A method for blocking the infection by flaviviruses, comprising the interference of the interaction of the viral envelope protein with the alpha-2 macroglobulin receptor identified in the sequence listing as Seq. ID. 3, or with the alpha-2 macroglobulin identified in the sequence listing as Seq. ID. 2.

2. A method according to claim 1 wherein the flavivirus is the dengue virus, the Yellow Fever Virus, the Japanese Encephalitis virus, the Tick-Borne Encephalitis virus, the Murray Valley Encephalitis virus, the West Nile Virus, the Kunjin virus, the Powasan virus, the Langat virus or the Saint Louis Encephalitis virus.

3. A method according to claim 1 wherein the sequence of the viral protein is at least 60% homologous to the sequence identified in the sequence listing as Seq. ID. 1.

4. A method according to claim 1 wherein a fragment of the sequence of the receptor for the virus is any of the fragments located between aminoacid residues 25 to 66, or 70 to 110, or 852 to 892, or 893 to 933, or 934 to 973, or 974 to 1013, or 1014 to 1053, or 1060 to 1099, or 1102 to 1142, or 1143 to 1182, or 2522 to 2563, or 2564 to 2602, or 2603 to 2641, or 2642 to 2690, or 2694 to 2732, or 2733 to 2771, or 2772 to 2814, or 2816 to 2855, or 2856 to 2899, or 2902 to 2940, or 3332 to 3371, or 3372 to 3410, or 3411 to 3450, or 3451 to 3491, or 3492 to 3533, or 3534 to 3572, or 3573 to 3611, or 3612 to 3649, or 3652 to 3692, or 3693 to 3733, or 3739 to 3778, of the sequence identified in the sequence listing as Seq. ID. 3.

5. A method according to claim 1 wherein a fragment of the sequence of the receptor for the virus is at least 60% homologous to any of the fragments located between aminoacid residues 25 to 66, or 70 to 110, or 852 to 892, or 893 to 933, or 934 to 973, or 974 to 1013, or 1014 to 1053, or 1060 to 1099, or 1102 to 1142, or 1143 to 1182, or 2522 to 2563, or 2564 to 2602, or 2603 to 2641, or 2642 to 2690, or 2694 to 2732, or 2733 to 2771, or 2772 to 2814, or 2816 to 2855, or 2856 to 2899, or 2902 to 2940, or 3332 to 3371, or 3372 to 3410, or 3411 to 3450, or 3451 to 3491, or 3492 to 3533, or 3534 to 3572, or 3573 to 3611, or 3612 to 3649, or 3652 to 3692, or 3693 to 3733, or 3739 or 3778, of the sequence identified in the sequence listing as Seq. ID. 3.

6. A method according to claim 1 wherein the sequence of the protein functioning as a carrier for the virus is at least 60% homologous to that identified in the sequence listing as Seq. ID. 2.

7. A method according to claim 1 wherein the agent that interferes the interaction of the viral envelope protein with the protein identified in the sequence listing as Seq. ID. 3 is an antibody, or a competitive ligand of the receptor, or the sequence identified in the sequence listing as Seq. ID. 26.

8. A method according to claim 1 wherein the agent that interferes the interaction of the viral envelope protein with the protein identified in the sequence listing as Seq. ID. 3 is obtained by chemical synthesis, or by recombinant DNA techniques, or from a natural source.

9. A method according to claim 1 wherein the agent that interferes the interaction of the virus with the receptor is any of the peptides identified in the sequence listing as Seq. IDs. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

10. A method according to claim 1 wherein the agent that interferes the viral interaction is a chemical modification of any of the peptides identified in the sequence listing as Seq. IDs. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18.

11. A method according to claim 10 wherein said peptide has a chemical structure described by the formula:

Nt-C1-F2-F3-F4-F5-F6-T7-T8-T9-T10-T11-T12-G13-G14-G15-G16-G17-C18-Ct where,

Nt;

is an optional N-terminal extension, formed by a chemical group covalently bonded to the N-terminal group, such as an acetylation, or a methylation, or any acylation, or a peptide, or polyethylene glycol.

C1;

Cysteine (forms a disulphide bridge with another Cysteine in C18), Lysine (Glutamic Acid/Aspartic Acid) covalently joined by an amide bond to the side chain of Glutamic Acid/Aspartic Acid (Lysine) at position C18.

F2;

Serine, Asparagine, Valine, Isoleucine, Threonine, Phenylalanine, Tyrosine.

F3;

Tyrosine, Asparagine, Isoleucine.

F4;

Isoleucine, Valine, Threonine, Phenylalanine, Tyrosine.

F5;

Valine, Isoleucine, Tyrosine, Threonine, Phenylalanine.

F6;

Isoleucine, Valine, Threonine, Phenylalanine, Tyrosine.

T7;

Glycine.

T8;

Valine, Alanine, Isoleucine, Serine, Threonine, Arginine.

T9;

Glycine, Glutamic Acid.

T10;

Glutamic Acid, Aspartic Acid, Threonine, Asparagine, Proline.

T11;

Lysine, Arginine, Asparagine, Glycine, Serine, Threonine, Glutamine.

T12;

Glutamine, Alanine, Arginine.

T13;

Isoleucine, Leucine, Valine, Phenylalanine, Threonine.

T14;

Lysine, Arginine, Asparagine, Threonine.

T15;

Leucine, Isoleucine, Valine, Phenylalanine, Tyrosine, Threonine, Histidine.

T16;

Asparagine, Aspartic Acid, Serine, Histidine, Glutamine.

T17;

Tryptophan, Phenylalanine, Tyrosine.

C18;

Cysteine (forms a disulphide bridge with another Cysteine at C1), Lysine (Glutamic Acid/Aspartic Acid) covalently joined by an amide bond to the side chain of Glutamic Acid/Aspartic Acid (Lysine) at position C1.

Ct;

is an optional C-terminal extension, a chemical group covalently bonded to the terminal carbonyl group, acetylation, methylation, peptide, polyethylene glycol, acylation.

12. A method according to claim 1 wherein the agent that blocks the infection interferes the interaction of the protein identified in the sequence listing as Seq. ID. 1 with one or more of the residues defined as a ligand binding patch of the protein identified in the sequence listing as Seq. ID. 3.

13. A method according to claim 1 wherein the agent interfering the viral interaction is the active principle of a pharmaceutical composition.

14. A method according to claim 1 wherein the interference of the interaction of the virus with the receptor comprises blocking the expression of the protein identified in the sequence listing as Seq. ID. 3.

15. A method according to claim 14 wherein the expression of the protein identified in the sequence listing as Seq. ID. 3 is blocked by RNA interference.

17. A method according to claim 1 wherein the agent interfering the interaction of the virus with the receptor is obtained by:

a) the incubation of the flavivirus receptor of sequence Seq. ID. 3 together with a preparation containing the viral envelope protein of sequence Seq. ID. 1 and the blocking compound;

b) the quantification of the viral envelope protein bound to the receptor; and

c) the comparison to the amount of receptor-bound viral envelope protein in the absence of the blocking compound.

18. A method according to claim 1 wherein the agent that interferes the interaction of the virus with the receptor is obtained by:

a) The use of the atomic coordinates of a model of the three-dimensional structure of a peptide having a sequence identified as Seq. IDs. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18 or a chemically modified version thereof, in a conformation according to the structure of an antiparallel beta hairpin that includes a beta turn in the connecting loop between the beta strands;

b) The use of the atomic coordinates of a model of the three-dimensional structure of one of the ligand binding domains of the sequence identified in the sequence listing as Seq. ID. 3, which has been determined experimentally or modeled by computational means;

c) A computational procedure of molecular docking that allows the reproduction of the atomic details of the interaction of the structures described in subsection a) with the structures described in subsection b);

or

d) The use of the procedure of computational docking described in subsection c) for the selection from a database of molecular structures of those compounds that reproduce the characteristics of the interaction described in subsection c), as blocking agents for the infection of the virus.

19. A method according to claim 1 wherein the agent that interferes the interaction of the viral envelope protein with the protein identified in the sequence listing as Seq. ID. 2 is an antibody, or a ligand for alpha-2 macroglobulin, or a peptide.

20. A method according to claim 1 wherein the agent that interferes the interaction of the viral envelope protein with the protein identified in the sequence listing as Seq. ID. 2 is obtained by chemical synthesis, or by recombinant DNA techniques, or from a natural source.

22. The use of the agents interfering the viral interaction according to claim 19 for the determination of the susceptibility of an organism to the infection by dengue virus.

16. A method for determining the susceptibility of a cell and/or an organism to the infection by dengue virus comprising interfering with an interaction of a viral envelope protein with alpha-2 macroglobulin receptor identified in the sequence listing as Seq. ID. 3, or with alpha-2 macroglobulin identified in the sequence listing as Seq. ID. 2.

21. (canceled)

23. Peptides identified in the sequence listing as Seq. IDs. 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 and 18, wherein said peptides are the agents that interfere the interaction of the virus with the cellular receptor.

24. Peptides according to claim 23, wherein said peptides are the agents that interfere the interaction of the virus with the cellular receptor identified in the sequence listing as Seq. ID. 3.

25. Peptides according to claim 23, wherein said peptides are the active principle of a pharmaceutical composition with antiviral properties.