Title:  Peptides that block viral infectivity and methods of use thereof

United States Patent:  6,258,932

Assignee:  Tripep AB (BE)

Filed:  August 9, 1999

The discovery of peptides in amide form that inhibit viral infection, including human immunodeficiency virus (HIV) infection is disclosed. Methods of use of peptides are also disclosed including use in medicaments for the treatment and prevention of viral infection, such as HIV infection.

SUMMARY OF THE INVENTION

The present invention is related to small peptides (two to ten amino acids in length) that inhibit viral infectivity. An intact capsid structure is of vital importance for the infectivity of a virion. A way to disrupt assembly of capsid protein macromolecules, that for their infectivity are dependent on di-, tri-, tetra-, or polymerization, is to construct small molecules that affect such protein-protein interactions. It was discovered that small peptides with their carboxyl terminus hydroxyl group replaced with an amide group have such an inhibiting effect on capsid-protein interactions. Thus, aspects of the present invention relate to modified small peptides that effect viral capsid assembly.

In desirable embodiments, the short peptides bind to a protein that is involved in capsomere organization and capsid assembly of HIV-1, HIV-2, and SIV and thereby inhibit and/or prevent proper capsid assembly and, thus, viral infection. The small peptides Gly-Pro-Gly-NH₂ (GPG-NH₂), Gly-Lys-Gly-NH₂ (GKG-NH₂), Cys-Gln-Gly-NH₂ (CQG-NH₂), Arg-Gln-Gly-NH₂ (RQG-NH₂), Lys-Gln-Gly-NH₂ (KQG-NH₂), Ala-Leu-Gly-NH₂ (ALG-NH₂), Gly-Val-Gly-NH₂ (GVG-NH₂), Val-Gly-Gly-NH₂ (VGG-NH₂), Ala-Ser-Gly-NH₂ (ASG-NH₂), Ser-Leu-Gly-NH₂ (SLG-NH₂), and Ser-Pro-Thr-NH₂ (SPT-NH₂) are the preferred species. These small peptides and peptidomimetics resembling their structure (collectively referred to as "peptide agents") are used in a monomeric or multimeric form. The peptide agents of the present invention are suitable for therapeutic and prophylactic application in mammals, including man, suffering from viral infection.

In one embodiment, a composition for inhibiting viral replication in host cells infected with a virus has an effective amount of a peptide in amide form having the formula X₁ X₂ X₃ -NH₂, wherein X₁, X₂, and X₃ are any amino acid and said peptide is not Gly-Pro-Gly-NH₂, and wherein said composition inhibits viral replication by interrupting viral capsid assembly. Desirably, X₃ of these peptides is glycine. Additionally, the compositions described above can include a peptide in amide form selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂.

In another related embodiment, the composition described above is a peptide in amide form that has the formula X₄ x 5 X₁ X₂ X₃ -NH₂, wherein X₄ and X₅ are any amino acid and any one or two amino acids can be absent. This embodiment can be a tripeptide having the formula X₁ X₂ X₃, wherein the sequence is found in the amino acid sequence of the capsid protein of the virus.

In some embodiments, the compositions described above are joined to a support and in other embodiments, the compositions described above are incorporated into a pharmaceutical having a pharmaceutically acceptable carrier. For example, the peptide in amide form can have the formula Gly-Lys-Gly-NH₂.and can be joined to a support. Further, the peptide in amide form can have a formula such as Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, or Ser-Leu-Gly-NH₂. Thes peptides can also be joined to a support.

Methods of inhibiting HIV replication in a host cell are also embodiments. One approach, for example, involves administering to a cell an effective amount of a peptide in amide form having the formula X₁ X₂ X₃ -NH₂, wherein X₁, X₂, and X₃ are any amino acid and said peptide is not Gly-Pro-Gly-NH₂. Accordingly, the peptide above can be selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂. The method described above can further include the step of administering an antiviral treatment selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. The peptide used in the method above can bejoined to a support or can be administered in a pharmaceutical comprising a pharmaceutically acceptable carrier.

In another embodiment, a composition for inhibiting HIV replication in host cells includes an effective amount of a peptide in amide form having the formula X₁ X₂ X₃ -NH₂, wherein X₁, X₂, and X₃ are any amino acid and said peptide is not Gly-Pro-Gly-NH₂ and wherein said composition inhibits HIV replication by interrupting assembly of the capsid. Desirably, X₃ is glycine in the peptides of this embodiment. Preferably, the peptide of this embodiment is selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂. Additionally, the peptide in amide form, described above, can have the formula X₄ X₅ X₁ X₂ X₃ -NH₂, wherein X₄ and X₅ are amino acids and wherein any one or two, amino acids is absent. These compositions can have a tripeptide X₁ X₂ X₃ that is found in the amino acid sequence of the capsid protein of HIV, for example. In some embodiments, these peptides are joined to a support and in other embodiments, these peptides are incorporated into a pharmaceutical comprising a pharmaceutically acceptable carrier.

In another method, an approach to inhibit viral replication in host cells is provided, which involves administering to said cells an effective amount of a peptide in amide form having the formula X₁ X₂ X₃ -NH₂, wherein X₁, X₂, and X₃ are any amino acid and said peptide is not Gly-Pro-Gly-NH₂. In this method, the peptide can be selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂. This method can also include the step of administering an antiviral treatment selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. Further, the peptide used in this method can be joined to a support or can be administered in a pharmaceutical comprising a pharmaceutically acceptable carrier.

In another method, an approach for interrupting viral capsid assembly is provided. This approach involves contacting a cell with an effective amount of a peptide in amide form having the formula X₁ X₂ X₃ -NH₂, wherein X₁, X₂, and X₃ are any amino acid and said peptide is not Gly-Pro-Gly-NH₂. Desirably, the peptide used in this method is selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂. In some embodiments, X₃ is glycine in the peptide used in this method. In other embodiments, the method employs a peptide in amide form having the formula X₄ X₅ X₁ X₂ X₃ -NH₂, wherein X₄ and X₅ are an amino acid and, wherein any one or two amino acids is absent. Still further, the method can involve the use of a tripeptide X₁ X₂ X₃ that is found in the amino acid sequence of a protein of the virus. Oftentimes the peptide of the method is joined to a support or is incorporated in a pharmaceutical.

Methods of identification of peptide agents are also provided. By one method, for example, a peptide agent for incorporation into a anti-viral pharmaceutical is identified by contacting a plurality of cells infected with a virus with an effective amount of a peptide agent, wherein said peptide is not Gly-Pro-Gly-NH₂, analyzing the virus for incomplete capsid formation, and selecting the peptide agent that induces incomplete capsid formation. This method can involve an analysis of capsid formation that employs microscopy and the virus can be selected from the group consisting of HIV-1, HIV-2, and SIV. Further, the peptide agent identified can be selected from the group consisting of a tripeptide, an oligopeptide, and a peptidomimetic. For example, the peptide agent above can be selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂. In a preferred embodiment, the peptide agent used in the method above has an amino acid sequence that corresponds to an amino acid sequence of p24.

In another embodiment, a method for identifying a peptide agent that binds to a viral protein is provided, which involves providing a viral protein, contacting the viral protein with an effective amount of a peptide agent, wherein said peptide agent is not Gly-Pro-Gly-NH₂, and detecting the formation of a complex comprising the viral protein and the peptide agent. As above, this method can involve the use of a viral protein that is from a virus selected from the group consisting of HIV-1, HIV-2, and SIV. Further, in some aspects, the peptide agent is selected from the group consisting of a tripeptide, an oligopeptide, and a peptidomimetic. Desirably, the method above employs a peptide agent is selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂. Additionally, a method of making a pharmaceutical is provided in which the peptide agent identified by the methods above are incorporated in a pharmaceutical.

Another approach to make a pharmaceutical is also provided, which involves administering to a cell an effective amount of a peptide in amide form having the formula X₁ X₂ X₃ -NH₂, wherein X₁, X₂, and X₃ are any amino acid and said peptide is not Gly-Pro-Gly-NH₂, detecting an inhibition of viral replication in the cell, and incorporating the peptide that causes inhibition of viral replication into the pharmaceutical. This method can involve the use of a peptide that is selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂. Further, the method above can involve the step of incorporating an antiviral compound selected from the group consisting of nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors into the pharmaceutical. Additionally, the method above can involve the step of incorporating a carrier into the pharmaceutical.

In another embodiment, a composition for inhibiting viral replication in host cells infected with a virus includes an effective amount of a peptide having the formula X₁ X₂ X₃ -R, wherein X₁, X₂, and X₃ are any amino acid and said peptide is not Gly-Pro-Gly-NH₂, wherein R is a modulation group attached to the carboxy-terminus of said peptide and R comprises an amide group or other moiety having similar charge and steric bulk and wherein said composition inhibits viral replication by interrupting viral capsid assembly. This composition can be a peptide selected from the group consisting of peptides having the formula Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂. Desirably, X₃ is glycine in these embodiments.

Additionally, the composition above can include a peptide that has the formula X₄ X₅ X₆ X₇ X₈ X₉ X₁0 X₁ X₂ X₃ -R, wherein X₄, X₅, X₆, X₇, X₈, X₉, and X₁0 are any amino acid and wherein any one, two, three, four, five, six, or seven amino acids is absent, wherein R is a modulation group attached to the carboxy-terminus of said peptide and R comprises an amide group or other moiety having similar charge and steric bulk. Preferably, the composition above includes a peptide X₁ X₂ X₃ that is found in the amino acid sequence of the capsid protein of the virus.

Claim 1 of 33 Claims

What is claimed is:

1. A peptide selected from the group consisting of Gly-Lys-Gly-NH₂, Arg-Gln-Gly-NH₂, Cys-Gln-Gly-NH₂, Lys-Gln-Gly-NH₂, Ala-Leu-Gly-NH₂, Gly-Val-Gly-NH₂, Val-Gly-Gly-NH₂, Ala-Ser-Gly-NH₂, Ser-Leu-Gly-NH₂, and Ser-Pro-Thr-NH₂.

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