Embodiments relate to the discovery that certain tripeptide amides and glycine amide can be used to inhibit viral infection, including human immunodeficiency virus (HIV) infection. More specifically, medicaments comprising said tripeptide amides and/or glycine amide and methods of using said compounds for the prevention and treatment of viral infection, such as HIV infection, are provided.

SUMMARY OF THE INVENTION

The present invention is related to molecules that inhibit viral infectivity, specifically replication of Human Imnmunodeficiency Virus (HIV). It was discovered that certain tripeptides and the amino acid glycine, with their carboxyl terminus hydroxyl group replaced with an amide group, have an inhibiting effect on the replication of viruses, such as HIV. It is contemplated that these molecules inhibit viral replication by affecting protein-protein interactions during capsid assembly and/or by interfering with virus budding.

In addition to glycine amide (G-NH₂), the tripeptide amides AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂ are the preferred species. These molecules and peptidomimetics resembling their structure (collectively referred to as "peptide agents") are used in a monomeric or multimeric form. Glycine amide and the tripeptide amides (i.e., peptide agents) are suitable for therapeutic and prophylactic application in mammals, including man, suffering from viral infection. Glycine amide or any one of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂ can be administered individually or the molecules can be provided in any combination (e.g., glycine amide can be provide with GLG-NH₂ or APG-NH₂ can be provided with GFG-NH₂ etc.)

In one embodiment, a composition for inhibiting viral replication in host cells infected with a virus has an effective amount of glycine amide and/or a peptide in amide form selected from the group of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. 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.

Methods of inhibiting viral replication in a host cell are also embodiments of the present invention. One approach, for example, involves administering to a cell an effective amount of glycine amide and/or a peptide in amide form selected from the group consisting of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. The method described above can be supplemented with 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 glycine amide and/or the tripeptide amide used in the method above can be joined 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 glycine amide and/or a peptide in amide form selected from the group consisting of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. In some embodiments, the glycine amide or the tripeptide amides are joined to a support and in other embodiments, these molecules are incorporated into a pharmaceutical comprising a pharmaceutically acceptable carrier.

In another method, an approach to inhibit HIV replication in host cells is provided, which involves administering to said cells an effective amount of glycine amide and/or a peptide in amide form selected from the group consisting of peptides of the formula AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. This method can also be supplemented by 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 glycine amide and/or tripeptide amide 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 glycine amide and/or a peptide in amide form selected from the group consisting of peptides of the formula AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. The glycine amide and/or the tripeptide amide can be joined to a support or incorporated in a pharmaceutical.

In another method, an approach for inhibiting proper viral budding is provided. This approach involves contacting a cell with an effective amount of glycine amide and/or a peptide in amide form selected from the group consisting of peptides of the formula AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. The glycine amide and/or the tripeptide amide can be joined to a support or incorporated in a pharmaceutical.

In still another method, an approach for interrupting HIV capsid assembly is provided. This approach also involves contacting a cell with an effective amount of glycine amide and/or a peptide in amide form selected from the group consisting of peptides of the formula AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. The glycine amide and/or the tripeptide amide of this method can be joined to a support or incorporated in a pharmaceutical.

In still another method, an approach for inhibiting proper HIV budding is provided. This approach also involves contacting a cell with an effective amount of glycine amide and/or a peptide in amide form selected from the group consisting of peptides of the formula AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. The glycine amide and/or the tripeptide amide of this method can be joined to a support or incorporated in a pharmaceutical.

Methods of identification of peptide agents that inhibit viral replication, specifically HIV replication are also provided. By one method, for example, a peptide agent for incorporation into an anti-viral pharmaceutical is identified by contacting a plurality of cells infected with a virus with an effective amount of a peptide agent, analyzing the virus for incomplete capsid formation or impaired viral budding, and selecting the peptide agent that induces incomplete capsid formation or induces impaired viral budding. This method can involve an analysis of capsid formation or viral budding that employs microscopy (e.g., electron microscopy) and the virus can be selected from the group consisting of HIV-1, HIV-2, and SWV. Further, the peptide agent identified can be selected from the group consisting of glycine amide, a tripeptide amide, and a peptidomimetic resembling glycine amide or a tripeptide amide. For example, the peptide agent above can be selected from the group consisting of glycine amide, AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂.

In another embodiment, a method of identifying a peptide agent that binds to a viral protein is provided. Some aspects of this method involve providing a viral protein, contacting the viral protein with an effective amount of a peptide agent, and detecting the formation of a complex comprising the viral protein and the peptide agent. Some methods use a viral protein that is from a virus selected from the group consisting of HIV-1, HIV-2, and SIV. Further, in some embodiments, the peptide agent is selected from the group consisting of glycine amide, a tripeptide amide and a peptidomimetic resembling glycine amide or a tripeptide amide. Desirably, the method above employs glycine amide and/or a peptide agent selected from the group consisting of AIG-NH₂, GFG-NF₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-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 making a pharmaceutical involves administering to a cell, especially a cell present in an animal such as a human, an effective amount of glycine amide or a peptide in amide form, described above, detecting an inhibition of viral replication in the cell, and incorporating the molecule that causes inhibition of viral replication into the pharmaceutical. This method can involve the use of glycine amide and/or a tripeptide amide selected from the group consisting of AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. Further, this method can be supplemented with administration of 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 be supplemented by incorporating a carrier into the pharmaceutical.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

It has been discovered that glycine amide and certain tripeptide amides prevent and/or inhibit viral infection. Such amino acid or peptides are useful in the treatment of viral disease, particularly in HIV/AIDS afflicted subjects, and as preventive agents for patients at-risk of viral infection, particularly HIV infection, and for use with medical devices where the risk of exposure to virus is significant.

The disclosure below demonstrates that glycine amide and certain tripeptides in amide form, such as AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂ inhibit the replication of viruses, for example HIV-1. Evidence of the inhibition of viral replication was found in viral infectivity assays that monitor the amount of capsid protein present in culture supernatant.

Several approaches to making biotechnological tools and pharmaceutical compositions comprising glycine amide and/or tripeptide amides and peptidomimetics that resemble these molecules (collectively referred to as "peptide agents") are given below. Preferred peptide agents are glycine and tripeptides with an amide group at their carboxy termini, and include the following: G-NH₂, AIG-NH₂, GFG-NH₂, GWG-NH₂, FLG-NH₂, GYG-NH₂, APG-NH₂, GLG-NH₂, and α-t-butylglycine-PG-NH₂. In some embodiments, the peptide agents are provided in monomeric form; in others, the peptide agents are provided in multimeric form or in multimerized form. Support-bound peptide agents are also used in several embodiments.

Pharmaceutical compositions comprising peptide agents are administered as therapeutics or prophylactics or both for the treatment and/or prevention of viral disease, particularly, HIV infection. In some embodiments, the pharmaceutical compositions comprising peptide agents are administered in combination with other antiviral treatments including nucleoside analogue reverse transcriptase inhibitors, nucleotide analogue reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and protease inhibitors. These small molecules are resistant to acid hydrolysis. A significant amount of tripeptide amides, for example, is effectively delivered to blood, plasma, and organ tissue when administered to test subjects. The administration of large doses of small peptides to test subjects is relatively nontoxic. (See U.S. Pat. No. 6,258,932, which is herein expressly incorporated by reference in its entirety).

Additionally, several methods of identifying a peptide agent that inhibits or prevents viral replication or interrupts viral capsid assembly or both are provided. By one approach, an effective amount of a peptide agent is contacted with cells infected with a virus and the cells are analyzed for viral replication or the presence of viral products. Accordingly, a capsid protein (e.g., p24) is contacted with a peptide agent, for example a peptide in amide form, as described above, and a complex comprising the capsid protein (e.g., p24) bound with the peptide agent is identified.

The amide form of the molecules listed in TABLE 1 were tested. Many of these molecules were selected and synthesized because they are modifications of sequences that correspond to HIV and/or SIV viral proteins. The tripeptide amides of TABLE 1 were synthesized according to the method disclosed in EXAMPLE 1 below, but could of course be synthesized by any method known in the art. Glycine amide was purchased from Bachem, Switzerland (product No. 4025766), whereas Glycine-OH was purchased from Merck, Germany (product No. 14201-250). GPG-NH₂ was also purchased from Isochem, France.

Claim 1 of 19 Claims

1. An antiviral composition for human administration comprising monopeptidic glycine amide and a compound that inhibits replication of HIV in the presence of monopeptidic glycine amide.

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.