Inhibitors of G protein-mediated signaling, methods of...

Details Inhibitors of G protein-mediated signaling, methods of... Inhibitors of G protein-mediated signaling, methods of...

2000-01-21

2003-05-06

Carlson, Karen Cochrane (Department: 1653)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Carbohydrate doai

C514S002600, C514S013800, C514S014800, C536S023500, C536S023100, C530S300000, C530S326000, C530S327000

Reexamination Certificate

active

06559128

ABSTRACT:

BACKGROUND OF THE INVENTION
Many biologically active molecules transduce their signals through heptahelical receptors called G protein coupled receptors (GPCRs), which interact specifically with heterotrimeric guanine nucleotide-binding proteins, called G proteins. The interaction of a G protein with its cognate GPCR results in the initiation of intracellular signaling events which, in turn, lead to a variety of important cellular responses (Hamm et al., 1996, Curr. Opin. Cell. Biol. 8: 189-196; Hamm, 1998, Science 273: 669-672). Stimulation of a GPCR by its appropriate agonist causes conformational changes within the receptor that lead to the interaction of the activated receptor with a specific heterotrimeric G protein. Thus, the GPCR-G protein interaction plays an important role in determining the specificity and temporal characteristics of a variety of cellular responses.
Heterotrimeric G proteins are composed of a single &agr; subunit complexed with the &bgr;&ggr; dimer. Molecular cloning has resulted in the identification of 18 distinct &agr; subunits, 5 &bgr; subunits, and 12 &ggr; subunits. G proteins are usually divided into four subfamilies G
i
, G
s
, G
q
, and G
12
based on the sequence similarity of the G&agr; subunit. Several lines of evidence suggest that the interaction between a given GPCR and its cognate G protein involves multiple sites of contact on both proteins. All three intracellular loops as well as the carboxyl terminal tail of the receptor have been implicated. The GPCR is thought to interact with all three subunits of the G protein. As the receptor-G protein interaction can be disrupted by a number of treatments that block the carboxyl terminus, including pertussis toxin-catalyzed ADP-ribosylation of G
i
and binding of monoclonal antibodies, the carboxy terminal region of the G&agr; subunit has been the most intensely investigated contact site. These studies have shown that the G&agr; carboxy terminal region is important not only to the interaction, but also plays a critical role in defining receptor specificity (Hamm et al., 1988, Science 241: 832-5; Osawa et al., 1995, J. Biol. Chem. 270: 31052-8; Garcia et al., 1995, EMBO 14: 4460-9; Sullivan et al., 1987, Nature 330: 758-760; Rasenick et al., 1994, J. Biol. Chem. 269: 21519-21525; West et al., 1985, J. Biol. Chem. 260: 14428-30; Conklin et al., 1993, Nature 363: 274-276; Conklin et al., 1996, Mol. Pharmacol. 50: 885-890). Furthermore, it has been shown that peptides corresponding to the carboxy terminal region of a G&agr; subunit can block GPCR signaling events (Hamm et al., 1988, Science 241: 832-5; Gilchrist et al., 1998, J. Biol. Chem 273: 14912-19).
Because many medically significant biological processes are mediated by G proteins and their downstream effector molecules, the GPCRs with which they interact, have been the focus of intense drug discovery efforts (Holler et al:, 1999, Cell. Mol. Life Sci. 55: 257-70; Farfel et al., 1999, New Engl. J. Med. 340: 1012-20). A number of therapeutic agents targeting GPCRs have been discovered. Traditionally, the agonist binding site on the GPCR is the point of intervention. However, for some GPCR's, classical antagonists have been difficult to identify. Such is the case for proteinase activated receptors (PAR), classical antagonists are ineffective due to the unique mechanism of enzymatic cleavage of the receptor and generation of a tethered ligand. In other cases, intrinsic or constitutive activity of receptors directly leads to pathology. Thus, alternative targets for blocking downstream consequences of GPCR signaling are needed. Agents are needed that can inhibit GPCR by blocking the receptor-G protein interface. The present invention satisfies these needs.
BRIEF SUMMARY OF THE INVENTION
The present invention includes an isolated nucleic acid comprising a minigene, wherein said minigene encodes a modified carboxy terminal G&agr; peptide, wherein said peptide blocks the site of interaction between a G protein and a G protein coupled receptor in a cell, such as a human cell. In addition, the minigene can further comprise one or more of a promoter, a ribosomal binding site, a translation initiation codon, and a translation termination codon.
In one embodiment, the nucleotide sequence of a minigene of the invention can be one of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, and 14.
In another embodiment, a minigene encodes a modified carboxy terminal G&agr; peptide having one of the following general formulas: MGX, MX, and MZX,
wherein M is a methionine amino acid residue,
wherein G is a glycine amino acid residue,
wherein Z is an amino acid residue other than a glycine amino acid residue, and
wherein X is a carboxy terminal G&agr; peptide which comprises an amino acid sequence of the carboxy terminus of a G&agr; subunit, and has the property of binding a G protein coupled receptor. In this embodiment, X can comprise from at least about three contiguous amino acids to at least about 54 contiguous amino acids, from at least about three contiguous amino acids to at least about eleven contiguous amino acids, and at least about eleven contiguous amino acids. In one embodiment, X comprises the seven contiguous terminal amino acid residues of the carboxy terminus of a G&agr; subunit. For example, the amino acid sequence of a modified carboxy terminal G&agr; peptide can be one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29.
The present invention also includes a composition comprising a modified carboxy terminal G&agr; peptide having a general formula selected from the group consisting of MGX, MX, and MZX,
wherein M is a methionine amino acid residue,
wherein G is a glycine amino acid residue,
wherein Z is an amino acid residue other than a glycine amino acid residue, and
wherein X is a carboxy terminal G&agr; peptide which comprises an amino acid sequence of the carboxy terminus of a G&agr; subunit, and has the property of binding a G protein coupled receptor. In this embodiment, X can comprise from at least about three contiguous amino acids to at least about 54 contiguous amino acids, from at least about three contiguous amino acids to at least about eleven contiguous amino acids, and at least about eleven contiguous amino acids. In one embodiment, X comprises the seven contiguous terminal amino acid residues of the carboxy terminus of a G&agr; subunit. For example, the amino acid sequence of a modified carboxy terminal G&agr; peptide can be one of SEQ ID NOs: 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, and 29.
Additionally, the invention includes a pharmaceutical composition comprising a pharmaceutically acceptable carrier and one of a modified carboxy terminal G&agr; peptide and a minigene encoding a modified carboxy terminal G&agr; peptide.
In one aspect, the invention encompasses a method of inhibiting a G protein-mediated signaling event in a cell. This method comprises administering to a cell, preferably a human cell, one of a modified carboxy terminal G&agr; peptide, and an isolated nucleic acid comprising a minigene which encodes a modified carboxy terminal G&agr; peptide, whereby following the administration, the carboxy terminal G&agr; peptide inhibits the G protein mediated signaling event in the cell.
Additionally, the invention includes a method of blocking the site of interaction between a G protein and a G protein coupled receptor in a cell. This method comprises administering to a cell, preferably a human cell, one of a modified carboxy terminal G&agr; peptide, and an isolated nucleic acid comprising a minigene which encodes a modified carboxy terminal G&agr; peptide, whereby following the administration, the modified carboxy terminal G&agr; peptide blocks the site of interaction between the G protein and the G protein coupled receptor in the cell.
Further, the invention includes a method of inhibiting one or more of migration, permeability, and proliferation of a cell. This method comprises administering to a cell, preferably a human cell, one of a modified carboxy terminal G&agr; pep

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