GRB2 SH3 binding peptides and methods of isolating and using...

Details GRB2 SH3 binding peptides and methods of isolating and using... GRB2 SH3 binding peptides and methods of isolating and using...

1996-02-16

2001-02-06

MacMillan, Keith D. (Department: 1627)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Peptide containing doai

C514S012200, C514S014800, C514S015800, C530S324000, C530S325000, C530S326000, C530S327000, C530S328000

Reexamination Certificate

active

06184205

ABSTRACT:

1. FIELD OF THE INVENTION
The present invention relates to SH3 binding peptides having a broad range of binding specificities. That is, certain members of the SH3 binding peptides disclosed bind with approximately the same facility with SH3 domains derived from different SH3 domain-containing proteins. Other members, in contrast, bind with a much greater degree of affinity for specific SH3 domains. The SH3 binding peptides are obtained from random peptide libraries that are also phage-displayed. Methods are described of obtaining the phage clones that bind to the SH3 domain targets and of determining their relevant nucleotide sequences and consequent primary amino acid sequence of the binding peptides. The resulting SH3 binding proteins are useful in a number of ways, including, but not limited to, providing a method of modulating signal transduction pathways at the cellular level, of modulating oncogenic protein activity or of providing lead compounds for development of drugs with the ability to modulate broad classes, as well as specific classes, of proteins involved in signal transduction.
2. BACKGROUND OF THE INVENTION
2.1. Src and the SH3 Domain
Among a number of proteins involved in eukaryotic cell signaling, there is a common sequence motif called the SH3 domain. It is 50-70 amino acids in length, moderately conserved in primary structure, and can be present from one to several times in a large number of proteins involved in signal transduction and in cytoskeletal proteins.
The protein pp60c-src represents a family of at least nine non-receptor protein tyrosine kinases (NR-PTKs). Members of this family share an overall structural organization comprising a series of catalytic and non-catalytic domains. In Src, a 14-amino-acid myristylation signal resides at the extreme amino-terminus, and is followed by a unique region that is not highly conserved among family members. Following this region are two highly conserved 60- and 100-amino-acid regions, the Src homology (SH) domains 3 and 2, respectively. SH2 and SH3 domains have been shown to play an important role in mediating protein-protein interactions in a variety of signaling pathways. Koch, C. A., et al., in
Science
(1991) 252:668-74. The carboxy-terminal half of Src contains the PTK catalytic domain, as well as a negative regulatory tyrosine (Y527) near the carboxy terminus. Phosphorylation of this residue (e.g., by Csk) results in the inhibition of PTK activity. Cooper, J. A., et al., in
Science
(1986) 231:1431-1434. Mutation of Y527→F generates forms of Src with increased PTK and oncogenic activity. Cartwright, C. A., et al., in
Cell
(1987) 49:83-91; Kmiecik, T. E., et al., in
Cell
(1987) 49:65-73; and Piwicna-Worms, H., et al., in
Cell
(1987) 75-82.
The fact that some mutations which result in increased Src PTK and transforming activity map to the Src SH2 (Seidel-Dugan, C., et al., in
Mol. Cell. Biol
. (1992) 12:1835-45; and Hirai, H. and Varmus, H. E. in
Mol. Cell. Biol
. (1990) 10:1307-1318) and SH3 domains (Seidel-Dugan, C., et al., supra; Hirai, H. and Varmus, H. E., supra; Superti-Furga, G., et al., in
Embo. J
. (1993) 12:2625-34; and Potts, W. M., et al., in
Oncogene Res
. (1988) 3:343-355) suggests a negative regulatory role for these domains. That phosphotyrosine residues within specific sequence contexts represent high affinity ligands for SH2 domains suggests a model in which the SH2 domain participates in Y527-mediated inhibition of PTK activity by binding phosphorylated Y527, thereby locking the kinase domain in an inactive configuration. Matsuda, M., Mayer, B. J., et al., in
Science
(1990) 248:1537-1539. This model is supported by the observation that phosphopeptides corresponding to the carboxy-terminal tail of Src bind active, but not inactive, variants of Src. Roussel, R. R., et al., in
Proc. Natl. Acad. Sci. U S A
(1991) 88:10696-700; and Liu, X., et al., in
Oncogene
(1993) 8:1119-1126.
The mechanism of SH3-mediated inhibition of Src PTK activity remains unclear. There is evidence that pY527-mediated inhibition of Src PTK activity involves the SH3 domain as well as the SH2 domain. Okada, M., Howell, et al., in
J. Biol. Chem
. (1993) 268:18070-5; Murphy, S. M., et al., in
Mol. Cell. Biol
. (1993) 13:5290-300; and Superti-Furga, G., et al., supra. Although these effects are thought to be a consequence of SH3-mediated protein-protein interactions, precisely how the Src SH3 domain exerts its negative regulatory effect is unclear. Identification of high affinity ligands for the Src SH3 domain could help resolve these issues.
2.2. Protein Tyrosine Kinases and the Immune Response
Src-related tyrosine kinases are expressed in a variety of cell types including those of the immune system (lymphocytes, T cells, B cells, and natural killer cells) and the central nervous system (neural cells, neurons, oligodendrocytes, parts of the cerebellum, and the like). Umemori, H. et al., in
Brain Res. Mol. Brain Res
. (1992) Dec. 16(3-4):303-310. Their presence in these cells and tissues and their interaction with specific cell surface receptors and immunomodulatory proteins (such as T cell antigen receptor, CD14, CD2, CD4, CD40 or CD45) suggest that these kinases serve an important role in the signalling pathways of not only the central nervous system but of the immune system, as well. See, e.g., Ren, C. L. et al., in
J. Exp. Med
. (1994) 179(2):673-680 (signal transduction via CD40 involves activation of Lyn kinase); Donovan, J. A. and Koretzky, G. A., in
J. Am. Soc. Nephrol
. (1993) 4(4):976-985 (CD45, the immune response, and regulation of Lck and Fyn kinases); and Carmo, A. M. et al., in
Eur. J. Immunol
. (1993) 23(9):2196-2201 (physical association of the cytoplasmic domain of CD2 with p56lck and p59fyn).
For instance, mice with disruptions in their Src-like genes, Hck and Fgr, possess macrophages with impaired phagocytic activity or exhibit a novel immunodeficiency characterized by an increased susceptibility to infection with Listeria monocytogenes. Lowell, C. A. et al., in
Genes Dev
. (1994) 8(4):387-398. Also, it has been shown that bacterial lipopolysaccharide (LPS) activates CD14-associated p56lyn, p68hck, and p59c-fgr, while inducing the production of lymphokines, such as TNF-alpha, IL-1, IL-6, and IL-8. Inhibition of the protein tyrosine kinases blocks production of TNF-alpha and IL-1.
2.3. SH3 Binding Peptides
As mentioned above, it has long been suspected that SH3 domains are sites of protein-protein interaction, but it has been unclear what SH3 domains actually bind. Efforts to identify ligands for SH3 domains have led to the characterization of a number of SH3-binding proteins, including 3BP1 and 2 (Ren, R., Mayer, et al., in
Science
(1993) 259:1157-61), SOS (Olivier, J. P., et al., in
Cell
(1993) 73:179-91; and Rozakis-Adcock, M., et al., in
Nature
(1993) 363:83-5), p85 PI-3′ Kinase (Xingquan, L., et al., in
Mol. Cell. Biol
. (1993) 13:5225-5232), dynamin (Gout, I., et al., in
Cell
(1993) 75:25-36), AFAP-110 (Flynn, D. C., et al., in
Mol. Cell. Biol
. (1993) 13:7892-7900), and CD42 (Barfod, E. T., et al., in
J. Biol. Chem
. (1993) 268:26059-26062). These proteins tend to possess short, proline-rich stretches of amino acids, some of which have been directly implicated in SH3 binding. A variety of consensus sequences have been proposed, although the similarity among proline-rich regions of different SH3-binding proteins tends to be fairly low. Also, attempts to build consensus sequences are likely complicated by the incorporation of data from proteins that bind different SH3 domains.
Thus, Cicchetti, P., et al., in
Science
(1992) 257:803-806, published their work relating to the isolation and sequencing of two naturally-occurring proteins that could be bound in vitro by the SH3 domain of the abl oncogene product. These workers found that SH3 domains bind short, proline-rich regions of such proteins. Subsequently, this same group disclosed further results (Ren, R. et al., supra) in which the SH3 binding sites of the SH3 binding proteins were localized to

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