&bgr;-sheet mimetics and methods relating to the use thereof

Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Heterocyclic carbon compounds containing a hetero ring...

Reexamination Certificate

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C514S384000, C530S323000, C530S332000, C548S263400

Reexamination Certificate

active

06372744

ABSTRACT:

TECHNICAL FIELD
This invention relates generally to &bgr;-sheet mimetics and, more specifically, to &bgr;-sheet mimetics which inhibit biologically active peptides and proteins.
BACKGROUND OF THE INVENTION
The &bgr;-sheet conformation (also referred to as a &bgr;-strand conformation) is a secondary structure present in many polypeptides. The &bgr;-sheet conformation is nearly fully extended, with axial distances between adjacent amino acids of approximately 3.5 Å. The &bgr;-sheet is stabilized by hydrogen bonds between NH and CO groups in different polypeptides strands. Additionally, the dipoles of the peptide bonds alternate along the strands which imparts intrinsic stability to the &bgr;-sheet. The adjacent strands in the &bgr;-sheet can run in the same direction (i.e., a parallel &bgr;-sheet) or in opposite directions (i.e., an antiparallel &bgr;-sheet). Although the two forms differ slightly in dihedral angles, both are sterically favorable. The extended conformation of the &bgr;-sheet conformation results in the amino acid side chains protruding on alternating faces of the &bgr;-sheet.
The importance of &bgr;-sheets in peptides and proteins is well established (e.g., Richardson,
Nature
268:495-499, 1977; Halverson et al.,
J. Am. Chem Soc.
113:6701-6704, 1991; Zhang,
J. Biol. Chem.
266:15591-15596, 1991; Madden et al.,
Nature
353:321-325, 1991). The &bgr;-sheet is important in a number of biological protein-protein recognition events, including interactions between proteases and their substrates, protein kinases and their substrates or inhibitors, the binding of SH2 domain containing proteins to their cognate phosphotyrosine containing protein targets, farnesyl transferase to its protein substrates, and MHC I and II and their antigenic peptides, and has been implicated in many disease states.
Inhibitors that mimic the &bgr;-sheet structure of biologically active proteins or peptides would have utility in the treatment of a wide variety of conditions. For example, Ras, the protein product of the ras oncogene, is a membrane bound protein involved in signal transduction regulating cell division and growth. Mutations in the ras gene are among the most common genetic abnormalities associated with human cancers (Barbacid, M. “ras genes,” 56:779-827, 1987). These mutations result in a growth signal which is always “on,” leading to a cancerous cell. In order to localize to the cell membrane, Ras requires prenylation of the cysteine within its C-terminal CaaX sequence by farnesyl transferase (FTase). (In the sequence CaaX “a” is defined as an amino acid with a hydrophobic side chain and “X” is another amino acid.) This post-translational modification is crucial to its activity. Peptidyl inhibitors of FTase with the sequence CaaX have been shown to block or slow the growth of tumors in cell culture and in whole animals (Kohl et al., “Selective inhibition of ras-dependent transformation by a farnesyltransferase inhibitor,”
Science
260:1934-1937, 1993; Buss, J. E. & Marsters, Jr., J. C. “Farnesyl transferase inhibitors: the successes and surprises of a new class of potential cancer chemotherapeutics,”
Chemistry and Biology
2:787-791, 1995).
SH2 domains, originally identified in the src subfamily of PTKs, are noncatalytic sequences and consist of about 100 amino acids conserved among a variety of signal transducing proteins (Cohen et al.,
Cell
80:237-248, 1995). SH2 domains function as phosphotyrosine-binding modules and mediate critical protein-protein associations (Pawson,
Nature
573-580, 1995). In particular, the role of SH2 domains has been clearly defined as critical signal transducers for receptor tyrosine kinases (RTKs such as EGF-R, PDGF, insulin receptor, etc.). Phosphotyrosine-containing sites on autophosphorylated RTKs serve as binding sites for SH2-proteins and thereby mediate the activation of biochemical signaling pathways (Carpenter, G.,
FAESEB J.
6:3283-3289, 1992; Sierke, S. and Koland,
J., Biochem.
32:10102-10108, 1993). The SH2 domains are responsible for coupling the activated growth-factor receptors to cellular responses which include alterations in gene expression, cell proliferation, cytoskeletal architecture and metabolism.
At least 20 cytosolic proteins have been identified that contain SH2 domains and function in intracellular signaling. The distribution of SH2 domains is not restricted to a particular protein family, but is found in several classes of proteins, protein kinases, lipid kinases, protein phosphatases, phospholipases, Ras-controlling proteins and some transcription factors. Many of the SH2-containing proteins have known enzymatic activities while others (Grb2 and Crk) function as “linkers” and “adapters” between cell surface receptors and downstream effector molecules (Marengere, L., et al.,
Nature
369:502-505, 1994). Examples of proteins containing SH2 domains with enzymatic activities that are activated in signal transduction include, but are not limited to, the src subfamily of protein tyrosine kinases (src (pp60
c-src
), abl, lck, fyn, fgr and others), phospholipase-C-&ggr; (PLC-&ggr;), phosphatidylinositol 3-kinase (Pl-3-kinase), p21-ras GTPase activating protein (GAP) and SH2 containing protein tyrosine phosphatases (SH-PTPase) (Songyang et al.,
Cell
72:767-778, 1993). Intracellular tyrosines are phosphorylated when surface receptors are engaged by diverse ligands for growth factor receptors, cytokine receptors, insulin receptor, and antigen-mediated signaling through T- or B-cell receptors. The phosphorylation of proteins at tyrosine residues is critical in the cellular signal transduction, neoplastic transformation and control of the cell cycle. Due to the central role these various SH2-proteins occupy in transmitting signals from activated cell surface receptors into a cascade of additional molecular interactions that ultimately define cellular responses, inhibitors which block specific SH2-protein binding are desirable as agents for a variety of potential therapeutic applications.
Disease areas in which tyrosine phosphorylation and inhibition of SH2 binding represent targets for drug development include the following:
Cancer
SH2 domains which mediate signaling are clearly significant elements in the regulation of oncogene and protooncogene tyrosine kinase activity and cellular proliferation (Carpenter,
Fed. Am. Soc. Exp. Biol. J.
6:3283-3289, 1992). The SH2 domains define an important set of substrates through which activated RTKs mediate signaling and through which nonreceptor tyrosine kinases associate with RTKs and are thus targets for anticancer drug development. The ability to block interaction of the RTK with the SH2-containing substrate using a mimetic inhibitor provides a means to abrogate signaling and thereby eliminate oncogenic activity. The biological significance is also illustrated by the v-crk oncogene, a protein composed almost entirely of SH domains, which is able to bring about cellular transformation by interacting with phosphotyrosine containing proteins. As above, the ability of inhibitors to block v-crk binding via its SH2 domain to other proteins would be expected to be effective as an anticancer agent.
Immune Regulation
Regulation of many immune responses is mediated through receptors that transmit signals through tyrosine kinases containing SH2 domains. T-cell activation via the antigen specific T-cell receptor (TCR) initiates a signal transduction cascade leading to lymphokine secretion and cell proliferation. One of the earliest biochemical responses following TCR activation is an increase in tyrosine kinase activity. In particular, T-cell activation and proliferation is controlled through T-cell receptor mediated activation of p56
lck
and p59
fyn
tyrosine kinases, as well as ZAP-70 and Syk (Weiss and Litman,
Cell
76:263-274, 1994) which contain SH2 domains. Additional evidence indicates that several src-family kinases (lck, blk, fyn) participate in signal transduction pathways leading from B-cell antigen receptors and hence may serve to integrate stimuli received from sever

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