The activation of cells by growth factors, mitogens and antigens resulting in proliferation and differentiation is often dependent on inducible tyrosine kinase activity. This tyrosine kinase activity increases the phosphotyrosine content of many receptor-like and cytoplasmic regulatory proteins. Often, the physical association of such regulatory proteins is mediated through those phosphotyrosine residues. The interaction between a regulatory protein and a tyrosine kinase is often a critical step in initiating and modulating cellular signal transduction pathways.
The receptor-mediated activation of cells is often dependent on inducible tyrosine kinase activity. Activation of tyrosine kinase signal transduction pathways result in a transient increase in intracellular tyrosine phosphorylation on selective subsets of regulatory proteins. This tyrosine phosphorylation of proteins provides multiple means of regulation.
For example, tyrosine phosphorylation may modulate the enzymatic activity of proteins, as in the case of p56lck (sometimes referred to herein as "Lck"), PLC.gamma., and P1-3-kinase. In addition to enzyme regulation through covalent modification, one of the major roles for tyrosine phosphorylation is to create a binding site for Src homology 2 domains ("SH2 domains"). SH2 domains are homologous motifs of approximately 100 amino acids, which recognize and bind to the phosphorylated sequences present on regulatory proteins and growth factor receptors (D. Anderson et al., Science, 250, pp. 979-82 (1990)). A particularly important subset of proteins containing SH2 domains is the Src family of proteins ("Src family").
One of the primary purposes of the Src family phosphoprotein/SH2 domain interaction is to initiate the association of proteins into an activation complex, often around the intracellular domain of the receptor itself. This role of the Src family SH2 domain mediates and organizes the ordered, physical assembly of the various proteins in the activation complex. The activity of a number of immunologically important Src family SH2 domain-containing proteins, including Src, Fyn, Fgr, Yes, Lyn, Hck and Lck, is mediated in this way. P56lck is of particular interest because it has been associated with the signal transduction cascade needed for T-cell activation mediated by the T-cell receptor (TCR) (D. B. Straus et al., Cell 70, pp. 585-93 (1992)).
Disrupting the interaction between Src family SH2 domains and their phosphotyrosine-bearing native ligands (referred to herein as "Src family SH2 domain inhibition") represents a unique therapeutic approach to immunomodulation and the treatment or prevention of disorders associated with aberrant cellular transduction modulated by Src family SH2 domain binding interactions, such as certain autoimmune and inflammatory disorders, cancer and diabetes. However, despite considerable effort being devoted to this field of endeavor, no suitable drug candidates have yet emerged.
One major hurdle in this drug discovery effort has been the necessary inclusion of a phosphorylated tyrosine residue (sometimes abbreviated herein as "pTyr"), or a phosphorylated analog thereof, to perform the crucial role of the native phosphotyrosine in the phosphotyrosine-containing ligands of Src family SH2 domain-containing regulatory proteins. However, agents containing phosphotyrosine, other phosphorylated (.alpha.-amino acid residues, or phosphorylated analogs thereof, are not optimal therapeutic agents because the presence of the phosphorylated moiety substantially impedes their cell penetrability. In addition, the phosphate moiety tends to be metabolically unstable and therefore, results in premature degradation of the therapeutic agent . In contrast, the absence of a phosphate moiety is often associated with agents having poor binding affinity and lack of potency. Prior attempts at producing therapeutic agents containing phosphotyrosyl mimetics have met with only marginal success, including the attempted use of the conformationally constrained pTyr analogue, N.sup..alpha. -acetyl pTyr amide (T. R. Burke, Jr. et al., J. Med. Chem., 38, pp. 1386-96 (1995)). Another attempt to produce an active compound containing a phosphotyrosyl mimetic replaced pTyr with (2-malonyl)Tyr (B. Ye et al., J. Med. Chem., 38, pp. 4270-75 (1995)). The production of compounds containing other types of phosphate mimics include the use of NO.sub.2, CH.sub.2 CONHOH, NH.sub.2, CH.sub.2 COOH, CH.sub.2 SO.sub.3 H, CH.sub.2 PO.sub.3 H.sub.2, CHOHPO.sub.3 H.sub.2, CF.sub.2 PO.sub.3 H.sub.2 and OPSO.sub.2 H.sub.2 (T. Gilmer et al., J. Biol. Chem., 269(50), pp. 31711-19 (1994)), and CH.sub.2 CH(COOH).sub.2 (Charifson et al., Biochemistry, 36, pp. 6283-93 (1997)). However, until now, no effective replacement or mimic for the critical phosphotyrosine residue has been reported in connection with a Src family SH2 inhibitor so that cellular or in-vivo activity was clearly demonstrated. Recently published PCT patent application WO 97/12903 shows the only published example of which we are aware of cellular activity of any phosphate mimic. Specifically, WO 97/12903 refers to peptidomimetics incorporating CF.sub.2 PO.sub.3 H.sub.2 and monoesters thereof (see Table 4 on page 112).
Another major hurdle to producing effective Src family SH2 domain inhibitors has been the high negative charge carried by native ligands of Src family SH2 domain binding proteins. For example, sequences containing pTyr-Glu-Glu-Ile have been reported as the optimal binding sequence for Src family SH2 domains (Z. Songyang et al., Cell, 72, pp. 767-78 (1993)). Five negatively charged groups are associated with this sequence. The presence of these multiple negative charges, although useful for binding affinity, is not amenable for use in a bioavailable drug substance. As most of the above-referenced documents reveal, attempts to replace all of these negative charges with neutral- or positively charged-amino acids or amino acid mimics met with limited success due to reduced binding affinity (Gilmer, et al., supra, ).
A limited number of reports discuss the use of particular Src family SH2 domain inhibitors in whole cells. However, the majority of these trials have required the use of artificial means (e.g., extraneous agents or special microinjection techniques) to allow the Src family SH2 domain inhibitors to successfully enter the cell (Xiao, et al., J. Biol. Chem. 269, pp. 21244-8 (1994) and Wange, et al., J. Biol. Chem. 270, pp. 944-8 (1995)). To date, aside from WO 97/12903, no reported Src family SH2 inhibitor has been shown to possess high activity levels in whole cells without cell permeabilizers, signal peptides, prodrugs and the like.
Accordingly, the need exists for Src family SH2 inhibitors that overcome the above-mentioned deficiencies.
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