Ligands for the Tyrosine Kinase p56^lck SH2 Domain:  Discovery of Potent Dipeptide Derivatives with Monocharged, Nonhydrolyzable Phosphate Replacements

Abstract

p56lck is a member of the src family of tyrosine kinases. Through modular binding units called SH2 domains, p56lck promotes phosphotyrosine-dependent protein-protein interactions and plays a critical role in signal transduction events that lead to T-cell activation. Starting from the phosphorylated dipeptide (2), a high-affinity ligand for the p56lck SH2 domain, we have designed novel dipeptides that contain monocharged, nonhydrolyzable phosphate group replacements and bind to the protein with KD's in the low micromolar range. Replacement of the phosphate group in phosphotyrosine-containing sequences by a (R/S)-hydroxyacetic (compound 8) or an oxamic acid (compound 10) moiety leads to hydrolytically stable, monocharged ligands, with 83- and 233-fold decreases in potency, respectively. This loss in binding affinity can be partially compensated for by incorporating large lipophilic groups at the inhibitor N-terminus. These groups provide up to 13-fold increases in potency depending on the nature of the phosphate replacement. The discovery of potent (2-3 microM), hydrolytically stable dipeptide derivatives, bearing only two charges at physiological pH, represents a significant step toward the discovery of compounds with cellular activity and the development of novel therapeutics for conditions associated with undesired T-cell proliferation.

Molecular Docking and High-Throughput Screening for Novel Inhibitors of Protein Tyrosine Phosphatase-1B

“…The dissimilarity of the inhibitors to phosphotyrosine suggests that inhibitors lacking a phosphate can be found for PTP1B and other tyrosine phosphatases and kinases, as has been shown by other investigators. 26,31,32 This will be important to the development of sensible drug candidates for this target. The dissimilarity of the HTS and docking hits came as a surprise, particularly in light of control experiments showing that randomly chosen selections of ACD and Pharmacia compounds typically show such similarity.…”

Identification of Novel Non-phosphorylated Ligands, Which Bind Selectively to the SH2 Domain of Grb7

“…Because the first screening with a random peptide phage display library resulted in a strong binding consensus, the biased library was designed to have 1) the same constrained structure as the random peptide library, 2) positions 10 and 12 fixed to display Y and N residues, respectively, and 3) position 11 biased for D, A, or E (50%). The biased library design is depicted as follows: X 4 -Cys-X 4 -Tyr 10 -(Asp/Ala/Glu/X) 11 -Asn 12 -X 3 -Cys-X 4 , referred to as the YXN-biased library. This library design biases positions 10, 11, and 12 in the displayed peptides with strongly conserved residues and allows for randomization in the other positions for further optimization.…”

“…These domains are attractive targets for developing targeted therapeutics, especially in the area of oncology and immunology. Our laboratory was the first to identify a nonphosphorylated ligand to the SH2 domain of Grb2 (3), and several other groups have also reported ligands targeting the SH2 domains of Grb2 (4,5), pp60c-src (6 -9), and p56lck (10,11). Our interests are to target SH2 domain proteins that play a role in signaling by the ErbB tyrosine kinase family of receptors, which comprises EGFR, ErbB2, ErbB3, and ErbB4.…”

2-(Oxalylamino)-Benzoic Acid Is a General, Competitive Inhibitor of Protein-tyrosine Phosphatases

“…It appears that the unique combination of a phosphate mimetic (the oxamic acid part), the o-carboxy group, and an aromatic ring in the OBA structure provides a significant improvement over other known carboxylic acid-based PTP inhibitors. Interestingly, Beaulieu and coworkers (44) recently reported the replacement of phosphate with the non-hydrolyzable oxamic acid group in peptide-based SH2 domain ligands. Although 20 times less potent than phosphate, these findings add to the notion of oxamic acid as a phosphate mimetic.…”