Ligands for the tyrosine kinase p56lck SH2 domain: discovery of potent dipeptide derivatives with monocharged, nonhydrolyzable phosphate replacements.

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.

Nonpeptidic, monocharged, cell permeable ligands for the p56lck SH2 domain.

p56lck is a member of the src family of tyrosine kinases and plays a critical role in the signal transduction events that lead to T cell activation. Ligands for the p56lck SH2 domain have the potential to disrupt the interaction of p56lck with its substrates and derail the signaling cascade that leads to the production of cytokines such as interleukin-2. Starting from the quintuply charged (at physiological pH) phosphorylated tetrapeptide, AcpYEEI, we recently disclosed (J. Med. Chem. 1999, 42, 722 and J. Med. Chem. 1999, 42, 1757) the design of the modified dipeptide 3, which carries just two charges at physiological pH. Here we present the elaboration of 3 to the nonpeptidic, monocharged compound, 9S. This molecule displays good binding affinity for the p56lck SH2 domain (K(d) 1 microM) and good cell permeation, and this combination of properties allowed us to demonstrate clear-cut inhibitory effects on a very early event in T cell activation, namely calcium mobilization.

Preorganized ligand arrays based on spirotetrahydrofuranyl motifs. Synthesis of the stereoisomeric 1,8,14-trioxatrispiro[4.1.4.1.4. 1]octadecanes and the contrasting conformational features and ionic binding capacities of these belted ionophores.

The cis,trans trispiro ether 4 is accessible from several synthetic directions as a consequence of a crossover in reaction selectivity when proceeding from nucleophilic attack on the cis dispiro ketone to oxygenation of the alpha,beta-unsaturated ester 17. Its cis,cis isomer 3 was obtained in 17 steps and 14.6% overall yield from 3, 5-dimethoxybenzoic acid by making use of the alicyclic side chain in N as a "conformational lock". Although 4 shows no measurable tendency to complex with alkali metal ions, 3 binds strongly to Li(+) and Na(+) ions, as well as to CH(3)NH(3)(+). Whereas the 3eq conformation is populated in the solid state and in solution, complex formation occurs readily. (13)C NMR studies have defined slow exchange limits as the 2:1 sandwich complex with lithium ion is initially formed and transformed progressively into a 1:1 species upon the addition of more LiClO(4). Only the 2:1 complex with sodium ion is formed during comparable titration with NaClO(4). Association constants, molecular mechanics calculations, and X-ray crystallographic studies provide insight into the binding capacity of this belted tridentate ionophore.