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.

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.

Phosphotyrosine-containing dipeptides as high-affinity ligands for the p56lck SH2 domain.

Src homology-2 (SH2) domains are noncatalytic motifs containing approximately 100 amino acid residues that are involved in intracellular signal transduction. The phosphotyrosine-containing tetrapeptide Ac-pYEEI binds to the SH2 domain of p56lck (Lck) with an affinity of 0.1 microM. Starting from Ac-pYEEI, we have designed potent antagonists of the Lck SH2 domain which are reduced in peptidic character and in which the three carboxyl groups have been eliminated. The two C-terminal amino acids (EI) have been replaced by benzylamine derivatives and the pY + 1 glutamic acid has been substituted with leucine. The best C-terminal fragment identified, (S)-1-(4-isopropylphenyl)ethylamine, binds to the Lck SH2 domain better than the C-terminal dipeptide EI. Molecular modeling suggests that the substituents at the 4-position of the phenyl ring occupy the pY + 3 lipophilic pocket in the SH2 domain originally occupied by the isoleucine side chain. This new series of phosphotyrosine-containing dipeptides binds to the Lck SH2 domain with potencies comparable to that of tetrapeptide 1.

Carboxymethyl-phenylalanine as a replacement for phosphotyrosine in SH2 domain binding.

The crystal structure of human p56(lck) SH2 domain in complex with an inhibitor containing the singly charged p-(carboxymethyl)phenylalanine residue (cmF) as a phosphotyrosine (Tyr(P) or pY) replacement has been determined at 1.8 A resolution. The binding mode of the acetyl-cmF-Glu-Glu-Ile (cmFEEI) inhibitor is very similar to that of the pYEEI inhibitor, confirming that the cmFEEI inhibitor has a similar mechanism of SH2 domain inhibition despite its significantly reduced potency. Observed conformational differences in the side chain of the cmF residue can be interpreted in terms of maintaining similar interactions with the SH2 domain as the Tyr(P) residue. The crystal structure of the free p56(lck) SH2 domain has been determined at 1.9 A resolution and shows an open conformation for the BC loop and an open phosphotyrosine binding pocket, in contrast to earlier studies on the src SH2 domain that showed mostly closed conformation. The structural information presented here suggests that the carboxymethyl-phenylalanine residue may be a viable Tyr(P) replacement and represents an attractive starting point for the design and development of SH2 domain inhibitors with better pharmaceutical profiles.

Neutrophil rolling altered by inhibition of L-selectin shedding in vitro.

The L-selectin adhesion molecule is involved in guiding leukocytes to sites of inflammation. L-selectin is cleaved by an unusual proteolytic activity at a membrane-proximal site resulting in rapid shedding from the cell surface. Although it has been demonstrated that L-selectin mediates, in part, the early event of leukocyte rolling under hydrodynamic flow, the contribution of shedding to L-selectin function has remained unknown. Here we show that hydroxamic acid-based metalloprotease inhibitors block L-selectin downregulation from the cell surface of stimulated neutrophils, without affecting Mac-1 mobilization or general neutrophil activation, and inhibit cleavage of L-selectin in a cell-free system. Unexpectedly, the hydroxamic acid-based inhibitors reduced neutrophil rolling velocity under hydrodynamic flow, resulting in increased neutrophil accumulation. These results suggest that L-selectin is cleaved in seconds--much faster than previously suspected--during the process of rolling under hydrodynamic flow, and that shedding of L-selectin may contribute significantly to the velocity of leukocyte rolling. L-selectin shedding during rolling interactions may be physiologically important for limiting leukocyte aggregation and accumulation at sites of inflammation.

Crystal structures of the human p56lck SH2 domain in complex with two short phosphotyrosyl peptides at 1.0 A and 1.8 A resolution.

src homology 2 (SH2) domains are modules of about 100 amino acid residues and bind to phosphotyrosine-containing motifs in a sequence-specific manner. They play important roles in intracellular signal transduction and represent potential targets for pharmacological intervention. The protein tyrosine kinase p56lck is a member of the src family and is involved in T-cell activation. The crystal structure of its SH2 domain with an 11-residue peptide showed that the phosphotyrosine and the Ile residue at the pY + 3 position are recognized by the SH2 domain. We present here the crystal structure of the SH2 domain of human p56lck in complex with the short phosphotyrosyl peptide Ac-pTyr-Glu-Glu-Ile (pYEEI peptide) at 1.0 A resolution. The structural analysis at atomic resolution reveals that residue Arg134 (alphaA2), which interacts with the phosphotyrosine side-chain, is present in two conformations in the complex. The structure at 1.8 A resolution of the complex with the phosphotyrosyl peptide Ac-pTyr-Glu-Glu-Gly (pYEEG peptide), which is 11 fold less potent, shows another binding mode for the pY + 3 residue as well as rearrangements of the side-chain of Arg196 (EF3) and one of the water molecules at the base of the pY + 3 pocket. The structure of the complex with the short pYEEI peptide at atomic resolution represents a good starting point for the design and optimization of new inhibitors. Comparative structural analysis of many different inhibitor complexes will be an important component of this drug discovery process.

Determination of receptor-ligand kinetic and equilibrium binding constants using surface plasmon resonance: application to the lck SH2 domain and phosphotyrosyl peptides.

Experimental and computational methods were developed for surface plasmon resonance (SPR) measurements involving interactions between a solution-binding component and a surface-immobilized ligand. These protocols were used to distinguish differences in affinity between the SH2 domain of lck and phosphotyrosyl peptides. The surface-immobilized ligand was the phosphotyrosyl peptide EPQpYEEIPIA, which contains a consensus sequence (pYEEI) for binding lck SH2. In the kinetic experiment, SPR phenomena were measured during association and dissociation reactions for a series of glutathione-S-transferase (GST)-SH2 concentrations, generating a set of SPR curves. A global computational analysis using an A + B<==>AB model resulted in single set of parameter estimates and statistics. In an abbreviated format, an equilibrium experiment was designed so that equilibrium constants (Keq) could be determined rapidly and accurately. A competitive equilibrium assay was developed for GST-SH2 in which Keq values for a series of phosphotyrosyl peptides (derived from the pYEEI sequence) varied over 3 orders of magnitude. Interestingly, these results highlighted the significance of the +1 glutamate in providing high-affinity binding to the SH2 domain. For most drug discovery programs, these Keq determinations are a sufficient measure of potency for the primary screen, with koff and kon determined in a secondary assay. Thus, the application of these techniques to SPR binding phenomena should prove valuable in the discovery and design of receptor-ligand antagonists.

Rapid, sensitive and efficient HPLC assays for HIV-1 proteinase.

The proteinase encoded by human immunodeficiency virus type 1 (HIV-1) cleaves peptide substrates of sequences derived from processing sites in HIV-1 gag-pol polypeptide. Based on this cleavage, assays that utilize HPLC to measure activity of HIV-1 proteinase are reported herein. In the assay first described, a baseline separation of unlabeled substrate and products is achieved with a run time of 10 min and UV detection. Enzyme concentrations as low as 1 nM, which is the lowest reported for an assay employing underivatized peptide substrate, are attained. Even more powerful, versatile and sensitive, a second method that takes advantage of a peptide substrate labeled at its N-terminus with the fluorescein derivative is described as well. Because of the fluorescein label, this method offers several superior features, including very fast analysis of substrate and product in less than 3 min and fluorescence detection which provides essentially total freedom from interference. Synthesis of fluorescein-labeled peptide substrate is accomplished by solid-phase peptide synthesis.

Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus.

The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.

Rhinovirus 3C protease catalyzes efficient cleavage of a fluorescein-labeled peptide affording a rapid and robust assay.

The 3C protease encoded by human rhinovirus type 2 catalyzes with equal efficiency cleavage of a peptide substrate with or without a fluorescein label attached to the amino acid at the P7' position. Substrates Ac-MEALFQGPLQYKDL-NH2 and MEALFQGPLQYKE(fluorescein)L are hydrolyzed with values of Vmax/KM of 970 M-1 s-1 and 1100 M-1 s-1, respectively. With the labeled substrate, HPLC achieves separation of substrate and product in 2.5 min. Separation in as little as 12 s is feasible. Fluorescein was derivatized so that it could be incorporated into peptides using automated solid-phase peptide synthesis.