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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.

The NS3 serine protease enzyme of the hepatitis C virus (HCV) is essential for viral replication. Short peptides mimicking the N-terminal substrate cleavage products of the NS3 protease are known to act as weak inhibitors of the enzyme and have been used as templates for the design of peptidomimetic inhibitors. Automated solid-phase synthesis of a small library of compounds based on such a peptidomimetic scaffold has led to the identification of potent and highly selective inhibitors of the NS3 protease enzyme.

Starting from palinavir (1), our lead HIV protease inhibitor, we have discovered a new series of truncated analogues in which the P(3)-P(2) quinaldic-valine portion of 1 was replaced by 2', 6'-dimethylphenoxyacetyl. With EC(50)'s in the 1-2 nM range, some of these compounds are among the most potent inhibitors of HIV replication in vitro, reported to date. One of the most promising members in this series (compound 27, BILA 2185 BS) exhibited a favorable overall pharmacokinetic profile, with 61% apparent oral bioavailability in rat. X-ray crystal structures and molecular modeling were used to rationalize the high potency resulting from incorporation of this structurally simple, achiral ligand into the P(3)-P(2) position of hydroxyethylamine-based HIV protease inhibitors.

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.

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.

The development of novel monobactam inhibitors of HCMV protease incorporating a carbon side chain at C-4 and a urea function at N-1 is described. Substitution with small groups at the C-3 position of the beta-lactam ring gave an increase in enzymatic activity and in stability; however, a lack of selectivity against other serine proteases was noted. The use of both tri- and tetrasubstituted urea functionalities gave effective inhibitors of HCMV protease. Benzyl substitution of the urea moiety was beneficial, especially when strong electron-withdrawing groups where attached at the para position. Modest antiviral activity was found in a plaque reduction assay.

Substrate hydrolysis by human cytomegalovirus (HCMV) protease is essential to viral capsid assembly. The interaction of HCMV protease and the N-terminal cleavage products of the hydrolysis of R- and M-site oligopeptide substrate mimics (R and M, respectively, which span the P9-P1 positions) was studied by NMR methods. Protease-induced differential line broadening indicated that ligand binding is mediated by the P4-P1 amino acid residues of the peptides. A well-defined extended conformation of R from P1 through P4 when complexed to HCMV protease was evidenced by numerous transferred nuclear Overhauser effect (NOE) correlations for the peptide upon addition of the enzyme. NOE cross-peaks between the P4 and P5 side chains placing these two groups in proximity indicated a deviation from the extended conformation starting at P5. Similar studies carried out for the M peptide also indicated an extended peptide structure very similar to that of R, although the conformation of the P5 glycine could not be established. No obvious variation in structure between bound R and M (notably at P4, where the tyrosine of the R-site has been suggested to play a key role in ligand binding) could be discerned that might explain the observed differences in processing rates between R- and M-sequences. Kinetic studies, utilizing R- and M-site peptide substrates for which the P5 and P4 residues were separately exchanged, revealed that these positions had essentially no influence on the specificity constants (kcat/KM). In sharp contrast, substitution of the P2 residue of an M-site peptide changed its specificity constant to that of an R-site peptide substrate, and vice versa.