Solid-phase synthesis of peptidomimetic inhibitors for the hepatitis C virus NS3 protease.

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.

Highly potent and selective peptide-based inhibitors of the hepatitis C virus serine protease: towards smaller inhibitors.

Structure-activity studies on a hexapeptide N-terminal cleavage product of a dodecamer substrate led to the identification of very potent and highly specific inhibitors of the HCV NS3 protease/NS4A cofactor peptide complex. The largest increase in potency was accomplished by the introduction of a (4R)-naphthalen-1-yl-4-methoxy substituent to the P2 proline. N-Terminal truncation resulted in tetrapeptides containing a C-terminal carboxylic acid, which exhibited low micromolar activity against the HCV serine protease.

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.

Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase with improved in vivo antiviral activity.

We have been investigating the potential of a new class of antiviral compounds. These peptidomimetic derivatives prevent association of the two subunits of herpes simplex virus (HSV) ribonucleotide reductase (RR), an enzyme necessary for efficient replication of viral DNA. The compounds disclosed in this paper build on our previously published work. Structure-activity studies reveal beneficial modifications that result in improved antiviral potency in cell culture in a murine ocular model of HSV-induced keratitis. These modifications include a stereochemically defined (2,6-dimethylcyclohexyl)amino N-terminus, two ketomethylene amide bond isosteres, and a (1-ethylneopentyl)amino C-terminus. These three modifications led to the preparation of BILD 1351, our most potent antiherpetic agent containing a ureido N-terminus. Incorporation of the C-terminal modification into our inhibitor series based on a (phenylpropionyl)valine N-terminus provided BILD 1357, a significantly more potent antiviral compound than our previously published best compound, BILD 1263.

Ureido-based peptidomimetic inhibitor of herpes simplex virus ribonucleotide reductase: an investigation of inhibitor bioactive conformation.

We have been investigating peptidomimetic inhibitors of herpes simplex virus (HSV) ribonucleotide reductase (RR). These inhibitors bind to the HSV RR large subunit and consequently prevent subunit association and subsequent enzymatic activity. This report introduces a new series of compounds that contain an extra nitrogen (a ureido function) at the inhibitor N-terminus. This nitrogen improves inhibitor binding potency 50-fold over our first published inhibitor series. Evidence supports that this improvement in potency results from a new hydrogen-bonding contact between the inhibitor and the RR large subunit. This report also provides evidence for the bioactive conformation around two important amino acid residues contained in our inhibitors. A tert-butyl group, which contributes 100-fold to inhibitor potency but does not directly bind to the large subunit, favors an extended beta-strand conformation that is prevalent in solution and in the bound state. More significantly, the bioactive conformation around a pyrrolidine-modified asparagine residue, which contributes over 30 000-fold to inhibitor potency, is elucidated through a series of conformationally restricted analogues.

Determination of the HIV protease inhibitor BILA 2185 BS in rat plasma by liquid-liquid extraction and high performance liquid chromatography photodiode array detector.

A novel series of hydroxyethylamine-based inhibitors of HIV protease which contain a substituted pipecolinic amide were developed. After preliminary screening, a representative of this series, compound BILA 2185 BS, demonstrated an IC50 value of 3.3 nM in the enzymatic assay and an EC50 value of 2.0 nM in cell culture. The plasma profile and bioavailability values for BILA 2185 BS in the rat will be presented. The analyte was isolated from rat plasma using a liquid-liquid extraction procedure. The analytical technique used utilizes a high performance liquid chromatography system with photodiode array detector. The range of the standard curve was from 10 to 5000 nM. Recovery values averaged 72.4 +/- 8.6% (mean +/- S.D.). The limit of detection for BILA 2185 BS was 6-12 nM.

Peptidomimetic inhibitors of herpes simplex virus ribonucleotide reductase: a new class of antiviral agents.

We have been investigating a new class of antiviral compounds effective against herpes simplex virus (HSV) in vitro and in vivo. Antiviral activity results from inhibition of HSV ribonucleotide reductase (RR). The inhibitors are designed as mimics of the RR small subunit C-terminus, a region essential for RR subunit association and consequently enzymatic activity. Inhibition results from specific binding of the inhibitor to the HSV RR large subunit thereby preventing subunit association. This report details the structure--activity studies that lead to the indentification of BILD 1263, a potent inhibitor of HSV RR subunit association (IC50, 0.2 nM) that also inhibits the replication of HSV types 1 and 2 in cell culture (EC50, 3 and 4 microM) and reduces the severity of HSV-1-induced keratitis in a murine ocular model. The discovery of inhibitors with in vitro antiviral results from a combination of improving inhibitor potency in a RR binding assay and modifying inhibitor physicochemical properties. The importance and possible role of the new structural modifications introduced into this inhibitor series is discussed.