Inhibition of herpes simplex virus type 1 ribonucleotide reductase by substituted tetrapeptide derivatives.

It is known that peptides corresponding to the C-terminus of the small subunit of herpes simplex virus type 1 and 2 ribonucleotide reductase can inhibit enzymatic activity by preventing the association of the enzyme's two subunits. In a quest for smaller, more potent inhibitors, we have conducted a structure activity investigation based on the pentapeptide H-Val-Val-Asn-Asp-Leu-OH. Potency increases of up to 4000 times (IC50 0.18 microM) have been achieved in an enzymatic assay by a combination of modifying the N-terminal valine to a diethylacetyl group, adding a methyl group to the beta-carbon of the adjacent valine, dialkylating the asparagine side-chain nitrogen and dimethylating the beta-carbon of the aspartic acid residue. In addition the relative contribution of various inhibitor functionalities to inhibitor potency has been investigated.

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.

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.

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.

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.

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.

Herpes simplex virus ribonucleotide reductase subunit association inhibitors: the effect and conformation of beta-alkylated aspartic acid derivatives.

Incorporating beta-alkylated aspartic acid derivatives into herpes simplex virus ribonucleotide reductase subunit association inhibitors can improve inhibitor potency up to 50 times over the corresponding inhibitors containing an unsubstituted aspartic acid. A combination of NMR studies, conformational analysis, and molecular mechanics calculations suggests that the beta-alkyl group improves inhibitor potency by favoring the bioactive conformation of the critical aspartic acid carboxyl group. Further support for this hypothesis is provided by a potent conformationally restricted aspartic acid derivative in which the carboxyl group is locked in the putative bioactive conformation.