Phage Selection of Chemically Stabilized α-Helical Peptide Ligands.

Short α-helical peptides stabilized by linkages between constituent amino acids offer an attractive format for ligand development. In recent years, a range of excellent ligands based on stabilized α-helices were generated by rational design using α-helical peptides of natural proteins as templates. Herein, we developed a method to engineer chemically stabilized α-helical ligands in a combinatorial fashion. In brief, peptides containing cysteines in position i and i + 4 are genetically encoded by phage display, the cysteines are modified with chemical bridges to impose α-helical conformations, and binders are isolated by affinity selection. We applied the strategy to affinity mature an α-helical peptide binding ß-catenin. We succeeded in developing ligands with Kd's as low as 5.2 nM, having >200-fold improved affinity. The strategy is generally applicable for affinity maturation of any α-helical peptide. Compared to hydrocarbon stapled peptides, the herein evolved thioether-bridged peptide ligands can be synthesized more easily, as no unnatural amino acids are required and the cyclization reaction is more efficient and yields no stereoisomers. A further advantage of the thioether-bridged peptide ligands is that they can be expressed recombinantly as fusion proteins.

Phage Selection of Peptide Macrocycles against ß-Catenin To Interfere with Wnt Signaling.

Upregulation of ß-catenin, the primary mediator of the Wnt signaling pathway, plays an important role in the tumorigenesis of several types of human cancer. Targeting ß-catenin to interfere with its ability to serve as a translational co-activator is considered an attractive therapeutic approach. However, the development of inhibitors has been challenging because of the lack of obvious binding pockets for ligands, and because inhibitors should not interfere with other ß-catenin functions. Only two ligands with known molecular interactions with ß-catenin have been developed so far, and are based on stabilized α-helical peptides. In this study, we screened a large combinatorial library of bicyclic peptides by phage display. Binders to different surface regions of ß-catenin were identified. The binding site of one group of ligands was mapped to the interaction region of the translational Wnt inhibitor ICAT (inhibitor of ß-catenin and Tcf), which is a prime target site on ß-catenin for therapeutic intervention, and to which no ligands could be developed before.

Protein disulfide isomerase-mediated disulfide bonds regulate the gelatinolytic activity and secretion of matrix metalloproteinase-9.

Matrix metalloproteinase-9 (MMP-9) is one of the major MMPs that can degrade extracellular matrix. Besides normal physiological functions, MMP-9 is involved in metastasis and tumor angiogenesis. Although several inhibitors of MMP-9 have been identified, in vivo regulators of MMP-9 activation are unknown. In the present study we intended to investigate novel therapeutic target protein(s) that regulate MMP-9 activation and/or secretion. We have identified protein disulfide isomerase as a novel upstream regulator of MMP-9. Mass spectrometric analysis of post-translational modification in MMP-9 confirmed six disulfide bonds in the catalytic domain and one disulfide bond in the hemopexin domain of MMP-9. Establishment of cells that overexpressed wild-type and mutant forms of MMP-9 revealed that 'cysteine-switch' and disulfide bonds within the catalytic domain are necessary for the secretion and intracellular trafficking of MMP-9. However, the disulfide bond of the hemopexin domain and other cysteines have no significant role in secretion. These insights into the secretion of MMP-9 constitute the basis for the development of potential drugs against metastasis.

Maximum likelihood analysis of mammalian p53 indicates the presence of positively selected sites and higher tumorigenic mutations in purifying sites.

The tumor suppressor gene TP53 (p53) maintains genome stability. Mutation or loss of p53 is found in most cancers. Analysis of evolutionary constrains and p53 mutations reveal important sites for concomitant functional studies. In this study, phylogenetic analyses of the coding sequences of p53 from 26 mammals were carried out by applying a maximum likelihood method. The results display two branches under adaptive evolution in mammals. Moreover, each codon of p53 was analyzed by the PAML method for presence of positively selected sites. PAML identified several statistically significant amino acids that undergo positive selection. The data indicates that amino acids responsible for the core functions of p53 are highly conserved, while positively selected sites are predominantly located in the N- and C-terminus of p53. Further analysis of evolutionary pressure and mutations showed the occurrence of more frequent tumorigenic mutations in purifying sites of p53.

Discovery of a small molecule PDI inhibitor that inhibits reduction of HIV-1 envelope glycoprotein gp120.

Protein disulfide isomerase (PDI) is a promiscuous protein with multifunctional properties. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction. The entry of HIV-1 into cells is facilitated by the PDI-catalyzed reductive cleavage of disulfide bonds in gp120. PDI is regarded as a potential drug target because of its reduction activity. We screened a chemical library of natural products for PDI-specific inhibitors in a high-throughput fashion and identified the natural compound juniferdin as the most potent inhibitor of PDI. Derivatives of juniferdin were synthesized, with compound 13 showing inhibitory activities comparable to those of juniferdin but reduced cytotoxicity. Both juniferdin and compound 13 inhibited PDI reductase activity in a dose-dependent manner, with IC(50) values of 156 and 167 nM, respectively. Our results also indicated that juniferdin and compound 13 exert their inhibitory activities specifically on PDI but do not significantly inhibit homologues of this protein family. Moreover, we found that both compounds can inhibit PDI-mediated reduction of HIV-1 envelope glycoprotein gp120.

The potential of protein disulfide isomerase as a therapeutic drug target.

Protein disulfide isomerase (PDI) is a multifunctional protein of the thioredoxin superfamily. PDI mediates proper protein folding by oxidation or isomerization and disrupts disulfide bonds by reduction; it also has chaperone and antichaperone activities. Although PDI localizes primarily to the endoplasmic reticulum (ER), it is secreted and expressed on the cell surface. In the ER, PDI is primarily involved in protein folding, whereas on the cell surface, it reduces disulfide bonds. The functions of PDI depend on its localization and the redox state of its active site cysteines. The ER-based functions of PDI are linked to cancer invasion and migration. Surface-associated PDI facilitates the entry of viruses, such as HIV-1, and toxins, such as diphtheria and cholera. Thus, based on its involvement in pathological events, PDI is considered a potential drug target. However, a significant challenge in the therapeutic targeting of PDI is discovering function-specific inhibitors for it. To this end, a wide range of therapeutic agents, such as antibiotics, thiol blockers, estrogenic compounds, and arsenical compounds, have been used, although few are bona fide specific inhibitors. In this review, we will describe the potential of PDI as a therapeutic drug target.