Total chemical synthesis of human interferon alpha-2b via native chemical ligation

Abstract

Interferon-alpha (IFNα) is a cytokine that orchestrates innate and adaptive immune responses and potently inhibits proliferation of normal and tumor cells. These properties have warranted the use of IFNα in clinical practice for the treatment of several viral infections and malignancies. However, overexpression of IFNα leads to immunopathology observed in the context of chronic viral infections and autoimmune conditions. Thus, it is desirable to develop therapeutic approaches that aim at suppressing excessive IFNα production. To that end, artificial evolution of peptides from phage display libraries represents a strategy that seeks to disrupt the interaction between IFNα and its cell surface receptor and thus inhibit the ensuing biological effects. Mirror-image phage display that screens peptide libraries against the D-enantiomer is particularly attractive because it allows for identification of proteolysis-resistant D-peptide inhibitors. This approach, however, relies on the availability of chemically synthesized D-IFNα composed entirely of D-amino acids. Here, we describe the synthesis and biological properties of IFNα2b of 165 amino acid residues produced by native chemical ligation, which represents an important first step toward the discovery of D-peptide antagonists with potential therapeutic applications.

Keywords: interferon; native chemical ligation; peptide synthesis; synthetic protein.

Fig 1

Strategy of native chemical ligation for the total chemical synthesis of human interferon alpha-2b protein, IFNα2b. The N-terminal 28-residue (red) and middle 69-residue peptide-thioester (green) fragments and C-terminal 68-residue fragment (blue) were synthesized by stepwise SPPS techniques using Boc-chemistry protocols. Folded synthetic IFNα2b is depicted by the NMR structure of human interferon alpha-2a (PDB code 1ITF) [35].

Fig 2

Native chemical ligation reactions monitored by analytical RP-HPLC and ESI-MS. All HPLC chromatograms were obtained at 40 °C on a Waters Symmetry 300 C18 column (4.6×150 mm, 5 μm) running a linear gradient of 5% - 65 % of acetonitrile containing 0.1% TFA at a flow rate of 1 ml/min over 30 min. (A) HPLC traces of the three synthetic peptide fragments, Thz-Cys(2–28)IFNα-COSR, Thz-Cys(30–97)IFNα-COSR and (98–165)IFNα, and their ESI-MS data (B). (C) The first ligation reaction monitored by HPLC and verified by ESI-MS (D). (E) The second ligation reaction and protein oxidative folding monitored by HPLC and verified by ESI-MS (F). As expected, protein folding shortens retention time of IFNα2b as its hydrophobic residues are buried in the folded structure. The determined molecular mass of folded synthetic IFNα2b of 19265.5 Da is within experimental error of the theoretical value of 19265.1 Da calculated on the basis of the average isotopic compositions of IFNα2b.

Fig 3

The CD spectrum of IFNα2b obtained at room temperature. The double negative peaks at 208 and 222 nm and single positive peak at 195 nm are indicative of alpha-helical secondary structure, consistent with the known structural features of IFNα.

Fig 4

Comparative analyses of the biological properties of recombinant and synthetic IFNα2b. (A): Antiviral activity of the two molecules was determined based on a visual read of the protection of A549 cells from cytopathic effect (CPE) due to viral challenge with encephalomyocarditis virus (ECMV). Recombinant and synthetic IFNα2b were titrated in duplicate samples, and protection from CPE due to ECMV infection was determined in comparison to untreated A549 cells (no IFNα), and to uninfected, untreated cells (no virus, no IFNα) controls. (B, C): Inhibition of cell proliferation. Peripheral blood mononuclear cells (PBMC) of healthy volunteers were plated in triplicate samples, and proliferation was stimulated with Staphylococcal enterotoxin B and IL-2 (Roche Biochemicals). Cells were cultured in the presence of recombinant or synthetic IFNα2b (0.01–10 ng/ml) for 3 days at 37°C. Proliferation was determined by incorporation of [³H]-Thymidine compared to untreated samples (B). In samples treated with 10 ng/ml IFNα2b, cellular proliferation was also measured by cell counts with trypan blue exclusion (C).

Fig 4

Comparative analyses of the biological properties of recombinant and synthetic IFNα2b. (A): Antiviral activity of the two molecules was determined based on a visual read of the protection of A549 cells from cytopathic effect (CPE) due to viral challenge with encephalomyocarditis virus (ECMV). Recombinant and synthetic IFNα2b were titrated in duplicate samples, and protection from CPE due to ECMV infection was determined in comparison to untreated A549 cells (no IFNα), and to uninfected, untreated cells (no virus, no IFNα) controls. (B, C): Inhibition of cell proliferation. Peripheral blood mononuclear cells (PBMC) of healthy volunteers were plated in triplicate samples, and proliferation was stimulated with Staphylococcal enterotoxin B and IL-2 (Roche Biochemicals). Cells were cultured in the presence of recombinant or synthetic IFNα2b (0.01–10 ng/ml) for 3 days at 37°C. Proliferation was determined by incorporation of [³H]-Thymidine compared to untreated samples (B). In samples treated with 10 ng/ml IFNα2b, cellular proliferation was also measured by cell counts with trypan blue exclusion (C).

Fig 4

Comparative analyses of the biological properties of recombinant and synthetic IFNα2b. (A): Antiviral activity of the two molecules was determined based on a visual read of the protection of A549 cells from cytopathic effect (CPE) due to viral challenge with encephalomyocarditis virus (ECMV). Recombinant and synthetic IFNα2b were titrated in duplicate samples, and protection from CPE due to ECMV infection was determined in comparison to untreated A549 cells (no IFNα), and to uninfected, untreated cells (no virus, no IFNα) controls. (B, C): Inhibition of cell proliferation. Peripheral blood mononuclear cells (PBMC) of healthy volunteers were plated in triplicate samples, and proliferation was stimulated with Staphylococcal enterotoxin B and IL-2 (Roche Biochemicals). Cells were cultured in the presence of recombinant or synthetic IFNα2b (0.01–10 ng/ml) for 3 days at 37°C. Proliferation was determined by incorporation of [³H]-Thymidine compared to untreated samples (B). In samples treated with 10 ng/ml IFNα2b, cellular proliferation was also measured by cell counts with trypan blue exclusion (C).