Retrovirally Delivered Random Cyclic Peptide Libraries Yield Inhibitors of Interleukin-4 Signaling in Human B Cells

Abstract

Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a “split and inverted” configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line ε transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides. Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a “split and inverted” configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line ε transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides. interleukin-4 green fluorescent protein blue fluorescent protein fluorescence-activated cell sorter doxycycline reverse transcriptase transforming growth factor-β mass spectrometry hemagglutinin matrix-assisted laser desorption ionization time-of-flight long terminal repeat heparin-binding epidermal growth factor Some of the most common scaffolds utilized in nature for producing high-affinity drug-like effectors are based on cyclized peptide architectures. Both naturally occurring and synthetically designed cyclic peptides have been successfully employed as drugs in man (1Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1947) Google Scholar, 2Vera M.D. Joullie M.M. Med. Res. Rev. 2002; 22: 102-145Crossref PubMed Scopus (137) Google Scholar, 3Trabi M. Craik D.J. Trends Biochem. Sci. 2002; 27: 132-138Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). These cyclic peptides often exhibit enhanced binding to macromolecules due to a restricted conformation space, and lowered conformational entropy loss upon binding and diminished proteolytic susceptibility (3Trabi M. Craik D.J. Trends Biochem. Sci. 2002; 27: 132-138Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar,4Schreiber S.L. Science. 2000; 287: 1964-1969Crossref PubMed Scopus (2226) Google Scholar). By utilizing the protein splicing and ligation properties of inteins, it is now possible to direct synthesis of diversity-oriented, cyclic peptide libraries in vivo (5Scott C.P. Abel-Santos E. Jones A.D. Benkovic S.J. Chem. Biol. 2001; 8: 801-815Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar).Here, we demonstrate the use of split inteins to create random cyclic peptides in mammalian cells using retroviral technology. Furthermore, we demonstrate the ability to test functionality of these in vivo cyclic peptide libraries in a genetic screen. For such a screen, we have chosen the IL-41 signaling pathway in the human B cell line, BJAB. IL-4 stimulation activates the germ line epsilon (ε) gene, a sterile transcript required for IgE isotype class switching (6Monticelli S. Vercelli D. Allergy. 2001; 56: 270-278Crossref PubMed Scopus (18) Google Scholar, 7Stavnezer J. Curr. Top. Microbiol. Immunol. 2000; 245: 127-168PubMed Google Scholar). Disruption of this pathway, or of ε promoter function, is a potential therapeutic approach for lowering IgE levels to ameliorate diseases such as allergy and asthma (8Oettgen H.C. Geha R.S. J. Allergy Clin. Immunol. 2001; 107: 429-440Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Some of the most common scaffolds utilized in nature for producing high-affinity drug-like effectors are based on cyclized peptide architectures. Both naturally occurring and synthetically designed cyclic peptides have been successfully employed as drugs in man (1Schreiber S.L. Crabtree G.R. Immunol. Today. 1992; 13: 136-142Abstract Full Text PDF PubMed Scopus (1947) Google Scholar, 2Vera M.D. Joullie M.M. Med. Res. Rev. 2002; 22: 102-145Crossref PubMed Scopus (137) Google Scholar, 3Trabi M. Craik D.J. Trends Biochem. Sci. 2002; 27: 132-138Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). These cyclic peptides often exhibit enhanced binding to macromolecules due to a restricted conformation space, and lowered conformational entropy loss upon binding and diminished proteolytic susceptibility (3Trabi M. Craik D.J. Trends Biochem. Sci. 2002; 27: 132-138Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar,4Schreiber S.L. Science. 2000; 287: 1964-1969Crossref PubMed Scopus (2226) Google Scholar). By utilizing the protein splicing and ligation properties of inteins, it is now possible to direct synthesis of diversity-oriented, cyclic peptide libraries in vivo (5Scott C.P. Abel-Santos E. Jones A.D. Benkovic S.J. Chem. Biol. 2001; 8: 801-815Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Here, we demonstrate the use of split inteins to create random cyclic peptides in mammalian cells using retroviral technology. Furthermore, we demonstrate the ability to test functionality of these in vivo cyclic peptide libraries in a genetic screen. For such a screen, we have chosen the IL-41 signaling pathway in the human B cell line, BJAB. IL-4 stimulation activates the germ line epsilon (ε) gene, a sterile transcript required for IgE isotype class switching (6Monticelli S. Vercelli D. Allergy. 2001; 56: 270-278Crossref PubMed Scopus (18) Google Scholar, 7Stavnezer J. Curr. Top. Microbiol. Immunol. 2000; 245: 127-168PubMed Google Scholar). Disruption of this pathway, or of ε promoter function, is a potential therapeutic approach for lowering IgE levels to ameliorate diseases such as allergy and asthma (8Oettgen H.C. Geha R.S. J. Allergy Clin. Immunol. 2001; 107: 429-440Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). We thank P. A. Achacoso, A. Martinez, and L. Dong for technical support; M. Aujay and M. Fox for DNA sequencing support; A. M. Friera, C. Young, and J. Warner for assistance with generation of screening cell line and screening strategy; and L. Tamayo and Carolyn Sousa for graphics and manuscript support.