Todd M. Kinsella

The nuclear replication and retention functions of the Epstein–Barr virus (EBV) have been utilized here to maintain retroviral constructs episomally within human cell-based retroviral packaging lines. These hybrid EBV/retroviral constructs are capable of producing helper-free recombinant retrovirus as soon as 48 hr and for at least 30 days after transfection into 293T-based ecotropic and/or amphotropic retroviral packaging cells. Viral titers greater than 107 TU/ml were obtained after puromycin selection of transfected retroviral packaging cells. This episomal approach to retroviral production circumvents some limitations inherent in transient and chromosomally stable retroviral producer systems, affording reproducibly rapid, large-scale, stable, and high-titer retrovirus production. Producing high-titer retrovirus can be a limiting factor in the successful application of retroviruses for gene transduction. A retroviral producer system capable of both rapid and long-term virus production would circumvent many of the restrictions inherent to chromosomal-stable and/or transient-based production methods. It is demonstrated here that replication and nuclear retention functions of the human Epstein–Barr virus (EBV) can confer stable episomal maintenance to retroviral constructs within human cell-based packaging lines. Such hybrid EBV/retroviral constructs permit both rapid and long-term production of high-titer retrovirus. Furthermore, it is demonstrated that these constructs are not prone to arrangements when maintained episomally in packaging cells and that viral stocks are helper-virus free.

Ligand-induced protein degradation has emerged as a compelling approach to promote the targeted elimination of proteins from cells by directing these proteins to the ubiquitin-proteasome machinery. So far, only a limited number of E3 ligases have been found to support ligand-induced protein degradation, reflecting a dearth of E3-binding compounds for proteolysis-targeting chimera (PROTAC) design. Here, we describe a functional screening strategy performed with a focused library of candidate electrophilic PROTACs to discover bifunctional compounds that degrade proteins in human cells by covalently engaging E3 ligases. Mechanistic studies revealed that the electrophilic PROTACs act through modifying specific cysteines in DCAF11, a poorly characterized E3 ligase substrate adaptor. We further show that DCAF11-directed electrophilic PROTACs can degrade multiple endogenous proteins, including FBKP12 and the androgen receptor, in human prostate cancer cells. Our findings designate DCAF11 as an E3 ligase capable of supporting ligand-induced protein degradation via electrophilic PROTACs.

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.

Modulation of mitochondrial function through inhibiting respiratory complex I activates a key sensor of cellular energy status, the 5'-AMP-activated protein kinase (AMPK). Activation of AMPK results in the mobilization of nutrient uptake and catabolism for mitochondrial ATP generation to restore energy homeostasis. How these nutrient pathways are affected in the presence of a potent modulator of mitochondrial function and the role of AMPK activation in these effects remain unclear. We have identified a molecule, named R419, that activates AMPK in vitro via complex I inhibition at much lower concentrations than metformin (IC50 100 nM vs 27 mM, respectively). R419 potently increased myocyte glucose uptake that was dependent on AMPK activation, while its ability to suppress hepatic glucose production in vitro was not. In addition, R419 treatment of mouse primary hepatocytes increased fatty acid oxidation and inhibited lipogenesis in an AMPK-dependent fashion. We have performed an extensive metabolic characterization of its effects in the db/db mouse diabetes model. In vivo metabolite profiling of R419-treated db/db mice showed a clear upregulation of fatty acid oxidation and catabolism of branched chain amino acids. Additionally, analyses performed using both (13)C-palmitate and (13)C-glucose tracers revealed that R419 induces complete oxidation of both glucose and palmitate to CO2 in skeletal muscle, liver, and adipose tissue, confirming that the compound increases mitochondrial function in vivo. Taken together, our results show that R419 is a potent inhibitor of complex I and modulates mitochondrial function in vitro and in diabetic animals in vivo. R419 may serve as a valuable molecular tool for investigating the impact of modulating mitochondrial function on nutrient metabolism in multiple tissues and on glucose and lipid homeostasis in diabetic animal models.