ABSTRACT
Type I IFNs are unusually pleiotropic cytokines that bind to a single heterodimeric receptor and have potent antiviral, antiproliferative, and immune modulatory activities. The diverse effects of the type I IFNs are of differential therapeutic importance; in cancer therapy, an enhanced antiproliferative effect may be beneficial, whereas in the therapy of viral infections (such as hepatitis B and hepatitis C), the antiproliferative effects lead to dose limiting bone marrow suppression. Studies have shown that various members of the natural IFN-alpha family and engineered variants, such as IFN-con1, vary in the ratios between various IFN-mediated cellular activities. We used DNA shuffling to explore and confirm the hypothesis that one could simultaneously increase the antiviral and Th1-inducing activity and decrease the antiproliferative activity. We report IFN-alpha hybrids wherein the ratio of antiviral:antiproliferative and Th1-inducing: antiproliferative potencies are markedly increased with respsect to IFN-con1 (75- and 80-fold, respectively). A four-residue motif that overlaps with the IFNAR1 binding site and is derived by cross breeding with a pseudogene contributes significantly to this phenotype. These IFN-alphas have an activity profile that may result in an improved therapeutic index and, consequently, better clinical efficacy for the treatment of chronic viral diseases such as hepatitis B virus, human papilloma virus, HIV, or chronic hepatitis C.
Subject(s)
Chronic Disease/therapy , DNA Shuffling , Directed Molecular Evolution , Interferon-alpha/genetics , Virus Diseases/therapy , Amino Acid Motifs , Amino Acid Sequence , Animals , Antiviral Agents/pharmacology , CHO Cells , Cricetinae , Cricetulus , Gene Library , HeLa Cells , Humans , Interferon-alpha/chemistry , Interferon-alpha/pharmacology , Models, Molecular , Molecular Sequence Data , Protein Binding , Pseudogenes , Th1 Cells/drug effectsABSTRACT
The herbicide glyphosate is effectively detoxified by N-acetylation. We screened a collection of microbial isolates and discovered enzymes exhibiting glyphosate N-acetyltransferase (GAT) activity. Kinetic properties of the discovered enzymes were insufficient to confer glyphosate tolerance to transgenic organisms. Eleven iterations of DNA shuffling improved enzyme efficiency by nearly four orders of magnitude from 0.87 mM-1 min-1 to 8320 mM-1 min-1. From the fifth iteration and beyond, GAT enzymes conferred increasing glyphosate tolerance to Escherichia coli, Arabidopsis, tobacco, and maize. Glyphosate acetylation provides an alternative strategy for supporting glyphosate use on crops.
Subject(s)
Acetyltransferases/genetics , DNA Shuffling , Directed Molecular Evolution , Glycine/analogs & derivatives , Glycine/toxicity , Herbicides/toxicity , Plants, Genetically Modified , Acetylation , Acetyltransferases/chemistry , Acetyltransferases/metabolism , Amino Acid Sequence , Bacillus/enzymology , Catalysis , Drug Resistance , Escherichia coli/genetics , Gene Library , Genetic Variation , Glycine/metabolism , Herbicides/metabolism , Kinetics , Molecular Sequence Data , Mutagenesis , Plants, Genetically Modified/drug effects , Plants, Genetically Modified/genetics , Recombinant Proteins/metabolism , Recombination, Genetic , Nicotiana/drug effects , Nicotiana/genetics , Nicotiana/growth & development , Transformation, Genetic , Zea mays/drug effects , Zea mays/genetics , Zea mays/growth & development , GlyphosateABSTRACT
Recombinant human interferon (IFN)-alpha was the first biotherapeutic agent approved by the US Food and Drug Administration for the treatment of a human malignancy. Its efficacy has also been demonstrated for treatment of several viral diseases. The human genome codes for 12 IFN-alpha proteins, with IFN alpha-1 and IFN alpha-2 accounting for the majority of the naturally occurring IFN-as. However, only subspecies of IFN alpha-2, recombinant human IFN alpha-2a and IFN alpha-2b, are commercially available in the United States. Other species of IFN-a may demonstrate equivalent or improved efficacy and have more tolerable side effects. This article describes ongoing preclinical and clinical studies of several new and modified IFN-alphas. A current phase I trial of a human recombinant IFN alpha-1 is described. Basic pharmacokinetics and clinical studies of polyethylene glycol (PEG) IFN alpha-2b are reviewed as well. Lastly, two novel types of IFN-a, one gene shuffled and one hybridized with human albumin, are summarized.
