title: Site-Specific Transglutaminase-Mediated Conjugation of Interferon α-2b at Glutamine or Lysine Residues.
abstract: Interferon α (IFN α) subtypes are important protein drugs that have been used to treat infectious diseases and cancers. Here, we studied the reactivity of IFN α-2b to microbial transglutaminase (TGase) with the aim of obtaining a site-specific conjugation of this protein drug. Interestingly, TGase allowed the production of two monoderivatized isomers of IFN with high yields. Characterization by mass spectrometry of the two conjugates indicated that they are exclusively modified at the level of Gln101 if the protein is reacted in the presence of an amino-containing ligand (i.e., dansylcadaverine) or at the level of Lys164 if a glutamine-containing molecule is used (i.e., carbobenzoxy-l-glutaminyl-glycine, ZQG). We explained the extraordinary specificity of the TGase-mediated reaction on the basis of the conformational features of IFN. Indeed, among the 10 Lys and 12 Gln residues of the protein, only Gln101 and Lys164 are located in highly flexible protein regions. The TGase-mediated derivatization of IFN was then applied to the production of IFN derivatives conjugated to a 20 kDa polyethylene glycol (PEG), using PEG-NH2for Gln101 derivatization and PEG modified with ZQG for Lys164 derivatization. The two mono-PEGylated isomers of IFN were obtained in good yields, purified, and characterized in terms of protein conformation, antiviral activity, and pharmacokinetics. Both conjugates maintained a native-like secondary structure, as indicated by far-UV circular dichroism spectra. Importantly, they disclosed good in vitro antiviral activity retention (about only 1.6- to 1.8-fold lower than that of IFN) and half-lives longer (about 5-fold) than that of IFN after intravenous administration to rats. Overall, these results provide evidence that TGase can be used for the development of site-specific derivatives of IFN α-2b possessing interesting antiviral and pharmacokinetic properties.
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua , via Francesco Marzolo 5, 35131 Padua, Italy.|CRIBI Biotechnology Centre, University of Padua , viale Giuseppe Colombo 3, 35121 Padua, Italy.
authors.forename: Barbara
authors.initials: B
authors.lastname: Spolaore
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua , via Francesco Marzolo 5, 35131 Padua, Italy.
authors.forename: Samanta
authors.initials: S
authors.lastname: Raboni
authors.affiliation: CRIBI Biotechnology Centre, University of Padua , viale Giuseppe Colombo 3, 35121 Padua, Italy.
authors.forename: Abhijeet A
authors.initials: AA
authors.lastname: Satwekar
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua , via Francesco Marzolo 5, 35131 Padua, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Grigoletto
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua , via Francesco Marzolo 5, 35131 Padua, Italy.
authors.forename: Anna
authors.initials: A
authors.lastname: Mero
authors.affiliation: Veneto Institute of Oncology IOV - IRCCS , via Gattamelata 64, I-35128 Padua, Italy.
authors.forename: Isabella Monia
authors.initials: IM
authors.lastname: Montagner
authors.affiliation: Veneto Institute of Oncology IOV - IRCCS , via Gattamelata 64, I-35128 Padua, Italy.|Department of Surgery, Oncology, and Gastroenterology, University of Padua , via Nicolò Giustiniani 2, 35124 Padua, Italy.
authors.forename: Antonio
authors.initials: A
authors.lastname: Rosato
authors.affiliation: Department of Pharmaceutical and Pharmacological Sciences, University of Padua , via Francesco Marzolo 5, 35131 Padua, Italy.|Veneto Institute of Oncology IOV - IRCCS , via Gattamelata 64, I-35128 Padua, Italy.
authors.forename: Gianfranco
authors.initials: G
authors.lastname: Pasut
authors.affiliation: CRIBI Biotechnology Centre, University of Padua , viale Giuseppe Colombo 3, 35121 Padua, Italy.
authors.forename: Angelo
authors.initials: A
authors.lastname: Fontana
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000998:Antiviral Agents / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D001665:Binding Sites; D005973:Glutamine / Q000737:chemistry*; D006801:Humans; D000077190:Interferon alpha-2; D016898:Interferon-alpha / Q000737:chemistry* / Q000493:pharmacokinetics / Q000494:pharmacology; D008239:Lysine / Q000737:chemistry*; D008958:Models, Molecular; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000737:chemistry; D017433:Protein Structure, Secondary; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000493:pharmacokinetics / Q000494:pharmacology; D013379:Substrate Specificity; D011503:Transglutaminases / Q000378:metabolism*; D018116:Vesiculovirus / Q000187:drug effects
chemical_list: D000998:Antiviral Agents; D000077190:Interferon alpha-2; D016898:Interferon-alpha; D011994:Recombinant Proteins; D005973:Glutamine; D011092:Polyethylene Glycols; D011503:Transglutaminases; D008239:Lysine