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-NH for 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.
Microbial transglutaminase (mTGase) is an enzyme that catalyzes site-specific protein derivatization at specific glutamines. mTGase-mediated conjugation with PEG-NH2 to granulocyte colony stimulating factor (G-CSF) yields a site selective mono-derivative conjugate involving Gln135. The same enzymatic reaction of mTGase, i.e. the transfer of the Gln acyl group to an amino donor, was investigated by reversing the substrates. A specific acyl donor PEG derivative was synthesized by coupling the Z-QG mTGase substrate to PEG. The mTGase-mediated conjugation of this PEG-ZQG in the presence of G-CSF generated a high-yield PEG-G-CSF conjugate in which the polymer was selectively coupled to Lys41 of the protein. The PEG-K41-G-CSF conjugate was compared with the PEG-Q135-G-CSF one obtained through mTGase conjugation of PEG-NH2 to Gln135. Biophysical characterization showed that the two positional isomers have similar behaviors, and pharmacokinetic studies in rats demonstrated that they have comparable half-life extensions. Overall, the study demonstrates that mTGase protein derivatization is linked to inherent advantages because it carries with it the possibility of targeting lysines or glutamines, in both cases with a high site-selective specificity
The use of therapeutic proteins is often impaired by their short in vivo half-lives. PEGylation has been exploited to enhance protein stability and to prolong the pharmacokinetic. The biophysical characterization of two site-specific mono-PEGylated forms of human growth hormone (hGH)--chemically N-terminal PEGylated hGH (PEG-Nter-hGH) and enzymatically Gln141 PEGylated hGH (PEG-Gln141-hGH) via transglutaminase--is outlined here and their pharmacodynamics are compared. The thermal stability of PEG-Nter-hGH was increased with respect to that of hGH and PEG-Gln141-hGH. Pharmacodynamic studies in rats showed that a single injection of the conjugates had a better or comparable potency with respect to a daily hGH on a week schedule in terms of weight gain, femoral length, and tibial diaphysis width.
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