title: A drug delivery strategy: binding enkephalin to asialoglycoprotein receptor by enzymatic galactosylation.
abstract: Glycosylation of biopharmaceuticals can mediate cell specific delivery by targeting carbohydrate receptors. Additionally, glycosylation can improve the physico-chemical (drug-like) properties of peptide based drug candidates. The main purpose of this study was to examine if glycosylation of the peptide enkephalin could facilitate its binding to the carbohydrate receptor, asialoglycoprotein. Firstly, we described the one-pot enzymatic galactosylation of lactose modified enkephalin in the presence of uridine-5'-diphosphogalactose 4-epimerase and lipopolysaccharyl α-1,4-galactosyltransferase. Stability experiments using human plasma and Caco-2 cell homogenates showed that glycosylation considerably improved the stability of enkephalin (at least 60% remained stable after a 2 hr incubation at 37°C). In vitro permeability experiments using Caco-2 cells revealed that the permeability of mono- and trisaccharide conjugated enkephalins was 14 and 28 times higher, respectively, than that of enkephalin alone (Papp 3.1×10-8 cm/s). By the methods of surface plasmon resonance and molecular modeling, we demonstrated that the enzymatic glycosylation of enkephalin enabled binding the asialoglycoprotein receptor. The addition of a trisaccharide moiety to enkephalin improved the binding of enkephalin to the asialoglycoprotein receptor two fold (KD = 91 µM). The docking scores from molecular modeling showed that the binding modes and affinities of the glycosylated enkephalin derivatives to the asialoglycoprotein receptor complemented the results from the surface plasmon resonance experiments.
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Michelle P
authors.initials: MP
authors.lastname: Christie
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Pavla
authors.initials: P
authors.lastname: Simerská
authors.affiliation: Institute for Glycomics, Griffith University, Southport, Queensland, Australia.
authors.forename: Freda E-C
authors.initials: FE
authors.lastname: Jen
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Waleed M
authors.initials: WM
authors.lastname: Hussein
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia.
authors.forename: Mohamad F M
authors.initials: MF
authors.lastname: Rawi
authors.affiliation: Institute for Glycomics, Griffith University, Southport, Queensland, Australia.
authors.forename: Lauren E
authors.initials: LE
authors.lastname: Hartley-Tassell
authors.affiliation: Institute for Glycomics, Griffith University, Southport, Queensland, Australia.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Day
authors.affiliation: Institute for Glycomics, Griffith University, Southport, Queensland, Australia.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Jennings
authors.affiliation: School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, Queensland, Australia; School of Pharmacy, The University of Queensland, Pharmacy Australia Centre of Excellence, Woolloongabba, Queensland, Australia.
authors.forename: Istvan
authors.initials: I
authors.lastname: Toth
mesh_terms: D037263:Asialoglycoprotein Receptor / Q000737:chemistry / Q000378:metabolism*; D018938:Caco-2 Cells; D002241:Carbohydrates / Q000737:chemistry; D016503:Drug Delivery Systems; D004745:Enkephalins / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006801:Humans; D008958:Models, Molecular; D010539:Permeability; D011485:Protein Binding; D011487:Protein Conformation; D020349:Surface Plasmon Resonance
chemical_list: D037263:Asialoglycoprotein Receptor; D002241:Carbohydrates; D004745:Enkephalins
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