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Volume 10, Issue 8 (Suppl) J Proteomics Bioinform, an open access journal
ISSN: 0974-276X Structural Biology 2017
September 18-20, 2017
9th International Conference on
Structural Biology
September 18-20, 2017 Zurich, Switzerland
Abdulrahman Alshehri et al., J Proteomics Bioinform 2017, 10:8(Suppl)
DOI: 10.4172/0974-276X-C1-0100
Generation of long acting therapies using glycosylated linkers
Abdulrahman Alshehri, R.J.Ross and I.R.Wilkinson
University of Sheffield, UK
Rationale: The current therapeutic drugs such as, growth hormone (GH), granulocyte colony-stimulating factor (GCSF) and
leptin require once-daily injections, which are inconvenient and expensive. Therefore, a number of approaches to reducing
therapeutic regimens clearance have been tried mainly through conjugation with another moiety. One such technology already
being employed is PEGylation; however this has been shown to be non-biodegradable and toxic. A previous study by Asterion
has shown that the use of glycosylated-linkers between two GH ligands to create protein-tandems resulted in their glycosylation
and an increased molecular weight (MW) whilst maintaining biological activity. The use of this technology using GCSF as an
example will be presented, but can be easily applied to other molecules such as leptin.
Hypothesis: The incorporation of variable glycosylated linkers between two GCSF ligands will create a construct with high
molecular weight and protected from proteolysis resulting in reduced clearance with out blocking bioactivity.
Methodology: GCSF tandems with linkers containing between 2-8 NAT glycosylation motifs and their respective controls
(Q replaces N in the sequence motif NAT so there is no glycosylation) were cloned, and sequenced. Following expression in
Chinese hamster ovary (CHO) cells, expressed protein was analysed by SDS-PAGE to confirm molecular weights. In vitro
bioactivity was tested using an AML-193 proliferation assay. Immobilised Metal Affinity Chromatography (IMAC) was used to
purify the protein. Pharmacokinetic and pharmacodynamics properties of the purified GCSF tandem proteins were measured
in normal Sprague Dawley rats with full ethical approval.
Results: Purified glycosylated tandems show increased molecular weight above that of controls when analysed by SDS-PAGE.
All GCSF tandems show increased bioactivity in comparison to native GCSF. Following intravenous administration to rats,
GCSF2NAT, GCSF4NAT, GCSF8NAT containing 2, 4 & 8 glycosylation sites respectively and GCSF8QAT (non‐glycosylated
GCSF tandem control) showed approximately 3‐fold longer circulating half‐life compared to that reported for the native GCSF
(1.79 hours). Both GCSF2NAT and GCSF4NAT show a significant increase in the percentage of neutrophils over controls at
12 hours post injection. This effect however is short lived as the counts at 24+ hours are not significantly different to controls.
GCSF8NAT shows an increase in the percentage of neutrophils that is only significant at 48 hours.
Conclusion: Results show that the use of glycosylated linkers to generate GCSF tandems results in molecules with increased
molecular weight, improved in vitro bioactivity, longer circulating half-lives and enhanced neutrophilic population when
compared to both native GCSF and the non-glycosylated tandem protein.
Biography
AbdulrahmanAlshehri working at security Forces Hospital, Riyadh, Saudi Arabia for 12 years. I did my Phd at sheffield University, UK. I have been working in creating
long acting therapies using different stratigies such as, glycosylated linkers. Using multiple techniquies like PCR, gene cloning, cell culture, pharmacodynamic and
pharmacokinetic to generate these therapies.
Figure1: An example of 2NAT glycosylation motifs
and its control 2QAT within a flexible linker (Gly4Ser)n
between two GCSF ligands. (A) The glycosylation motif
2NAT inserted to the linker (glycosylated linker). (B) Non-
glycosylation motif 2QAT control.