Advanced Bioconjugation Technologies: A Dual-drug Candidate for Treatment of Type 2 Diabetes

Bioconjugation is a powerful tool for the introduction of diverse functionalities to biomolecules, enabling the modulation of their properties. This is important for a wide array of applications, including targeted drug delivery and the study of cellular processes. However, site-selective bioconjugation remains challenging, especially when targeting one specific natural amino acid residue in a wild-type protein due to high abundance of each amino acid.
In this thesis, two novel methods for sequence-specific, site-selective bioconjugation of serine has been investigated. Both strategies utilize phosphor-based electrophilic coupling partners for reaction through either a pyrophosphorylation (Strategy 1) or phosphorylation (Strategy 2). A library of peptides was synthesized and screened for reactivity to identify a sequence, amenable to sequence-specific bioconjugation. It was observed that Strategy 2 displayed superior reactivity and selectivity when using fluorophosphonates as electrophiles, with consistent conversion being above 90 % on the key sequence with little to no reaction on control peptides. The high conversion allows for use in recombinant proteins with the key sequence installed.
Two dual-drug candidates were designed consisting of a GLP-1 receptor agonist (GLP-1RA) and a GIP receptor (GIPR) antagonist for the treatment of Type 2 Diabetes (T2D). Combination treatment targeting the receptors of the two incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose dependent insulinotropic polypeptide (GIP), have shown great promise for treatment obesity-related diabetes. The dual-drug candidate I connects a peptide GIPR antagonist to a small molecule GLP-1RA through a lipase-cleavable linker for the stable release of the drug in plasma. Various linkers have been synthesized and are awaiting enzyme studies for the determination of release profiles.
For dual-drug candidate II a GIPR antagonist peptide containing the key sequence was reacted with the fluorophosphonate to introduce an azido-handle in 76 % conversion. The final step of the synthesis of the dual-drug candidate II would be the attachment of an alkyne functionalized GLP-1RA peptide.
In close relation to T2D, insulin resistance or dysfunction of insulin signaling in the brain is observed in patients suffering from Alzheimer’s Disease (AD). This has led to AD being classified as Type 3 Diabetes (T3D). GLP-1RAs have shown promise as neuroprotective agents, but access to the brain is hampered by the low penetrability of the brain barriers. Two fusion proteins, utilizing a novel blood-cerebrospinal fluid barrier (BCSFB) shuttle peptide, were tested in vivo in mice models using lean and diet-induced obesity (DIO) mice. The fusion proteins, consisting of a GLP-1RA and the shuttle peptide, showed significantly decreased acute food intake compared to the GLP-1RA alone, indicating that the shuttle peptide improves brain delivery.