Applications of sortase A in disulfide-rich peptide engineering

Abstract

While the global peptide drug market has been constantly growing, the intrinsic limitations of peptides, such as chemical and physical instability and poor oral bioavailability, continue to require innovative peptide engineering to facilitate the uptake of peptides by the pharmaceutical industry. In recent years, plant-derived cyclotides have been investigated for this purpose because cyclotides are remarkably stable against chemical, enzymatic or thermal attack. The stability of cyclotides is derived from their unique structure, which comprises a head-to-tail cyclized backbone and three disulfide bonds forming a knotted core. Although the overall structure of cyclotides is rigid, certain loops are ‘plastic’ enough to accommodate their synthetic replacement with a foreign peptide epitope. More than a dozen published studies have already demonstrated that bioactive peptide sequences can be grafted onto cyclotide scaffolds and thereby stabilized, while maintaining activity. Following lessons learnt from studies of cyclotides, other naturally occurring disulfide-rich cyclic peptides, including sunflower trypsin inhibitor I (SFTI-1) and θ-defensins, have been studied for similar purposes. Furthermore, cyclization has been applied to disulfide-rich neurotoxin peptides, derived from cone snails or scorpions, and has resulted in improved therapeutic potential. This thesis investigates a range of these natural peptides for their role as drug templates and explores novel approaches to produce them. The benefits of engineering cyclotides and other disulfide-rich peptides for therapeutic purposes can be maximized by efficient production systems. In this thesis, an efficient strategy to produce disulfide-rich cyclic peptides is investigated using the enzyme sortase A. Sortase A, originally discovered in Staphylococcus aureus, recognizes a penta-amino acid motif, LPXTG, and cleaves the peptide bond between Thr and Gly to form a thioacyl intermediate. This intermediate undergoes nucleophilic attack by an N-terminal poly-Gly sequence, forming a new peptide bond between the Thr and N-terminal Gly. This site-specific ligation (transpeptidation) activity of sortase A allowed the successful cyclization of a synthetic cyclotide precursor in a straightforward and safe manner compared to traditional chemical methods. The semi-enzymatic method was also readily applied to produce other disulfide-rich cyclic peptides, including SFTI-1, cVc1.1, and cyclic chlorotoxin. Furthermore, by considering cyclotides in a structural model, cyclization of the κ-conotoxin PVIIA originating from the venom of Conus purpurascens was also explored. Interestingly, κ-PVIIA and other conopeptides, which belong to the O1 gene superfamily of conotoxins, are topologically very similar to cyclotides except that their backbones are linear. In this thesis, cyclization of κ-PVIIA was studied as a proof-of-concept for other conopeptides in the O1 superfamily. Furthermore, this study provides insights into successful cyclization by suggesting factors to be considered to obtain the desired peptide activity. Even with enzymatic cyclization, chemical synthesis of disulfide-rich peptides is not as cost-effective as recombinant expression in large scale manufacturing. Therefore, recombinant expression of cyclotides, which are 28-37 amino acid long, is desirable for them to be attractive as potential therapeutics. In this thesis, bacterial periplasmic expression of kalata B1 precursor was investigated. To aid solubility of hydrophobic kalata B1 in the bacterial periplasm, maltose binding protein (MBP) was used as a fusion protein and sorting motifs were placed at both N and C terminus of kalata B1 sequence for subsequent cleavage from MBP and head-to-tail cyclization of kalata B1. Lastly, sortase A-mediated engineering was used to enable targeted delivery of cyclotides via conjugation to a single-domain antibody fragment (VHH). A non-natural amino acid, azidolysine, was incorporated during cyclotide synthesis, and bacterially expressed anti MHC Class II VHH was equipped with cyclooctyne using sortase A to enable a click reaction between them. The conjugation resulted in the improvement in immunogenicity of cyclotides by targeting antigen presenting cells. Overall, this strategy facilitates the application of a targeted delivery of cyclotides for therapeutic purposes.