Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation

Abstract

Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery—either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Recent advances in enzyme-catalysed macrocyclisation include discovery of new enzymes or design of new engineered enzymes. Here, we describe the engineering of a self-cyclising “autocyclase” protein, capable of performing a controllable unimolecular reaction for generation of cyclic biomolecules in high yield. We characterise the self-cyclisation reaction mechanism, and demonstrate how the unimolecular reaction path provides alternative avenues for addressing existing challenges in enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple, alternative way to access a vast diversity of macrocyclic biomolecules.

An Autocatalytic Peptide Cyclase Improves Fidelity and Yield of Circular Peptides In Vivo and In Vitro

“…Another limitation was that sortase required a low concentration of autocatalyst because dimer formation was universal in sortase products, whereas only the best native substrate (patellin 2) was dimerized in the auto-G constructs, and dimerization was otherwise not observed . We propose that dimerization is facilitated when, as found in sortase, a part of the substrate is also a recognition sequence for cyclization.…”

“…This work was inspired by natural autocatalytic enzymes, such as the BURP cyclases, which connect aromatic amino acids in plant peptides, and the N -methyltransferases from omphalotin biosynthesis. − In addition, while we were completing this work, an engineered autocatalytic cyclase was reported that uses sortase as the cyclization enzyme . Sortase has several advantages in comparison with auto-G enzymes.…”

Designing nanodiscs as versatile platforms for on-demand therapy

“…A study reported in 2023 obtained covalently circularized NDs (cNDs) through the engineering of a self-cycling "autocyclase" protein, compared with both linear and cyclised NDs equipped with imaging agents, the in vivo imaging results showed that cNDs have better biodistribution and metabolism. 126 These NDs function as powerful tools for reporting membrane biochemical reactions, making them valuable in various applications. 43 4.5.2 Magnetic field nanoscalpel.…”

Enhancing Stability and Albumin Binding Efficiency of α-Conotoxin GI through Fatty Acid Modification

“…This unique property suggests that the α-conotoxin GI has the potential to be developed into a new type of muscle relaxant with the ability to ameliorate clinical symptoms of dystonia in the future. Nonetheless, it is essential to note that α-conotoxin GI, being a peptide drug, is prone to degradation by different proteases and has a high clearance rate in the bloodstream and kidneys, which results in poor stability in vivo and multiple limitations in practical applications. , Various chemical modifications have been utilized to enhance the stability of peptides and overcome associated challenges, such as cyclization, C-terminal modification, PEGylation, glycosylation, and fatty acid modification. − Among these strategies, fatty acid modification has shown unique advantages in enhancing the albumin binding capacity of drugs, improving drug stability, and prolonging half-life. − Therefore, it is considered an effective strategy to enhance peptide stability and extend the circulation time of peptide drugs. , …”