Eliminates Peptide Synthesis Bottlenecks to Deliver Highly Stable Macrocyclic Biologic Therapeutics

Background

The development of peptide macrocycles and covalently constrained proteins is a critical area of focus in both the pharmaceutical and industrial sectors. In drug discovery, structurally diverse cyclic peptides are highly sought after for high-throughput screening against challenging biological targets. Furthermore, there is a significant need to enhance the physical properties of therapeutic proteins and industrial enzymes, such as those used in laundry detergents and food processing. Covalent cyclization addresses this need by dramatically improving essential characteristics like serum half-life, protease resistance, and overall thermal stability, allowing these molecules to function effectively under harsh physiological or chemical conditions.

Despite the clear advantages of constrained peptides, current manufacturing methods face significant limitations. Traditional solid-phase peptide synthesis is the standard approach, but it is inherently difficult to scale and lacks cost-effectiveness for large-scale production. Furthermore, chemical synthesis struggles to accommodate large, structured protein domains or full globular proteins without disrupting their delicate tertiary structures and biological functions. Existing techniques also offer limited flexibility in generating diverse macrocyclic linkages, often requiring complex, multi-step chemical modifications. Consequently, generating expansive, structurally diverse cyclic libraries remains a cumbersome and inefficient bottleneck for researchers.

Technology

This technology is a versatile enzymatic platform utilizing the ATP-grasp enzyme ThfB to catalyze peptide and protein cyclization. It employs engineered substrates with a modular “stem-and-loop” architecture. The stem contains specific donor residues and acidic acceptor residues, while the loop accommodates sequences ranging from short peptides to full globular proteins. By varying these residues, the platform forms diverse covalent linkages, including ω-ester, ω-amide, and ω-thioester bonds. Thioester bonds uniquely enable downstream native chemical ligation or spontaneous rearrangements for head-to-tail cyclization. The process is highly adaptable, functioning both in vivo via Escherichia coli co-expression and in vitro.

This solution is differentiated by providing a scalable, cost-effective alternative to traditional solid-phase peptide synthesis that accommodates exceptionally large protein domains. Unlike conventional methods, it cyclizes entire functional proteins without disrupting their tertiary structure or biological activity. The enzyme’s remarkable substrate promiscuity allows for the rapid generation of structurally diverse, covalently constrained macrocyclic libraries for high-throughput drug discovery. By structurally constraining these molecules, the technology dramatically improves their serum half-life, protease resistance, and thermal stability. Ultimately, it delivers profound advantages for developing robust therapeutics and stabilizing industrial enzymes designed to withstand harsh environmental conditions.

Benefits

• Significantly enhances the thermal stability, protease resistance, and serum half-life of therapeutic proteins and industrial enzymes.
• Provides a scalable and cost-effective alternative to traditional solid-phase peptide synthesis.
• Features a highly versatile “stem-and-loop” architecture that can accommodate large sequence insertions, including full globular proteins, without disrupting their biological function or tertiary structure.
• Offers flexible production options, allowing the cyclization process to be performed either in vivo through bacterial co-expression or in vitro using purified components.
• Supports the installation of diverse chemical bonds (ester, amide, and thioester) and enables advanced downstream modifications, such as Native Chemical Ligation (NCL) and head-to-tail cyclization.
• Facilitates the rapid generation of structurally diverse and highly constrained macrocyclic peptide libraries for high-throughput drug discovery and screening.

Applications

• Macrocyclic peptide drug discovery
• Therapeutic protein stability enhancement
• Industrial enzyme thermal stabilization
• Biologic drug half-life extension
• High-throughput peptide library screening

Additional info

This enzymatic platform utilizes the substrate-promiscuous ATP-grasp enzyme ThfB from *Thermobifida fusca* to catalyze peptide and protein cyclization. Using engineered stem-and-loop substrates, ThfB facilitates side-chain-to-side-chain or head-to-tail linkages, forming stable $\omega$-ester, $\omega$-amide, or $\omega$-thioester bonds. This versatile system enables *in vivo* or *in vitro* generation of structurally constrained macrocycles and stabilized proteins for therapeutic and industrial applications.