Cyclic Peptide Design

What is the Cyclic Peptide design?

The construction of cyclic peptides is a potent strategy in drug discovery, biomolecular engineering, and synthetic biology. The process entails the creation of peptides that assume a cyclic conformation, conferring stability, resistance to degradation, and selective binding to molecular targets. The cyclic configuration, formed by covalent connections at the peptide termini or among side chains, augments their bioactivity and therapeutic efficacy. Cyclic peptides has various advantageous characteristics, including high binding affinity, target selectivity, and low toxicity, rendering them a compelling option for medicinal development. Over 40 cyclopeptide pharmaceuticals are being utilized in clinical settings, with an average of approximately one novel cyclopeptide medicine introduced to the market year. The predominant number of therapeutically authorized cyclic peptides originate from natural sources, including antibacterial medicines and human peptide hormones. Innovative methodologies grounded on rational design and in vitro evolution enable the de novo development of cyclic peptide ligands to address targets for which natural solutions are unavailable.

Fig.1 Visual depiction of the procedure for cyclic peptide design. (Thakkar R., et al., 2023)

1. The advantages of Cyclic Peptide

(1) Increased stability and bioavailability: The cyclic configuration safeguards peptides against proteolytic degradation, hence enhancing their stability in biological contexts. This is particularly crucial for therapeutic applications that need prolonged circulation periods. Cyclic peptides have greater resistance to breakdown in the circulation and gastrointestinal tract than linear peptides, rendering them more appropriate for oral or systemic administration.

(2) Improved binding affinity: Cyclic peptides are very selective and have a high affinity for proteins due to their huge surface area. Cyclic peptides' limited structural flexibility allows them to pre-organize their active conformation, which in turn increases their binding affinity to molecular targets. Because of this, they may effectively activate or inhibit certain proteins. In addition to improving their binding characteristics, the reduced entropy loss during binding is another benefit of macroring structures' limited conformational flexibility.

(3) Less toxic: There is often minimal to no toxicity in cyclic peptides due to the innocuous amino acid makeup.

(4) Easy to produce: Aside from being amenable to processing, characterization, and modification, cyclic peptides are also simple to synthesize via automated chemical synthesis.

Cyclic peptides design strategies

In the field of cyclic peptide design, two primary methods are now in use. Intentional alterations to known cyclic analogues, which may or may not possess affinity for a target, constitute the first method. To ensure that the linear peptide's activity is preserved after cyclization, rational design necessitates an understanding of the function of each residue. In order to generate better molecules for diverse targets, some have manipulated or grafted natural cyclotides or altered cyclic hormones (such as melanocortin, gonadotropin releasing hormone, or somatostatin). In several instances, improved pharmacological drugs were synthesized by cyclizing linear peptides; these compounds frequently targeted protein-protein interactions (PPIs). When there is limited information regarding the target (such as a receptor) or the ligand (the CP to be created), the second method is employed: (a) screening approaches to find biological function; (b) synthesis of libraries based on linear peptides; and (c) mutagenesis studies driven by trial and error, of which make up this second strategy, which employs combinatorial syntheses to identify lead compounds. The problem of library creation has been examined by several organizations; the most popular ways for creating screening libraries include phage display and combinatorial approaches. These two methods are compatible and can be used in combination. Optimizing lead compounds found by combinatorial synthesis to find the optimal analogue can be achieved using reasonable techniques. Iterative replacement of each amino acid can also optimize peptides developed by rational procedures. Examples of cyclic peptides with pharmacological action have been successfully produced by combining the two procedures.

Fig.2 Main approaches to create cyclic peptides. (Claro B., et al., 2018)

Approaches to the preparation of cyclic peptides (CPs)

1. The genetic methods include phage display, intein-based CPs, and mRNA display.

(1) Phage display technique involves inserting a gene that encodes a protein of interest into a phage coat protein gene. This causes the phage to possess the gene for the protein or peptide on its inside, while also displaying it on its surface. The N-, middle-, or C-termini of coat proteins typically include peptides that are not intended to be cyclic. Nevertheless, disulfide bridge formation can be utilized to achieve cyclization. One major drawback of this approach is that phage display can only be used with naturally occurring ribosomal amino acids. The utilization of phage display to produce screening libraries is a crucial aspect of this technology. Additionally, computational techniques and databases have been useful in the research of phage displays.

(2) Inteins are protein sequences that are excised during maturation. SICLOPPS (split-intein circuit ligation of peptides and proteins) is an effective method for creating CP libraries utilizing a trans-intein DnaE protein. The CP derived from SICLOPPS adopts a generic structure of cyclo (XA1A2A3...An), with X representing either Cys or Ser. The primary drawbacks of these approaches are the necessity for a Cys (or Ser) residue in the sequences, the restriction to ribosomal amino acids, and the fact that CPs can only be synthesized in vivo.

(3) mRNA method, recently modified CPs may be generated via the in vitro approach of mRNA display, which involves displaying peptides or proteins attached to the encoding mRNA. Issues with its efficiency, inability to complete the cyclization procedure in sequences containing numerous Lys residues, and the formation of unwanted dimers between two mRNA peptide hybrid molecules facilitated by the crosslinker are among its limitations.

Fig.3 Genetic methods to produce cyclic peptides (CPs). (Claro B., et al., 2018)

2. The synthetic method for cyclic peptides includes chemical synthesis strategies in solution and solid-phase.

It is common practice to manufacture individual peptides using the solution-phase peptide synthesis technique. Peptide synthesis relies on the selective deprotection of orthogonally protected precursors at certain functional groups, which forms the basis of popular synthetic techniques. However, solid-phase peptide synthesis has nearly entirely replaced solution-phase synthesis in most research labs because to the lengthy synthesis time and the tedious purification, which typically reduces overall yields. The fundamental benefit of solid-supported peptide synthesis and macrocyclizations is that adequate purification is generally achieved with only simple washing and filtering. One typical method for anchoring the linear precursor to a solid support for peptide cyclization is the side chain of a trifunctional amino acid. To build the lin ear peptide, deprotect the N- and C-termini, cyclize head-to-tail, and cleave the product from the solid support, a protecting-group method with at least three orthogonal dimensions is needed. The synthesis of big polypeptides is now made easier and more efficient with the introduction of new, innovative, and uncomplicated techniques that include solid-phase peptide synthesis followed by in-solution fragment coupling.

After the carboxyl group of the first amino acid is attached to the solid support, the NPG-amino protecting group, is removed. Following this, the activated carboxyl group of the second amino acid is associated with the amino group, and this process continues for the subsequent amino acids. In addition to the selective removal of the amino group protectors from the side chains, the resins themselves cleave. The cyclization process begins with an amino group on the side chain and ends with a carboxyl group at the C-terminus. The protected groups' side chains are deprotected to complete the cyclization.

Fig.4 Cyclic peptide side chain features improving permeability. (Goetz., et al., 2019)

Fig.5 Cyclic peptide backbone features improving permeability. (Goetz., et al., 2019)

Fig.6 Visual representation of the solid-state synthesis of a side chain-to-tail cyclic peptides. (Claro B., et al., 2018)

3. Cyclization strategy for cyclic peptides

There are a number of ways to cycle a peptide after the chain is complete: Cyclization can be accomplished in three ways: (A) A peptide bond between the C-terminus and N-terminus allows for head-to-tail cyclization, which is convenient but limited in options; (B) Bioactive functional groups in the amino acids of interest can influence biological activity; cyclization within biologically active groups, such as a disulfide bond among cysteine residues or an amide bond between the side chains of lysine and aspartic or glutamic acid, or the N-or C-terminus; and (C) Add amino acids or other building blocks to the bioactive sequence in order to achieve cyclization without disrupting it. Their widespread introduction is due to the fact that specialized libraries may be produced without altering the sequence of interest.

Fig.7 Typical peptide cyclization. (Buckton L K., et al., 2021)

Fig.8 Alternative peptide cyclization strategies. (Qvit N., et al., 2016)

References

1. Qvit N., et al., Development of a backbone cyclic peptide library as potential antiparasitic therapeutics using microwave irradiation, Journal of Visualized Experiments: Jove, 2016 (107).
2. Goetz., et al., Cyclic Peptide Design, New York, NY: Springer New York, 2019.
3. Thakkar R., et al., Computational design of a cyclic peptide that inhibits the CTLA4 immune checkpoint, RSC Medicinal Chemistry, 2023, 14(4): 658-670.
4. Buckton L K., et al., Cyclic peptides as drugs for intracellular targets: the next frontier in peptide therapeutic development, Chemistry-A European Journal, 2021, 27(5): 1487-1513.
5. Claro B., et al., Design and applications of cyclic peptides[M]//Peptide applications in biomedicine, biotechnology and bioengineering. Woodhead Publishing, 2018: 87-129.