Peptides, cyclic or linear, are polymers made of amino acid residues connected by peptide bonds. A cyclic peptide, like a linear one, contains both amide C and N atoms, but it does not have a N- or C-terminal end due to its closed ring conformation. These topological differences significantly impact biological activities, stability, and interactions with biological targets.
Structurally, cyclic peptides differ from linear peptides. Cyclic peptides have physicochemical properties, structural preferences and biological activities which are not often solely the consequence of their closed topology, but of a different organization of these properties compared to the corresponding linear peptide. Linear peptides are unstructured polypeptides with open topology and N- and C-termini, which in solution can freely sample an ensemble of conformations: although this property can sometimes be useful to account for induced-fit binding to targets, it is more often responsible for structural and conformational heterogeneity, proteolytic lability, and other liabilities associated with "non-druggability". In cyclic peptides, on the other hand, an intra-molecular covalent bond (head-to-tail amide or side chain-to-side chain) turns the linear polypeptide into a mechanical closed-chain system with drastically reduced degrees of freedom, tending to occupy a restricted and defined number of low-energy conformations. As a result, the ring conformation, whose topology is thermodynamically preorganized in the absence of N- and C-termini, is forced to be compact (globular), which often makes these molecules much more protease stable than their linear counterparts. Ring formation can also direct, or even strongly favor, the appearance of local structural motifs such as a β-turn, a γ-turn, or a helical fragment. The stabilizing effect of the intramolecular hydrogen bonds and the improved side-chain packing that can result in a cyclic peptide are not stable in the linear analogue. The absence of termini also removes the zwitterionic character of linear peptides, which often represents an obstacle to transport across cell membranes, and the constrained topology of cyclic peptides can effectively mask the backbone polar amides from solvent exposure, lowering the desolvation cost for target binding. The consequence is a class of compounds that generally possess improved metabolic stability, selectivity, and affinity, and therefore are attractive chemical scaffolds to target protein–protein interactions and other intracellular targets.
Fig. 1 Cyclic peptides and their building blocks.1,5
The structural constraint of cyclic peptides directly affects their function. Cyclic peptides are conformationally restricted due to covalent linkage from N to C terminus. This means that, unlike linear peptides that can adopt a wide range of conformations in solution by rotation about their phi and psi angles, cyclic peptides are pre-organized into a specific or limited number of conformations. This is because the N and C termini must be within close proximity for bond formation to occur. This constraint is transmitted through the peptide and enforces certain secondary structures such as β-turns, γ-turns, or compact helices. Molecular dynamics simulations suggest that cyclic peptides adopt stable 3D structures with many intramolecular hydrogen bonds between the backbone atoms. The reduction in flexibility results in a rigid structure due to cooperative stabilization of hydrogen bonds and side chain interactions. This structure can also result in improved resistance to proteolytic enzymes, which tend to cleave amide bonds. Digestion by exopeptidases, which cleave off amino acids at the N- and C-termini, are effectively halted by the closed nature of cyclic peptides. Endopeptidases, which cleave peptide bonds internal to the peptide chain, are also hindered by the reduced backbone flexibility of cyclic peptides, resulting in limited proteolysis. Constrained ring size can also lead to conformational bias where steric hindrance from the ring can stabilize otherwise unfavorable cis peptide bonds. This effect is particularly notable in small rings. Larger cyclic peptides, however, maintain a degree of segmental flexibility that can be useful for induced-fit binding. Cyclotides, a class of cyclic peptides that contain a disulfide knot in addition to the cyclic backbone, can be doubly constrained by both elements. This can result in hyperstable structures with high resistance to denaturation by heat and chemicals. The degree of conformational constraint can be adjusted by varying ring size, by addition of conformational biases such as proline or D-amino acids, or by additional crosslinks, which can be useful for fine-tuning the degree of flexibility in a cyclic peptide to reach a certain biological activity. The rigid conformation also leads to high affinity for target binding. Cyclic peptides can bind with high affinity to target proteins because of their constrained structure.
Fig. 2 Scaffold-based cyclic peptide synthesis.2,5
One of the main advantages of cyclic peptides is their entropy gain, which is associated with their preorganized structure. This feature provides them with better binding thermodynamics and specificity. Entropy changes often cause major losses in binding affinities in the transition from the unbound to the bound state. In particular, the cyclic form of the peptide helps the drug to reduce the degree of conformational entropy of the unbound peptide. The greater this degree, the greater the entropy loss of this transition. Since most of the peptides have a linear form, in the free state they occupy a larger conformational ensemble than in the bound state, which is associated with the loss of degrees of freedom. The number of microstates, on the other hand, is large for an unfolded molecule and is small for a folded molecule. Therefore, by taking on a specific conformation in the cyclic form, peptides can significantly reduce the entropy penalty that occurs in the process of transition. In addition, the cyclic form often encodes a specific turn motif and precise three-dimensional orientation of side chains that complement the target protein surface, especially the binding groove. Macrocyclic peptides can also template water networks that are released in a more ordered state upon binding, which also contributes favorably to the entropy of the system. Protein–protein interfaces are also large and flat. For this reason, small molecules cannot inhibit most protein-protein interactions, as they cannot effectively cover a large interface with sufficiently strong interactions. Cyclic peptides, on the other hand, being more extended and rigid structures, are capable of interacting with more than one site of the interface in a multivalent manner, thus increasing both their affinity and selectivity. Because of the rigidity, the structures are less likely to bind to off-targets, as incorrect geometry or electrostatics will not be able to make significant conformational changes to achieve better binding in the bound state. This is a feature that linear peptides are more prone to utilize, thus causing selectivity issues. For example, this can be observed for the integrin receptor binding.
The resistance to enzymatic degradation that cyclic peptides present in comparison to their linear counterparts is another key pharmacological property which is directly related to their topology. Two well‑studied examples of this are cyclosporin and the plant cyclotides. In the case of the non‑ribosomal cyclic peptide cyclosporin, a fungal natural product and successful immunosuppressant, it is a combination of features that account for the increased stability. The macrocycle contains multiple N‑methylated amide bonds as well as D‑alanine and non‑proteinogenic side chains. The combination of these features leads to a peptide backbone that is sterically hindered, and the classical serine and metalloprotease recognition sequences are occluded from the solvent. This protection from the nucleophilic attack of proteases confers a long serum half‑life, and indeed, cyclosporin is orally available in spite of its high molecular weight. In this case, cyclization additionally protects the vulnerable N‑ and C‑termini that are the common target of exopeptidases for linear peptides.
A family of naturally occurring, plant backbone‑cyclized cystine‑knot peptides, the cyclotides, are another well‑studied example of how resistance to proteolytic degradation can be acquired by cyclic peptides. The combination of a head‑to‑tail cyclized backbone and three knotted disulfide bonds leads to a hyper‑rigid scaffold that is sterically crowded. The backbone amides are completely occluded from the solvent by a hydrophobic core, preventing proteases from gaining access to cleave them. In fact, cyclotides have been shown to be completely resistant to degradation by trypsin, pepsin, and thermolysin even after incubation for several hours, and this increased protease resistance appears to be a generic feature of the cyclotide framework rather than being dependent on the specific residues present. Therefore, both the backbone and the disulfide‑stabilized core of cyclotides appear to contribute to the steric hindrance observed with cyclotides that prevent enzymatic degradation. The closed backbone prevents threading of the peptide into the active site of a protease, while the disulfide‑stabilized native fold precludes local unfolding required for substrate recognition and cleavage. The structural features of cyclotides grant stability across multiple environments such as digestive fluids and serum making them suitable for drug development scaffolds.
Pharmacokinetics involves the processes of absorption, distribution, metabolism, and excretion of a drug. The impact of the conformational stability in cyclic peptides for their pharmacokinetic profile can be explained by their high conformational stability. The increased conformational stability generally leads to favorable systemic availability and resistance towards proteolysis that may give rise to prolonged plasma half-life than their linear analogs. In addition, the constrained conformation of cyclic peptides could also affect their membrane permeability and bioavailability. The physicochemical properties and balance of lipophilicity and hydrophilicity in cyclic peptides also play a role in the membrane permeability of the compound, which could further affect their pharmacokinetic properties.
An inherent level of lipophilicity is one reason for cyclic peptide permeability, the product of polarity masking and structure preorganization that facilitates membrane permeation. Linear peptide structures show an inherent limitation in drug delivery when compared to small molecule drugs because their flexible backbone exposes polar amide groups to the solvent environment and prevents passive membrane diffusion. However, in cyclic peptides, intramolecular hydrogen bonding between the hydrogen bond donors and acceptors present in the backbone of linear peptides makes these polar groups buried. This decreases the cost of desolvation required for a molecule to cross a membrane, making the surface of the peptide more hydrophobic on average. But, too much hydrophobicity and the peptide will start aggregating in aqueous solution or stick to membrane proteins and other cellular targets non-specifically, and not enough hydrophobicity and the molecule may not be driven across the membrane. For example, cyclosporin, is largely made up of N‑methylated amides and as a result, the peptide backbone of cyclosporin is largely devoid of hydrogen bond donors and acceptors, which increases its overall lipophilicity, and is therefore able to be passively transported across the intestinal epithelium in the gut. In this case, the macrocyclic structure of cyclosporin forces a globular conformation that minimizes the polar surface area exposed to the membrane. The molecule can also transiently adjust its conformation to fit the membrane's hydrophobic core. Conformational rigidity, provided in part by the ring, is also important; it restricts overall backbone motion, but the macrocycle still has some ability to segmentally "breathe," allowing for transient exposure of lipophilic patches of the macrocycle that can engage in van der Waals interactions with acyl chains in the lipid bilayer, to help with flip‑flop. The relationship between lipophilicity and permeability is not linear but rather parabolic. The presence of D‑amino acids or non‑canonical residues can help modulate this lipophilicity for the purposes of tuning the ability of a cyclic peptide to passively permeate a membrane. They can do this by disrupting the hydrogen‑bonding pattern and thus the stereoelectronic environment that would have otherwise made the peptide stay in the aqueous phase.
Cyclic peptides often have far greater systemic stability and longer plasma half‑lives than their linear analogues, both of which are positive pharmacokinetic traits, and may even enable oral delivery. The increased systemic half‑life is often due to a combination of protease resistance and slower renal clearance. First, the "closed backbone" of cyclic peptides protects them against enzymatic degradation by serum proteases and peptidases, which would otherwise rapidly degrade the peptide into smaller fragments within minutes of injection. Enzymatic stability means that a higher percentage of the injected dose is able to reach target tissue(s), prolonging its effect. For example, cyclosporin has a long systemic half‑life and therapeutic immunosuppressive levels can be achieved with daily oral dosing. This is because cyclosporin is very stable, i.e. resistant to enzymatic degradation, both in the GI tract and the systemic circulation, due to its macrocyclic structure and the N‑methylations and D‑amino acid, which prevent its recognition by both brush‑border enzymes and serum proteases, respectively, allowing it to survive first‑pass metabolism. Engineered cyclotides and other backbone‑cyclized peptides also have long half‑lives in serum stability assays, with trypsin, chymotrypsin, and elastase resistance, contributing to their serum stability. Second, the globular structure of cyclic peptides may lead to lower rates of glomerular filtration than linear peptides of a similar molecular weight, since the effective hydrodynamic radius is smaller, and serum protein binding may be affected. The combination of slower renal clearance and enzymatic protection leads to an increased duration of action and fewer dosing events, which is crucial for patient compliance. The oral delivery of cyclic peptides is also enabled by the dual protection described in the previous paragraph, which allows them to survive both the acidic pH and proteolytic environment of the stomach and small intestine, and to cross the intestinal epithelium, either by passive diffusion or carrier‑mediated transport (described above). This blurs the line between small molecule and biologics drugs, potentially allowing cyclic peptides to have the functional diversity and target specificity of peptides with the pharmacokinetic properties (such as oral delivery) of most small molecule drugs.
Macrocyclic peptides have emerged as a powerful class of molecules due to their high affinity and target selectivity, which is similar to antibody. The constrained and rigid structures of macrocyclic peptides allow their functional groups to assume a shape that is complimentary to target proteins, leading to higher affinity. The rigidity of macrocyclic peptides also allows for less entropy loss upon binding. In addition, the different scaffolds that macrocyclic peptides can adopt allow their binding pockets to be tuned to specific epitopes, resulting in high selectivity. These properties make them ideal for PPI inhibition, as the ability to target shallow and featureless protein surfaces is a potent tool for the manipulation of PPIs, which can lead to the modulation of more complex biological processes. The ability of macrocyclic peptides to target protein-protein interactions has seen success in targeting PPIs associated with various disease processes, most notably cancer, and as such has potential for therapeutic use in a wide range of medical applications.
Macrocyclic peptides have the potential to have antibody mimicking binding affinity and target selectivity. The conformational constraint imposed by cyclic peptides can enable them to present functional groups in a defined spatial orientation, akin to the antigen-binding sites of antibodies. This structural rigidity can enhance their ability to form precise and complementary interactions with target molecules, resulting in high binding affinity. The lack of terminal flexibility in cyclic peptides can reduce entropic penalties associated with binding, further contributing to their strong target engagement. Additionally, the diverse structural scaffolds available for macrocyclic peptides allow for the engineering of binding pockets that closely match the specific epitope, leading to high target selectivity. These properties make macrocyclic peptides appealing alternatives to antibodies in cases where smaller size, increased stability, or simpler production is desired. Combining antibody-like specificity with the advantages offered by cyclic peptides opens up new opportunities for therapeutic agents targeting complex biological interfaces. For instance, cyclic RGD peptides such as cRGDfV exhibit enhanced selectivity and affinity for integrin αvβ3 compared to linear RGD peptides.
Macrocyclic peptides have exhibited significant success in the development of inhibitors of protein-protein interactions (PPIs). PPIs have become increasingly difficult drug targets for small molecules as their large interaction surfaces are typically flat with minimal binding pockets. This is unlike most small molecule targets, which are usually deep pockets on a protein surface. Due to their flat surface, PPIs often have shallow, featureless surfaces that are not selective for specific ligands. Macrocyclic peptides, on the other hand, can be selected to specifically bind these areas. This provides a means to selectively inhibit protein-protein interactions, with great potential for PPIs involved in cancer and other diseases. For example, an allosteric inhibitor based on a macrocyclic peptide, D4-2, was selected to bind to the immunoglobulin-like domain of SIRPα, which restores the macrophage phagocytosis of cancer cells and has been shown to have a synergistic effect with other forms of treatment. A similar concept has been used to develop macrocyclic peptides based on protein kinase substrates. The resulting macrocyclic peptides mimic the protein interaction domain of the kinase substrate, thus acting as a competitive inhibitor and decreasing kinase activity. These macrocyclic peptides can be used as alternatives to small molecule kinase inhibitors, which often have poor target selectivity because of the high homology between protein kinase active sites.
The design and use of macrocyclic peptides are not without limitations. One significant challenge is the complexity of their synthesis. Macrocyclic peptides often require intricate synthetic strategies to construct their cyclic structures, which can involve multiple steps of protection and deprotection of functional groups. This complexity can lead to increased side reactions and lower overall yields. The ring-closing reaction, a critical step in macrocyclization, can also be challenging, especially for larger rings or those requiring specific stereochemistry. Factors such as ring size, cyclization topology, amino acid configuration, and the presence of turn-inducing residues must be carefully considered when selecting a synthetic approach. Over the years, various methodologies have been developed to address these challenges, but the synthesis of some macrocyclic peptides remains notoriously difficult. Another limitation is the low solubility of some macrocycles. Certain macrocyclic peptides exhibit poor solubility in water, which can affect their bioavailability and limit their therapeutic potential. This issue can be mitigated through chemical modifications, such as the introduction of hydrophilic groups or the use of solubility-enhancing excipients. However, these modifications can sometimes compromise the peptide's stability or binding affinity. The balance between solubility and other desired properties is therefore critical and must be carefully optimized. The cost of production is also a significant challenge. The complex synthesis processes and the need for specialized reagents and equipment contribute to the high production costs of macrocyclic peptides. This can limit their accessibility for widespread use, particularly in resource-limited settings. Advances in synthetic methodologies and the development of more efficient production processes are necessary to reduce costs and make macrocyclic peptides more economically viable for various applications.
| Parameter | Cyclic Peptides | Linear Peptides |
| Structural Rigidity | Conformationally locked, preorganized, stable secondary structures. | Highly flexible, dynamic conformers, transient structures |
| Proteolytic Stability | High resistance: no free termini, steric shielding, long half-life | Low: vulnerable to exo/endopeptidases, rapid degradation |
| Membrane Permeability | Moderate to good with optimization (N-methylation, D-amino acids) | Generally poor due to exposed polar groups |
| Synthetic Accessibility | Challenging: cyclization complications, potential oligomerization | Straightforward: standard SPPS, high yields |
| Production Cost | High: expensive reagents, complex purification, low yields | Low: inexpensive reagents, simple purification |
| Target Selectivity | High: rigid pharmacophore, precise complementarity | Lower: conformational adaptability, potential promiscuity |
| Pharmacokinetic Profile | Extended half-life, potential oral bioavailability, reduced clearance | Short half-life, rapid clearance, parenteral administration |
| Typical Applications | PPI inhibitors, oral therapeutics, antimicrobial agents, stable scaffolds | Research tools, diagnostics, short-lived applications |
Cyclic peptides are a highly diverse and critically important class of drug molecules in the current era of drug and functional peptide design, forming a natural pharmacological bridge between small molecules and larger proteins. The covalent constraints imposed by their cyclic nature result in significantly increased proteolytic stability and longer in vivo half‑lives, often making them far more drug‑like than their linear counterparts. In addition to their stability, cyclic peptides tend to feature well‑defined secondary structures, leading to high selectivity and affinity. These factors combined have led to their increased prominence in recent years. Clinically approved drugs are available, including small molecules that are orally bioavailable. Peptide cyclization can render a therapeutic protein more stable, enabling chronic dosing modes that are currently not available for peptides. These lead to further therapeutic opportunities such as oral dosing for treating metabolic, inflammatory, and cancer targets. Cyclic peptides are better able to recapitulate the surfaces of protein–protein interactions. Inhibiting such surfaces is challenging using small molecules, therefore providing further opportunity to apply cyclic peptides against oncogenic signaling proteins, as well as viral fusion proteins. The synthetic accessibility of cyclic peptides continues to improve, driven by technological advances in chemoenzymatic peptide synthesis, enzymatic protein cyclization, and in silico peptide design. As such, while cyclic peptides have historically been dismissed as either unsuitable drug candidates or unscalable, this is not the case with current state‑of‑the‑art methods for cyclic peptide design, synthesis, and production. In the future, it is expected that cyclic peptide synthesis will be further streamlined, in large part driven by machine learning‑based sequence design, and high‑throughput synthesis on automated peptide synthesizers, accelerating discovery timelines and lowering overall costs.
The unique structural benefits of cyclic peptides—enhanced stability, binding affinity, and resistance to degradation—make them an ideal choice for modern therapeutic and industrial applications. If you're evaluating whether to transition from linear to cyclic formats, our technical team can support your decision-making and development pipeline.
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