Cyclic Cell Penetrating Peptides: Definition, Structure, Mechanism and Applications
* Please kindly note that our products and services can only be used to support research purposes (Not for clinical use).
Online Inquiry
What are the cyclic cell penetrating peptides?
Cell-penetrating peptides (CPPs) are short oligopeptides of 5 to 30 amino acids, capable of transporting diverse cargos and traversing biological membranes. Cyclic CPPs are a distinct category of peptides engineered for effective cellular membrane penetration, ensuring stability and specificity. Cyclic CPPs, in contrast to their linear counterparts, possess chemically connected N- and C-termini that create a stable ring structure, hence improving cellular absorption and minimizing degradation.
Intracellular Drug Delivery Using Cyclic Cell-Penetrating Peptides. (Park S E., et al., 2019)
Types and structures of cyclic CPPs
Cationic cyclic CPPs
Cationic peptides encompass polylysine and natural protamines. The cationic cyclic CPPs comprise cTAT, FITC-[R3], FITC-[R4], FITC-[R5], FITC-[R6], FITC-[R7], FITC-[R8], FITC-[R9]. The cyclic TAT peptide is mostly cationic, including six arginine and two lysine residues. It is hypothesized that positively charged residues establish robust electrostatic contacts with the negatively charged membrane of cells. Furthermore, several studies on the binding affinities of cationic cyclic CPPs demonstrate their significant affinity for various anionic entities located at the extracellular surface, such as phosphate groups of membrane lipids, nucleolin protein, and proteoglycans like heparin sulfate.
Amphipathic cyclic CPPs
In the majority of cationic cyclic peptides, arginine and lysine residues are aggregated, but in amphipathic peptides, they are uniformly dispersed throughout the sequence. Moreover, amphipathic peptides are abundant in hydrophobic residues, including tryptophan, leucine, valine, phenylalanine, and alanine. The binding of amphipathic cyclic CPPs to the plasma membrane and their subsequent intracellular uptake differs significantly from that of cationic cyclic CPPs. Amphipathic peptides are characterized by their robust contact with the hydrophobic regions of the membrane and their incorporation into the lipid bilayer, a phenomenon not seen in cationic cyclic cell-penetrating peptides, and regarded as the primary interaction force.
Structure of cationic and amphiphilic cyclic CPPs. (Park S E., et al., 2019)
Cell Penetrating Peptides at Creative Peptides
Advantages of cyclic CPPs over linear CPPs
Higher cell permeability
Cyclic CPPs have been directly compared to their linear analogues in terms of the degree of cell permeability they exhibit. Cell permeability was shown to be consistently better with the cyclic CPP variation compared to the identical sequence linear variant. In the presence of doxorubicin, nucleoside reverse inhibitors, and negatively charged fluorescent phosphopeptide, Mandal's team found that peptides rich in arginine and tryptophan were able to pass through cells. Cyclic CPPs with a high concentration of arginine and hydrophobic tryptophan residues are far more permeable to cells than linear CPPs.
Higher resistance to proteolysis
A novel approach to designing cell-penetrating peptides involves increasing the stiffness of CPPs by cyclization. Several investigations have shown that CPP cyclization is responsible for their resistance to proteolysis. The cellular uptake of cFΦR4 peptide analogues, which were designed and synthesized by Qian's team by altering the stereochemistry and/or peptide sequence, was studied. The researchers used synthetic amino acids and varied the sequence lengths of the analogues. The results showed that the cyclic peptides rich in arginine residues, [FΦRRRRQ], where Φ stands for l-2-naphthylalanine, had excellent proteolytic stability, as confirmed by the pharmacokinetic studies conducted on mice. The stability of cyclic peptides is connected to the cysteine knot structure, and cyclization mainly decreases the peptides' vulnerability to thermal unfolding, according to Heitz's group's investigation into the function of a cyclic backbone in providing stability to peptide frameworks. According to some, cyclization decreases proteolysis by protecting components from proteolytic enzymes and by removing reactive C- and N-termini.
Improved target receptor affinity
When compared to their linear forms, CPPs with a cyclic structure have a far higher affinity for the target receptor. The improved affinity is probably because cyclized rings are more rigid and can tolerate a more constant conformation, making them better at binding to static target sites. A more efficient drug carrier for entering cells was created by Ichimizu's team using a number of palmitoyl-poly-arginine CPPs. In comparison to the linear variations tested, the cyclic peptide palmitoyl-cyclic-(D-Arg)12, which is composed of d-dodecaarginines, proved to be an exceptionally efficient cellular uptake facilitator.
Exhibit efficient endosomal escape
Research on cyclic CPPs has shown that they have several useful properties, including increased intracellular absorption, proteolysis resistance, and the ability to evade endosomal degradation. To illustrate the point, mechanistic investigations by Mandal's team demonstrated that fluorescently tagged F′-[W5R4K] was swiftly taken up into cell nuclei even in the absence of endosome-derived vesicles. In order to determine the impact of ring size on intracellular uptake, Parang's group produced several analogues of cyclic [WR]n peptides. Those cyclic CPPs that need to escape the endosome to leave the resultant endosome will greatly benefit from this endosome-independent cellular absorption. It was demonstrated in the aforementioned study by Qian's team that [FΦRRRRQ] binds directly to phospholipids in cell membranes, primarily enters cells by endocytosis, and effectively evades endosomes.
The advantages of cyclic CPPs compared to their linear counterparts. (Sajid M I., et al., 2021)
Custom Services at Creative Peptides
Translocation mechanism of cyclic CPPs
Endocytosis (pinocytosis and phagocytosis included), diffusion, active transport, direct penetration/translocation, and other cellular entry mechanisms are all possible for the cyclic CPPs. Several cyclic CPPs are translocated by endocytosis, with the most common routes being clathrin-mediated, caveolae-mediated, and clathrin/caveolae independent. Changing experimental settings suggest that multiple processes may be at work. For penetratin-like CPPs abundant in arginine and lysine residues, the inverted micelle approach has been suggested as a direct translocation mechanism. These peptides are able to cross biological membranes at lowered temperatures; moreover, they do not require energy to penetrate and do not bind to any receptors on the membrane. Penetratin peptides, which contain positively charged residues, first create a micelle by binding to the negatively charged phospholipids found in the cell membrane. After some time, the micelle moves into the cytoplasm and discharges its cargo, which includes the cyclic CPPs. Additional relevant translocation mechanisms for cationic antibacterial cyclic CPPs include the carpet model. The biological membrane is covered like a carpet by these peptides, which attach to the negatively charged membrane. When these peptides reach the membrane, they disrupt its stability, which in turn causes pores to develop, opening the door for cyclic CPPs and cargos.
Passive diffusion allows for the entry of tiny cyclic peptides with hydrophobic side chains, which are less than or equal to 10 amino acids in length. In addition to having at least two hydrophobic residues and three arginines, the cyclic peptide should have a tiny ring with no more than nine amino acids. Controlled amphipathic cyclic CPPs improve endosomal escape efficiency and endocytic absorption by binding efficiently with biological and endosomal membranes. It is also possible for these amphipathic cyclic CPPs to penetrate the biological membrane and enter the cell directly; however, the chemical mechanism of this penetration remains unknown. It is possible for the other cyclic CPPs to bind nonspecifically to the proteins on the surface of biological membranes and enter the cell through active transport. The entrance of cyclic CPPs into cells is facilitated by these processes.
There was an alternative route for arginine-rich peptides (RRPs) like TAT peptide to the nucleolus and cytoplasm, bypassing the endocytotic pathway. By blocking the endocytic pathways, they conducted studies with several cell lines, including BHK21-tTA/anti-CHC and mouse endothelioma cells. Ignoring these endocytic routes did not prevent the transduction of TAT peptide into the nucleolar and cytoplasmic compartments.
Summary of the translocation method employed by cyclic CPPs for cellular entry. (Sajid M I., et al., 2021)
Cyclization strategies for producing cyclic cell penetrating peptides
Two common methods of cyclization are forming amide bonds between the N-terminus α-amine and C-terminus α-carboxyl functional groups, and bridging two cysteine side chains. When lysine and aspartic acid produce an amide, the glutamic acid side chain, or a staple peptide undergoes cyclization; however, this process is only relevant to cyclic CPPs. An amide link between the C-terminus α-carboxyl group and the N-terminus α-amine group allowed Pfaff's team to disclose in 1994 the cyclization of peptides.
Cyclization is achieved when the N- or C-terminal cysteine side chains of peptides are bridged. When synthesizing cyclic CPPs, a disulfide bridge is preferred over the more traditional method of cyclization including an amide bond. The reversibility and ease of disulfide bridge cyclization make it a useful tool for improving the conformational definition of peptides in reductive environments. According to Lättig-Tünnemann's group, arginine-rich peptides and TAT peptides undergoing cyclization using the disulfide bridge technology outperformed their linear counterparts in terms of absorption efficiency. The cyclic CPPs generated by Qian's group were discovered to be efficiently taken up by HeLa cell lines and to display endosomal escape in another study that used the same cyclization strategy but with the disulfide bridging technique and the addition of cysteine residues at the ends.
Applications of Cyclic Cell-Penetrating Peptides
Gene therapy
Although siRNA-mediated gene silencing has been extensively studied as a novel cancer treatment option, the challenge of effective intracellular delivery of siRNA has not yet been overcome. The potential of cyclic CPP in siRNA delivery has been boosted by many findings. As an example, Shirazi's team investigated the use of gold nanoparticles coated with cyclic CPPs to improve the transport of siRNAs. Researchers in this work found that conjugating [WR]5-AuNPs with cyclic-peptide-based nanoparticles increased the delivery of small interfering RNA molecules in HeLa cells by a factor of 2 to 3.8 compared to incubating the cells with siRNA alone after 24 hours. Research on the cytotoxicity of these conjugates shown that they were quite safe when compared to other carrier systems, such lipofectamine. Similar to this, Welch et al. demonstrated in vitro and in vivo that disulfide-constrained cyclic peptides (Ac-C(FKFE)2CG-NH2 and Ac-C(WR)4CG-NH2) could be used to deliver short interfering RNAs (siRNAs) to the cytosol of mouse lungs. This finding suggests that these cyclic CPPs could be useful in the development of efficient siRNA delivery agents, which disrupt the expression of genes associated with diseases.
Drug delivery
The transportation of macromolecules and other hydrophilic medicinal compounds is a formidable obstacle, however cyclic CPPs show tremendous promise as a medication transporter. Their potential to enhance the efficacy of already available anticancer and antibacterial medicines has been suggested by many research. The potential uses of several cyclic CPPs as molecular transporters have also been thoroughly investigated by Parang and others. Take [WK]5 as an example; it has shown to be a reliable vehicle for delivering model anti-HIV medications. A cell-impermeable payload was efficiently delivered using [WH]5. [CR]4 showed improved cellular absorption of a phosphopeptide with a negative charge, and [HR]4 proved to be a functional molecular transporter.
Antibacterial agents
Oh's team discovered that certain amphiphilic cyclic peptides and analogues had strong antibacterial effects against microorganisms that were resistant to many drugs. The cyclic peptide [R4W4] showed the strongest antibacterial action against MRSA among all the peptides tested, with a minimum inhibitory concentration (MIC) of 2.67 μg/mL. Following several cyclic ring modifications utilizing different hydrophobic and cationic amino acids, it was discovered that four arginine and four tryptophan amino acids exhibited antibacterial action. Curiously, cyclic [WR]4 lacks antibacterial properties yet is able to penetrate cells due to its four alternating arginine and tryptophan residues. They discovered a fluorescently labeled-[R4W4] peptide that might be utilized to deliver antibiotics that are not permeable to cells. In order to prove that this antimicrobial peptide had a molecular transporter capability, scientists combined it with tetracycline and found that it was four to eight times more effective in killing MRSA germs than tetracycline alone. The authors hypothesized that cyclic CPPs, when administered in conjunction with antibiotics, may successfully suppress MDR bacteria.
Target the nucleus
Drawing inspiration from FR235222, a naturally occurring compound, Hilário's group created a cyclic tetrapeptide. They then attached a bioactive and fluorescent triazole aminocoumarin as a cargo model and tested the tetrapeptide's ability to penetrate the cell membrane and cause cell death. This work shown that this cyclic CPP can transport tiny bioactive compounds to both the cytosol and the nucleus.
Summary of Biomedical applications of cyclic CPPs. (Park S E., et al., 2019)
References
- Park S E., et al., Cyclic cell-penetrating peptides as efficient intracellular drug delivery tools, Molecular pharmaceutics, 2019, 16(9): 3727-3743.
- Sajid M I., et al., Applications of amphipathic and cationic cyclic cell-penetrating peptides: Significant therapeutic delivery tool, Peptides, 2021, 141: 170542.
- Qian Z., et al., Discovery and mechanism of highly efficient cyclic cell-penetrating peptides, Biochemistry, 2016, 55(18): 2601-2612.
- Dougherty P G., et al., Enhancing the cell permeability of stapled peptides with a cyclic cell-penetrating peptide, Journal of medicinal chemistry, 2019, 62(22): 10098-10107.
