Cell penetrating peptides

One of the major problems in cell biology is the inefficient transfer of peptides or proteins across cellular membranes. In order to deliver a peptide or a protein from the outside to the inside of intact cells, a carrier is generally required. Cell Penetrating Peptides (CPPs, also known as Cell Permeable Peptides or as Protein Transduction Domains, PTDs), are carriers with small peptide domains (generally less than 40 amino acids) that can easily cross cell membranes. Multiple cell permeable peptides have been identified that facilitate cellular uptake of various molecular cargo, ranging from nanosize particles to small chemical molecules. Cell penetrating sequences can be used as extensions to peptide sequences thereby making them more permeable to cell membranes, or cell penetrating peptides can be attached to other cargo molecules to enhance their cellular uptake.

Synthesis of Cell Penetrating Peptides

When cell penetrating sequences are used as extensions to a peptide cargo, we apply for the synthesis of the entire molecule in general either straight SPPS synthesis, or alternatively use chemical ligation strategies. Chemical ligation strategies are especially preferred in case the cargo peptides are quite large themselves (>50 amino acids).

When using ligation strategies the cargo peptide and the CPP are synthesized independently and the appropriate chemical handles are introduced at the chosen position during the peptide synthesis (click here for more information about the possible ligation chemistries). It is also possible to conjugate non-peptidic cargo molecule to CPP’s if those molecule bear a functional group suitable for ligation. Frequently applied ligations strategies for CPP’s involve oxime ligation, thiomaleimide coupling and Click chemistry, but strategies like isothiocyanate ligation, NHS coupling or NCL can be applied as well.

Pepscan has over the years been working with a wide range of CPPs, such as HIV-TAT, Oligo-Arg and many more. Originally most CPPs were composed of only natural amino acids. Yet in recent years research groups have found it beneficial to include unusual or non-natural amino acids, peptide branches and other modifications to improve existing CPPs or to create new ones. Each modification must be carefully designed to avoid problems that could lead to low synthesis yields, poor solubility or aggregation. With Pepscan you can rely on thorough expertise in peptide chemistry and a broad range of modifications to also synthesize such novel designer CPPs.

Frequently used CPPs

The most commonly used CPP is the HIV-TAT sequence, placed at the N-terminal part of a peptide. This is one of the many options for making peptides more permeable to cells. There are multiple other cell penetrating sequences, of which only a smalls selections is shown in the table below. For a comprehensive review of currently available CPPs see Reissman (J.Pept. Sci. 2014; 20: 760-784).

Name Sequence
HIV-TAT GRKKRRQRRRPQ
Oligo-Arginine RRRRRRRR
MPG Ac-GALFLGFLGAAGSTMGAWSQPKKKRKV-cya
PEP-1 Ac-KETWWETWWTEWSQPKKKRKC-cya
EB1 LIKLWSHLIHIWFQNRRLKWKKK
Transportan GWTLNSAGYLLGKINLKALAALAKKIL
p-Antp RQIKIWFQNRRMKWKK
hCT(18-32) KFHTFPQTAIGVGAP-NH2
KLA seq KLALKLALKALKAALKLA

A limitation of most of the CPPs is their lack of cell or tissue selectivity, which limits their use in clinical development.

Cell or tissue specificity of CPPs

Over time several receptors were found to be involved in the internalization of certain CPPs. These include receptor types as the chemokine receptors and the family of integrins. This can provide some degree of cell and tissue specificity, depending on the incidence of the corresponding receptor and its density at the target cell.

Recently, a new type of penetrating peptide has been found, that has intrinsic tissue selective properties. Azurin p18 and Azurin p28 are partial sequences of Azurin, a protein which preferentially targets human cancer cells.

The search for tissue specific transduction peptides has started to generate an increasing number of tissue specific CPP’s, mainly obtained via phage display selections. These so called “cell-penetrating-homing peptides” not only recognize specific types of cells, but also have the capacity to translocate across its cellular membrane. A number of cell penetrating homing peptides are summarized in the table below.

CPP name Target tissue Sequence
AGR Prostate carcinoma CAGRRSAYC
LyP-2 Skin and cervix tumor CNRRTKAGC
REA Prostate, cervix, breast carcinoma CREAGRKAC
LSD Melanoma, osteocarcinoma CLSDGKRKC
HN-1 Head and neck squamous cell carcinoma TSPLNIHNGQKL
CTP Cardiac myocytes APWHLSSQYSRT
HAP-1 Synovial tissue SFHQFARATLAS
293P-1 Keratocyte growth factor SNNNVRPIHIWP

Selected References for Cell Penetrating Peptides

Kuenzl et al:  Metab. Eng (2016) – PMID 27989807. Overcoming the membrane barrier: Recruitment of γ-glutamyl transferase for intracellular release of metabolic cargo from peptide vectors

Martin et al: Oncogene (2016) – PMID 27375016. Androgen deprivation leads to increased carbohydrate metabolism and hexokinase 2-mediated survival in Pten/Tp53-deficient prostate cancer

Milech et al: Sci Rep (2015) – PMID 26671759. GFP-complementation assay to detect functional CPP and protein delivery into living cells

Chu et al: Nanomedicine(2015) – PMID 25193363. Rational modification of oligoarginine for highly efficient siRNA delivery: structure-activity relationship and mechanism of intracellular trafficking