Cell Penetrating peptides 1. Structute of cell penetrating peptides. 2. Arginine rich RNA binding peptides. 3. Arginine rich DNA binding peptides 4. Peptides used for in RPE drug delivery 5. What are the possible mechanisms of cell penetrating peptides / cell membrane interaction. 6. What properties are essential for the peptide to be CPP. 7. What cargos can cell penetrating peptides deliver to cells.

8. Basic peptides: sequence / activity studies. a. Influence of components surrounding basic domain b. Influence of cargo c. Influence of peptide exposure d. Influence of hydrophobicity e. Influence of chemical linkage between peptide and cargo f. Influence of peptide density 9. Some peptides and peptide simulating polymers to be synthesized. 10. Some possible research areas to study peptides activity.

Representation of the occurrence for the study and the use of various cell penetrating peptides

Tat 44%

Ant 23%

Others

20%

Poly+ 7%

VP22 4%

Tpt 2%

Cell penetrating peptide

Sequence

PenetratinpAntp

H-RQIKIWFQNRRMKWKK-OH

Tat peptide H-GRKKRRQRRRPPQ-OH

HSV-1 VP22 peptide

H-DAATATRGRSAASRPTERPRAPARSASRPRRVD-OH

MAP H-KLALKLALKALKAALKLA-NH2

Transportan H-GWTLNSAGYLLGKINLKALAALAKKIL-NH2

MPG H-GALFLGWLGAAGSTMGAPKKKRKV-OH

Pep-1 H-KETWWETWWTEWSQPKKKRKV-OH

Syn B1Syn B3

SAP

pVEC

H-RGGRLSYSRRRFSTSTGR-OHH-RRLSYSRRRF-OH

H-VRLPPPVRLPPPVRLPPP-OH

H-LLIILRRRIRKQAHAHSK-OH

Pβ H-GALFLGFLGAAGSTMGAWSQPKKKRKV-OH

Poly-lysine H-(K)n-OH

Poly-arginine H-(R)n-OH

Loligo-mers Lysine branched peptides (each branch is derived from the import signal and NLS)

Inv3 H-TKRRITPKDVIDVRSVTTEINT-OH

Cell penetrating peptides structures

Arginine-rich RNA binding peptides

Sequence №Of positivecharges

№OfArgResi-dues

Trans-locationeffici-ency(Tat+++)

HIV-1 Rev-(34-50)

H-TRQARRNRRRRWRERQR-OH 10 10 +++

FHV Coat- (35-49)

H-RRRRNRTRRNRRRVR-OH 11 11 +++

BMV Gag-(7-25)

H-KMTRAQRRAAARRNRWTAR-OH 8 7 +++

HTLV-II Rex-(4-16)

H-TRRQRTRRARRNR-OH 8 8 +++

CCMV Gag-(7-25)

H-KLTRAQRRAAARKNKRNTR-OH 9 6 ++

P22 N-(14-30) H-NAKTRRHERRRKLAIER-OH 9 6 ++

λ n-(1-22) H-MDAQTRRRERRAEKQAQWKAAN-OH 7 5 +

φ 21 N-(12-29)

H-TAKTRYKARRAELIAERR-OH 7 5 +

Yeast PRP6-(129-144)

H-TRRNKRNRIQEQLNRK-OH 7 5 +

Human U2AF-(142-153

H-SQMTRQARRLYV-OH 3 3 -

Arginine rich RNA binding peptides

Arginine-rich DNA binding peptides

Sequence №Of positivecharges

№OfArgResi-dues

Trans-locationeffici-ency(Tat+++)

Human cFos-(139-164)

H-KRRIRRERNKMAAAKSRNRRRELTDT-OH 12 9 +++

Human cJun-(231-252)

H-RIKAERKRMRNRIAASKSRKRKLERIAR-OH 14 9 +++

Yeast GCN4-(231-252)

H-KRARNTEAARRSRARKLQRMKQ-OH 10 7 +++

Arginine-rich DNA binding peptides

Peptides used for in RPE drug delivery

HSV-1 VP22 peptide

H-DAATATRGRSAASRPTERPRAPARSASRPRRVD-OH

1. The peptide is 33 amino acid residues long

2. Peptide contains 9 arginine residues

3. Most of arginine residues are separated by 1-4 non basic amino acid residues.

4. Guanidine moieties are distributed more or less evenly along the peptide chain except of 6-aa N-terminus

5. 7 amino acid residues from 33 bear hydroxyl function; it might be important for the secondary structure formation

 Different steps in CPP-mediated intracellular delivery.

. 1. Interaction of the CPP (represented as a green bar) with the cell-surface proteoglycans (in red).

2. Endocytic pathway.

3a. Degradative route to lysosomes in clathrin-mediated endocytosis.

3b. CPP ultimately reach the Golgi apparatus (in purple) or endoplasmic reticulum (ER, in grey) in caveolin-mediated endocytosis.

3c. Endosomal release

Aspects of CPP interaction with cell membrane

Cell penetrating peptides

Peptide-lipid interaction Antp – interacts to negatively charged membranes.Transporatn - interacts to membranes independently of charge

Receptor binding Antp – receptor independent pass wayTat - receptor independent pass way

The role of heparan sulfate Tat and other basic peptides – dependent of heparan sulfate

Primary structure: basic character of most CPPs 1.CPPs apparently do not share common motifs, apart from 4-8

arginines2.CPPs rich in Lys get transduced with much less efficiency then Arg rich3.Charge alone is not the sole driving force4.An arginine octamer appears to present the optimal length for an efficient transduction (polymers of Orn; Lys and His are not transduced, indicating that guanidine group is a critical structure component for biological activity.

What are the possible mechanisms of cell penetrating peptides /

cell membrane interaction.?

Secondary structure: amphiphilic α-helices 1.In general, cationic amphiphilic α-helical peptides, which display both hydrophilic and hydrophobic sities are

efficiend transducers of DNA into cells2.Peptides consisting exclusively of Trp and Arg residues, which generate an amphiphatic molecule express tranducibility.3.Mastoparan, transportan, melittin and mangainin: increase of membrane permeability correlates with an increase of α-helical structure.4.In membranes with low anionic charge, the dependence on peptide helicity for peptide - bilayer interaction increases.5.For Antp and related peptides the necessity of α-helical structure is not clear at all. All the data are discrepant. 6.Flexibility of the molecules is also important for transduction characteristics.

Energy dependence1.Tat peptide transduction is energy independent?2.Poly- arginine peptide is energy dependent?

Direct membrane penetration

Tat penetrates the cellular membrane directly???

Inverted micelle formation 1.For Antp peptide a crucial step in transduction is a formation of an inverted micelle in the membrane?

2.Research on Antp interaction with artificial lipid bilayer shows that it does not penetrate into the hydrophobic core of the lipid membrane causing measurable disruption, do not form or channel or even an α-helical structure. That refutes the hypothesis of inverted micelle formation?

Penetration by endocytosis 1.SynB and Ant penetrate into cell by an adsorptive-mediated endocytosis.

2.The endocytic mechanism of Tat-plasmid complex uptake and Arg9-mediated protein delivery were

confirmed.3.The protein transduction facilitation by basic peptides might only be caused by the strong attachment of the cationic peptide side chains to the membrane, possibly mediated by HSPGs, from which the molecules would be taken up by constitutive endocytosis.

What properties are essential for the peptide to be CPP

NH

ONH

NH

NH2

NH

ONH2

1. Positively charged amino acids

Arginyl

Lysyl

3. The hydrogen bonding properties of the peptide backbone do not seem to be important for cellular uptake.

4. Flexibility of the linear alkyl chain appeared to be important for cellular uptake.

5. The linear structure of Arg-rich peptides is of no importance for traslocation.

1. It has been postulated that high degree of positive charges, due to content of Lys and Arg residues is important for initial step of internalisation.

2. The optimal number of Arg residues seems to be 7 – 15.

Hydrophobicity

N

O

N

O

N

O

N

O

N

O

N

Alanyl Valyl Leucyl

Phenylalanyl Tryptophanyl

1. The cellular uptake of penetratin has been described to be dependent on the presence of a central hydrophobic core.

2. For Tat peptide, the attachment of a small hydrophobic molecule, biotin, to the peptide caused a 6-fold increase in cellular uptake.

3. Upon varying the content of hydrophobic Leu and hydrophilic Lys amino acids in Hel amphipathic peptides, it was found that the peptide with the highest proportion of hydrophobic amino acids was the most efficient at delivering DNA 4. Octaarginine was lipophilised at the N-terminus with a stearyl, lauryl or cholesteryl group, and it was observed that the derivative bearing the stearyl group had the best transfection efficiency properties.

Amphipathicity

+1. In the context of peptides, the amphipathicity may result from the primary or secondary structure. Peptides with primary amphipathicity are assembled from a hydrophilic and a hydrophobic region which are normally divided by a spacer domain. On the other hand, secondary amphipathicity is achieved when the peptide conformation is such that all of the polar residues point to one side and the non-polar ones to the opposite side.

+2. CPP amphipathicity as a result of secondary structure could be divided into two main groups: secondary amphipathicity due to α-helical conformation or secondary amphipathicity from PP II structure.

+3. When three positions of the peptide Tat(47–57) were substituted by α-helical promoting Ala, a 5-fold internalisation increase was reached.

-4. Detecting uptake by HPLC, it was initially established that amphipathicity was crucial for internalisation, but afterwards, using an online protocol for confocal laser scanning microscopy including a washing step, it was shown that amphipathicity is not essential, as both amphipathic and nonamphipathic peptides (MAP analogs) crossed cell-membranes.

Cargo Cell penetrating peptides able to deliver cargo into cells

Oligonucleotides (DNA, plasmid DNA, phosphorothioates, siRNA)

Antp, Tat, (Tat)2 and (Tat)3

oligomers, Tat-liposome associated, loligomer 4, MPG, Pep1, oligolysines

PNA Antp, transportan, Tat

Biologically active peptides Polyarginine, Tat, Antp, HSV-1 VP22

Proteins Tat, Antp, HSV-VP22, poly-ornitine, poly-lysine, poly-arginine

Enzymes HSV-VP22, Tat, poly-arginine, poly-lysine

Nanoparticles Tat

Small molecules SynB1, penetratin, poly-lysine, Antp, Tat, MAP, transportan, loligomers

What cargos can cell penetrating peptides deliver to cells.

?

Basic peptides: sequence / activity studies

Influence of components surrounding basic domain

++ ++ + ++ +

1. No clear influence of the additional moiety attached either to N-terminal or C-terminal end of the “core” basic domain could be related to a variable ability of the basic peptide to mediate cellular uptake.

2. Comparison of uptake is rather difficult since the studies were performed on different cell lines employing different experimental protocoles.

Influence of cargoInfluence of peptide exposure

++ ++ + ++ + + ++ + ++ +

+

+

+ + ++ +

1. For a large cargoes (liposomes, nanoparticals) exposure of peptide can play a crucial role: only cargoes with appropriate exposure of the peptide are taken up by cells.

2. For oligonucleotides the peptide has to be attached to the 5’-terminus to achieve better biological activity.

Influence of hydrophobicity

O O OO O

O OO O O

1. Length of the spacer between guanidine group and α-carbon must be not less than 4 methylene groups: reduction of the side chain length down to 2 methylene groups leads a significant decrease of cell association.

2. The insertion of aminocaproic acid groups within the peptide backbone showed a stronger cell association.

3. The substitution of two Trp residues for two Phe residues in Antennapedia peptide sequence led to the complete loss of translocation properties, although Phe residues show a relatively higher hydrophobicity than Trp residues.

Influence of chemical linkage between peptide and cargo

SS

++ ++ + ++ +

++ ++ + ++ +

1. Surprisingly, the nature of the linkage between peptide and its cargo has not been deeply investigated. Nevertheless the nature of this linkage might appear to be critical, if one thinks about biological properties of cargo.

2. A labile bond between peptide and cargo expected to perform better.

3. Most convenient bond formation for this purpose could be a disulfide linkage between the peptide and cargo.

4. Permanent linkage between antisense oligonucleotide and basic peptide might increase biological activity of the cargo.

5. It was shown, that the cell uptake was more efficient for the stable link constructs, but nearly identical antisense activity was found for both stable and labile constructs.

Influence of peptide density

1. The requirement of several peptide molecules to promote cell uptake of large structures was mentioned, but no evaluation of the translocation property in accordance with the number of attached peptides was considered so far.

2. Content of guanidinium groups per complex appeared to be important: dimeric or tetrameric repeats of linear peptide complexed improved overall transfection efficiency.

3. Branched peptide constructs was shown to be much more effective for plasmid cell delivery with larger peptide repeats than corresponding lower brunched structures.

4. The given cargo requires an optimal number of peptides to be efficiently taken up by cells.

5. However, when tested in vivo complexes with several peptides displayed a weaker cell selectivity.

++ ++ + + + +++ ++ + + + +

++ ++ + + + +

++ ++ + + + +

++ ++ + + + +

Some peptides and peptide simulating polymers to be synthesized.

SH SH

SH

SH

SH

On

O

NH3

On

O

NH3

NH

NH

OO

NH3

n

OO

NH

n

NH

NH

SH

SH SH

SH SH

SH SH

SH SH

SH

SH

SH

Peptides to be synthesized

1. H-CGRKKRRQRRRPPQ-OH 2. H-CGRKKRWWRQRRRPPQ-OH

3. H-CGRKKRRQRRWWRPPQ-OH

15. H-CGRKKRAhxRQRRRPPQ-OH

14. H-CGRKKRRQRRAhxRPPQ-OH

Amino / guanidino group rich polymers (mw: 2000-3000)

4. H-CGRKKRRQRWWRRPPQ-OH

5. H-CGRKKRWWRQRWWRWWRPPQ-OH 6. H-CGRKKRAARQRRRPPQ-OH

7. H-CGRKKRRQRRAARPPQ-OH 8. H-CGRKKRRQRAARRPPQ-OH

9. H-CGRKKRAARQRAARAARPPQ-OH 10. H-CGRKKRRQRRLLRPPQ-OH

11. H-CGRKKRLLRQRRRPPQ-OH 12. H-CGRKKRRQRLLRRPPQ-OH

13. H-CGRKKRLLRQRLLRLLRPPQ-OH

16. H-CGRKKRRQRAhxRRPPQ-OH

17. H-CGRKKRAhxRQRAhxRAhxRPPQ-OH

Peptides synthesized

Selection of peptides for thebest cell penetrating ability(3 best + 2 worst)

Peptide conjugates with polymers

Peptide conjugates with oligonucleotides

Studies on lipidbiolayer

Testing withliposome models

Testing on different cell lines

HS lacking cellsGAG lacking cellsWild cell linesFluorescent labled

Some possible research areas to study peptides activity.