Present state of membrane permeablepeptides and its application

Literature seminar (2018. 1. 20)
M1 Hiroki Horigome

Contents

1. Current status of pharmaceutical products andcell permeable peptides.
2. Classification of Cell-Penetrating Peptides (CPPs)
2-1. Cationic CPPs
2-2. Amphipathic CPPs
2-3. Hydrophobic CPPs
3. Applications of CPPs

1. Current status of pharmaceutical products
and cell permeable peptides.

Transition of pharmaceuticals to larger compounds
Small molecule

Medium molecule

Antibody drugs

Structure

ex. Alectinib
ex. Ciclosporin
Molecularweight500 <

500 ～ 2000≒ 150000

Specificity

Low

HighHighSideeffect

HighLow

LowCell membrane : barrier function
Hydrophobic

Cell-Penetrating
Peptides (CPPs)

Hydrophilic© 2010 Nature Education All rights reserved.

Specialized proteins in the cell membrane regulate the concentrationof specific molecules inside the cell.

HIV-1 TAT protein

Ref) Green, M. and Loewenstein, P.M. Cell 1988, 55, 1179–1188
Ref) Vivès, E. et al. J. Biol. Chem. 1997, 272, 16010–16017

Strategy by CPPs
Hydrophobic

Cell-Penetrating
Peptides (CPPs)

Hydrophilic© 2010 Nature Education All rights reserved.

Specialized proteins in the cell membrane regulate the concentrationof specific molecules inside the cell.

Strategy by CPPs
Hydrophobic

CPPs

Hydrophilic© 2010 Nature Education All rights reserved.

Specialized proteins in the cell membrane regulate the concentrationof specific molecules inside the cell.

2. Classification of Cell-Penetrating Peptides (CPPs)
2-1. Cationic CPPs
2-2. Amphipathic CPPs
2-3. Hydrophobic CPPs

Various Cell-Penetrating Peptides (CPPs)① Cationic② Amphipathic③ HydrophobicRef) Daniela Rossi et al. Trends in Pharmacological Sciences 2017, 38, 406-424

2. Classification of Cell-Penetrating Peptides (CPPs)
2-1. Cationic CPPs
2-2. Amphipathic CPPs
2-3. Hydrophobic CPPs

① Cationic CPPs

TAT (Green, M. and Loewenstein in 1988)

Polyarginines (Futaki, S. in 2001)

Ref) Futaki, S. et al. J. Biol. Chem. 2001, 276, 5836–5840

① Cationic CPPs
Abundant basic amino acids will be cationic
under physiological environment.
So they interact with phospholipid head groups ofcell membrane.
Especially, arginine can form hydrogen bond with it.

Ref) Morris, M.C. et al. Biol. Cell 2008, 100, 201–217

Endocytosis

Endosome

CPPs can be more positive and interact with the negatively
charge components of the endosomal membrane.
This binding causes stiffening of the membrane, determining its
rupture and the release of the vesicle’s contents.

Ref) van den Berg, A. and Dowdy, S.F. Curr. Opin. Biotechnol. 2011, 22, 888–893

Polyarginines

Electrostatic interaction : 25 - 30%
Hydrogen bond : 70 – 75%

Polyarginines can interact with the membrane by hydrogen bond.
So, arginine-rich CPPs may enter the cell via a nonendocytoticmechanism.
Ref) Hitz, T. et al. Biochemistry 2006, 45, 5817–5829

Escape from endosome : Proton-sponge mechanism

Endosome< Proton-sponge mechanism >Ref) D. W. Pack, A. S. Hoffman, S. Pun, and P. S. Stayton. Nat. Rev. Drug Discov. 2005, 4(7), 581–593

Escape from endosome : TAT-HA2
HA2 peptide
pH 5pH 7.4

N-terminal 20 amino acidsof the influenza virus
hemagglutinin protein

The deeper insertion of the V-shaped
structure destabilizes the membrane.
So, TAT-HA2-fusion protein can escapefrom endosome.

Ref) Han, X., Bushweller, J.H., Cafiso, D.S., Tamm, L.K. Nat. Struct. Biol. 2001, 8, 715–720
Ref) David, Y., Vila-Perello, M., Verma, S., Muir, T. W. Nat. Chem. 2015, 7, 394

Escape from endosome : agents to help endosomal escape
Only using TAT-HA2 as a reagent can help
endosomal escape.
FITC-TAT trapped in endosome.
－ TAT-HA2＋ TAT-HA2

Ref) T. Sugita et al. Biochemical and Biophysical Research Communications 2007, 363, 1027–1032

Escape from endosome : agents to help endosomal escape

TAT-Cre trapped in endosome.

When TAT-Cre enters in cells, itrecombine EGFP reporter gene.

Ref) Wadia, J. S., Stan, R. V., Dowdy, S. F. Nat. Med. 2004, 10, 310-315

Escape from endosome : agents to help endosomal escapedfTAT = dimeric fluorescent TAT

dfTAT is more endosomolytic andcause lysis of endosomal membranethan monomeric TAT.

Ref) J. P. Pellois et al. Nat. Meth. 2014, 11, 861-867

Escape from endosome : agents to help endosomal escapeBafilomycin (V-ATPase inhibitor)
inhibited the delivery by dfTAT.

Endocytosis and endosomal
acidification are importantfor cytosolic penetration.

Ref) J. P. Pellois et al. Nat. Meth. 2014, 11, 8, 861-867

2. Classification of Cell-Penetrating Peptides (CPPs)
2-1. Cationic CPPs
2-2. Amphipathic CPPs
2-3. Hydrophobic CPPs

② Amphipathic CPPs
Amphipathic CPPs contain both polar (hydrophilic)and nonpolar (hydrophobic) regions of amino acids.

Ref) Daniela Rossi et al. Trends in Pharmacological Sciences 2017, 38, 406-424

② Amphipathic CPPs
Amphipathic CPPs can enter the cell directlyin addition to endocytosis.

Ref) Morris, M.C. et al. Biol. Cell 2008, 100, 201–217

Amphipathic CPPs can enter cells directly

MPG
Lipofectamine

Inhibitors that interfere with the endosomal pathwayRef) Gilles Divita et al. Nuc. Acid. Res. 2003, 31, 11, 2717-2724

MPG and Pep-1

HIV glycoprotein 41

tryptophan-rich cluster
Hydrophobic domain

LinkerHydrophilic

improves the flexibility and the integrity of boththe hydrophobic and the hydrophilic domains
Ref) Morris, M.C. et al. Biol. Cell 2008, 100, 201–217
Ref) Gilles Divita et al. Nuc. Acid. Res. 2003, 31, 11, 2717-2724

HIV glycoprotein 41

HIV glycoprotein 41

HIV gp41 interacts with the host cellular membrane.

Ref) Morris, M.C. et al. Biol. Cell 2008, 100, 201–217
Ref) Gilles Divita et al. Nuc. Acid. Res. 2003, 31, 11, 2717-2724

Amphipathic CPPs can enter the cell directly(1) Formation of the carrier—cargo complexes.
(2) Interaction of the carrier–cargo nanoparticles with the cell surface proteoglycans(3) Interaction with the glycans and phospholipid head groups(4) Direct interaction with the lipid phase of the cell membrane(5) Complexes are released into the cytoplasmor (6) is targeted to the nucleus or to specific organelles.
Ref) Morris, M.C. et al. Biol. Cell 2008, 100, 201–217

TP10TP10 :AGYLLG K INLKALAALAKKIL-amide
Galanin

Mastoparan

Neuropeptide

Helical structure

Sectional view of TP10Ref) Ulo Langel et al. The FASEB Journal 2018, 12, 1, 67-77

Mechanism of TP10 / membrane interaction① attach to membrane② change to helical structure③ insert to inter-membrane

TP10③①－ TP10②¥＋ TP10Ref) Ulo Langel et al. Nat. Bio. Tech. 1998, 16, 857-861
Ref) Ulo Langel et al. Biophysical Journal 2007, 92, 2434-2444

L17E : agents to help endosomal escape
L17E is an agent to help endosomal escape derived fromAmphipathic CPP.
M-lycotoxin, which disrupts cell membrane, derived fromthe venom of the wolf spider Lycosa carolinensis27.
Prof. Futaki @ Kyoto Uni.

M-lycotoxinRef) Shiroh Futaki et al. Nat. Chem. 2017, 9, 751–761

L17E : agents to help endosomal escapeRef) Shiroh Futaki et al. Nat. Chem. 2017, 9, 751–761

L17E : agents to help endosomal escapeAlexa488–dextran

L17E mediated cytosolic delivery.
Ref) Shiroh Futaki et al. Nat. Chem. 2017, 9, 751–761

2. Classification of Cell-Penetrating Peptides (CPPs)
2-1. Cationic CPPs
2-2. Amphipathic CPPs
2-3. Hydrophobic CPPs

③ Hydrophobic CPPs
As yet only a limited number of hydrophobic peptides has been discovered andtheir internalization mechanisms have been poorly studied compared with thecationic and amphipathic classes.
However, it has been proposed that this family of peptides could spontaneouslytranslocate across membranes in an energy-independent manner.

C105Y is a synthetic peptide (CSIPPEVKFNKPFVYLI) based on the amino acidsequence corresponding to residues 359–374 of α1-antitrypsin.
Ref) M. Rhee and P. Davis J. Biol. Chem. 2006, 281, 6, 1233-1240

3. Applications of CPPs

Assembly of complex CPP structures

CPP doesn’t change the nature of cargo.
But, only a few CPPs are applicable tothis method.
Amphipathic CPPs are likely to be applicablebecause of their hydrophobic interaction withcargo.
Ref) Siegmund Reissmann J. Pept. Sci. 2014, 20, 760–784Comparison between CPPs

Cationic

Amphipathic

Hydrophobic

Uptakemechanism

Endocytosis

Direct insertion

Direct insertion

Concentration
toleranceHigh

ModerateLow

・ Big size compounds(ex. antibody)・ Acidically weakcompounds

Applicability

・Some agents can help
endosomal escape・ Non-covalent
complex with cargo

Unknown※ Actually, which one is better depends on the nature of thecompound and cell which you want to introduce.

Enhancing the metabolic stability of CPPs
Peptidase degrades CPP.

・The membrane permeation
efficiency drops.
・The localization of cargochanges.

Endosome① Changing L-form amino acid to D-form

Stabilization of CPPs

② N-methylation③ Cyclization

Ref) van den Berg, A. and Dowdy, S.F. Curr. Opin. Biotechnol. 2011, 22, 888–893
Ref) Siegmund Reissmann J. Pept. Sci. 2014, 20, 760–784

①Changing L-form amino acid to D-form②N-methylation
hCT(9 – 32) : Amphipathic CPP
only CF (A)

(B)
(C)

(D)
(E)
(F)

(G)
(H)

Ref) R. Rennert et al. Biochimica et Biophysica Acta 2006, 1758, 347 – 354

③ Cyclization
Cyclic TAT
Cyclic R10

Linear TAT

Ref) Cardoso C. et al. Nat. Com. 2011, 2, 453, 1–6Ref) Cardoso C., Hackenberger R. et al. Nat. Chem. 2017, 9, 762–771
Ref) Cardoso C., Hackenberger R. et al. Angew. Chem. Int. Ed. 2015, 54, 1950 –1953

Cyclic TAT and R10
Cyclic TAT

Cyclic R10

Cyclic CPPs have enhanced cell permeability than linear CPPs
because of their metabolic stability.
Ref) Cardoso C. et al. Nat. Com. 2011, 2, 453, 1–6
Ref) Cardoso C., Hackenberger R. et al. Nat. Chem. 2017, 9, 762–771
Ref) Cardoso C., Hackenberger R. et al. Angew. Chem. Int. Ed. 2015, 54, 1950 –1953

Cyclic Amphipathic CPP

F and Φ(L-2-naphthylalanine) enhances themembrane transduction activity of CPPs.

Ref) Dehua Pei et al. ACS Chem. Biol. 2013, 8, 423−431
Ref) Dehua Pei et al. Biochemistry 2014, 53, 4034−4046

Cyclic Amphipathic CPP

Ref) Dehua Pei et al. ACS Chem. Biol. 2013, 8, 423−431
Ref) Dehua Pei et al. Biochemistry 2014, 53, 4034−4046

Cyclic Amphipathic CPP(A) Nuclear stain with DRAQ5(B) Green fluorescence of internalized cFΦR4-FITC(C) Red fluorescence of rhodamine B-dextran(D) A merge of panels A−C
Ref) Dehua Pei et al. ACS Chem. Biol. 2013, 8, 423−431
Ref) Dehua Pei et al. Biochemistry 2014, 53, 4034−4046

Bicyclization
CPP and cargo, whichwere cyclic, regain thelinear character in cell.
Many peptide ligands
must be in their linear
conformations to bebiologically active.

NEMO-binding domain (NBD)
reduces aberrant NF-κB activity.
NF-κB is a transcription factor
that controls the expression ofnumerous gene products.
Ref) Dehua Pei et al. Angew.Chem. Int.Ed. 2017, 56,1525 –1529

Bicyclization①③②① : Peptides were labeled with fluorescein FITC on thesidechain of a C-terminal lysine. Mean fluorescenceintensity (MFI) values showed their cellular uptake.
② : Cells transfected with a luciferase reporter gene
under the control of NF-κB were first treated withvarying concentrations of a peptide and then TNFα.
TNFα activates NF-κB.
③ : The metabolic stability of peptides was tested byincubating the peptides in human serum.
Ref) Dehua Pei et al. Angew.Chem. Int.Ed. 2017, 56,1525 –1529

Application for Preclinical and Clinical Evaluations

Ref) Daniela Rossi et al. Trends in Pharmacological Sciences 2017, 38, 406-424

Amphipathic CPPs interact with lipids
P294 : Ac-GALFL GFLGAAGSTM GAWSQ PKKKR KV-Cya

DMPC :DMPG :

C1-1 : [ P294 + DMPG ]C1-2 : [ P294 + DMPC ](1,2-dimyristoyl-sn-glycero-3-phosphocholine)(1,2-dilauroyl-sn-glycero-3-phosphocholine)

P294 forms stable 1:1 complexes withlipids. And hydrophobic interaction isessential for forming complex.

weakens the hydrophobic interactions

Ref) Richard B. Cole et al. Anal. Chem. 2005, 77, 1556-1565

Proline-rich CPPs
It was discovered that …

・ N-terminal γ-zein domain’s octamer (VHLPPP)8 can interact with cell membranes.
・ A peptide containing just proline residues P14 can internalize in cells.
Pro content of 50% is enough tomaintain a helical structure.

Ref) J. Fernandez-Carneado, M.J. Kogan, S. Castel, E. Giralt, Angew. Chem. Int. Ed. Engl. 2004, 43, 1811–1814
Ref) Pujals, S. and Giralt, E. Adv. Drug Deliv. Rev. 2008, 60, 473–484

Proline-rich CPPs
CF-(VRLPPP)8@ 37 ℃

@ 4℃

CF-(VRLPPP)8 internalize in cell by endocytosis.

Ref) J. Fernandez-Carneado, M.J. Kogan, S. Castel, E. Giralt, Angew. Chem. Int. Ed. Engl. 2004, 43, 1811–1814
Ref) Pujals, S. and Giralt, E. Adv. Drug Deliv. Rev. 2008, 60, 473–484

Poly-proline as CPP

Poly-proline

Good interaction
Anionic cell-surface
glycanRef) H. Wennemers et al. Angew.Chem.Int. Ed. 2017, 56,122 –126

MPG and Pep-1

HIV glycoprotein 41

tryptophan-rich cluster
Hydrophobic domain

LinkerNuclearLocalization
Signal(Hydrophilic)

Ref) Morris, M.C. et al. Biol. Cell 2008, 100, 201–217
Ref) Gilles Divita et al. Nuc. Acid. Res. 2003, 31, 11, 2717-2724

Nuclear Localization Signal

NLS binds to importin.

cytoplasm
The complex ofNLS and importin
enters into
nucleoplasm.

nucleoplasm

Ran-GTP cleavage thecargo from importin.

The NLS of MPG is essential
for nuclear translocation.
Ref) Morris, M.C. et al. Biol. Cell 2008, 100, 201–217
Ref) Gilles Divita et al. Nuc. Acid. Res. 2003, 31, 11, 2717-2724