In Situ Self-assembly of Peptides
for Cancer Therapy
Literature seminar #2
2024/6/6
M1 Mayo YamazakiContents
Contents

l Introductionn What is the significance of self-assembly of peptides?
n ALP-instructed self-assembling peptidesl Design of self-assembling peptides selectively targeting cancer cells
1. D-peptides vs. L-peptides
2. The number of the enzyme recognition sites
3. Addition of sites that react with other enzymesl Summary & Perspectives
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Contents
Contents

l IntroducGonn What is the signiﬁcance of self-assembly of pepGdes?
n ALP-instructed self-assembling pepGdesl Design of self-assembling pepGdes selecGvely targeGng cancer cells
1. D-pepGdes vs. L-pepGdes
2. The number of the enzyme recogniGon sites
3. AddiGon of sites that react with other enzymesl Summary & PerspecGves
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Advantage of self-assembly for therapy
Challenges in the therapeutic effects of drugs

Introductionl Weak drug-target interaction→ Limitation of therapeutic effectsl Drug resistance of target proteins
Advantage of self-assembly of peptides for therapyl Self-assembling in situ
l Multivalent interactionsl Extended durationl Rapid response→ Improvement for pharmacokineticsl Mechanical stressControlling noncovalent local
interac.ons by self-assembly→ Development of supramolecularassemblies func.oning in vivo 2024/6/6

Wang, H. et al. Adv. Mater. 2018, 30, 1703444, Xu, B. et al. Acc. Chem. Res. 2008, 41, 315–326, Zhou, J., Xu, B. et al. J. Am. Chem. Soc. 2016, 138, 3813–3823

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What causes self-assembly in vivo environment
Introductionl Specific pH
l Redox environmentl Presence of specific enzymes
Strategy
Introducing biological reaction sites
in the design of precursors
for self-assembling peptides→ Peptides can self-assemble in a
While reactive oxygen species (ROS) are mainly generated in the mitochondria, as well as, to a lesser degree, by the membrane-bound specific environment
NADPH oxidase (NOX), the cellular reducing agent glutathione is present in high concentration (10 mM) throughout the cytosol. During
the endocytic pathway, the pH inside the vesicles decreases significantly from 6.3 in the early endosomes to 5.5 in the late endosomes
to 4.7 in the lysosomes, whereas the cytosolic pH is typically neutral, at around 7.2.

Weil, T. et al. Nat. Rev. Chem. 2022, 6, 320–338

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5EISA; Enzyme-instructed self-assembly
Introduction

Biomimetic multi-step process① Enzymatic transformation of non-self-assembling precursors
to self-assembling molecules② Self-assembling of molecules
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Xu, B. et al. Bioconjugate Chem. 2015, 26, 987–999

6Higher expression of ALP on cancer cells
Introduction

Alkaline phosphatase (ALP,Protein tyrosine phosphatase)l On the cell membranel Highly expressed in somecancer cells→ bio-marker of cancer

Challenges in targeting ALPs for therapy
ALPs play a crucial role in the liver and brain!
Inhibitor of ALP will lead side effects→ undruggable…

EISA strategy
Using ALP as a trigger for
self-assembly of peptides
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Weil, T. et al. Nat. Rev. Chem. 2022, 6, 320–338

Feng, Z., Xu, B. et al. Chem. 2019, 5, 2442-2449

Stolbach, L. L. et al. Nature. 1968, 219, 697-699

7Mechanism of self-assembly of pepDdes
Introductionl Nap-FF (2-(naphthalen-2-yl)acetic-Phe-Phe)ü π-π stacking
ü Hydrogen bonding

ONHHNOOl β-sheet byNap-FF
l Electrostaticl Hydrogen bondingl HydrophobicØ Aromatic (π-π stacking)Ulijn, R. V. et al. Chem. Soc. Rev. 2014, 43, 8150-8177

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Yang, Z., Xu, B. et al. J. Am. Chem. Soc. 2006, 128, 3038–3043

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Design of ALP-instructed self-assembling peptidesl Nap-FFpY

Introduction

Phospho-Tyr :Trigger for enzyme-instructed hydrogelation

Hydrophiliccf) Nap−FFGEpY

Precursor
ü More soluble

ALP← TyrHydrogelator
ü More hydrophobic← Nap−FFü Self-assembly in waterü Forming nanofibers
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Yang, Z., Xu, B. et al. Angew. Chem. Int. Ed. 2007, 46, 8216–8219

Yang, Z., Xu, B. et al. J. Am. Chem. Soc. 2006, 128, 3038–3043

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Design of self-assembling peptides selectively targetingcancer cells
How can we design self-assembling peptides that selectively target cancer cells?
1Introduction

32

1. D-peptides vs. L-peptides
2. The number of the enzyme recognition sites
3. Addition of sites that react with other enzymes
Li, J., Xu, B. et al. J. Am. Chem. Soc. 2013, 135, 9907–9914 Zhou, J., Xu, B. et al. J. Am. Chem. Soc. 2016, 138, 3813–3823
Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384

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Contents
Contents

l Introductionn What is the significance of self-assembly of peptides?
n ALP-instructed self-assembling peptidesl Design of self-assembling peptides selectively targeting cancer cells
1. D-peptides vs. L-peptides
2. The number of the enzyme recognition sites
3. Addition of sites that react with other enzymesl Summary & Perspectives
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D-peptides vs. L-peptides
2-(naphthalen-2-yl)acetic-Phe-Phe (NapFF)

L-peptides

L-peptides

Phospho-Tyr : Trigger for enzyme-instructed hydrogelation

D-peptides

D-peptides

D-peptides

Binding of the phosphate precursors (presented as CPK model: yellow,phosphorus; red, oxygen.) to the active site of ALP (presented as solidribbons). (A) l-Peptide based precursor (1a) and (B) d-peptide based precursor(1b) binding to the phosphatase. (C) Top view and (D) side view of 1a (green)and 1b (dark pink) in the active site. Purple atoms represent the parts of 1aand 1b that would occupy the same space in the active site.

Crystal structure of ALP and pep>desü Phosphate groups on L-pep>des or
D-pep>des are able to bind theac>ve site of ALP.

Active sites of ALP
Li, J., Xu, B. et al. J. Am. Chem. Soc. 2013, 135, 9907–9914

u Expect to be more
biostable precursor

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12Self-assembling ability of D-peptides← 31P NMR shows the conversion of 1.0 wt % of (A) 1a and (B) 1b catalyzed by the phosphatase (0.02 U/mL) at
pH 7.6 at 3 min and 4, 12, 24, and 48 h. Time-dependent rheology study of 1.0 wt % of (C) 1a and (D) 1bcatalyzed by the phosphatase (0.02 U/mL) at pH 7.6.

L-peptides

D-peptides

D-peptides

Optical images and TEM images of the hydrogels formed by using ALP (1.0 U/mL) to treat 0.4 wt % of (A) 1a and (B) 1b at pH 7.6.

ü Hydrogelators L-pep>des and D-pep>des self-assemble toform long, ﬂexible, and uniform nanoﬁbers.
ü Precursors L-peptides and D-peptides undergo
dephosphorylation at similar rates upon being
treated with ALP.
Li, J., Xu, B. et al. J. Am. Chem. Soc. 2013, 135, 9907–9914

Chirality of these two hydrogelators→ little difference in the properties of the resulting hydrogels
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D-peptides can resist proteinase
D-peptides

L-peptides

D-peptides

ü D-pep.des resists proteinases↔ L-pep.des undergoes proteoly.c hydrolysis
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Li, J., Xu, B. et al. J. Am. Chem. Soc. 2013, 135, 9907–9914

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Short Summary of D-peptides
D-pep>desü D-peptides can act as a substrate of phosphatase.
ü D-peptides can self-assemble by enzyme-instruction.
ü D-peptides are intrinsically resistant to proteolytic hydrolysis.
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Li, J., Xu, B. et al. J. Am. Chem. Soc. 2013, 135, 9907–9914

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Contents
Contents

l Introductionn What is the significance of peptide self-assembly?
n ALP-instructed self-assembling peptidesl Design of self-assembling peptides selectively targeting cancer cells
1. D-peptides vs. L-peptides
2. The number of the enzyme recognition sites
3. Addition of sites that react with other enzymesl Summary & Perspectives
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The number of phospho-Tyr in one pepDde
Enzyme recognition sites
Two phospho-tyrosine residues

One phospho-tyrosine residue

Molecular structures of the precursors (a-2p, b-2p, c-2p, a-1p, b-1p, and c-1p) that have one or two phosphotyrosine residues and the corresponding
self-assembling d-peptides (i.e., hydrogelators a, b, and c).

Zhou, J., Xu, B. et al. J. Am. Chem. Soc. 2016, 138, 3813–3823

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Self-assembling ability with ALP
Enzyme recognition sites
TEM images of aggregates/nanofibers
in the solutions of different precursors(a-2p, a-1p, b-2p, b-1p, c-2p, and c-1p)
or nanofibers in the hydrogels formedby treating the solutions of theprecursors with alkaline phosphatase(ALP). C = 0.5 wt %, pH = 7.4, [ALP] = 1
U/mL. Insets are optical images of thesolutions of the precursors and thehydrogels formed after enzymatic
dephosphorylation. The scale bar is
100 nm.
(B) Time-dependent rheometry toshow the gelation points (that is, at thegel state, where storage modulus (Gʹ)
dominates loss modulus (Gʺ)) ofdifferent precursors treated with ALP(0.05 U/ml). C = 0.5 wt %.

ü Two phospho-tyr residues enhance the aqueous
solubility of the precursors.
ü +ALP→ All can self-assemble in water to form nanofibers.

ü DFDYDFDY exhibits higher ability to self-assemble(due to the orientation of DFs)ü One phosphorylated precursors form hydrogels
much faster than their corresponding two
phosphorylated precursors
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Zhou, J., Xu, B. et al. J. Am. Chem. Soc. 2016, 138, 3813–3823

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Selectivity for cancer cells
Enzyme recognition sites(B) IC50 of different precursors/hydrogelators against different cell lines after 48-h incubation.

Cell line

Cell origin

ALP expression levels

HeLa

cervical cancer cellHigher

SK-OV-3drug-resistantovarian carcinoma
cellA little higher

A2780cisü The precursors relatively selectively inhibited thesurvival of cancer cells over normal cells.
ü HeLa, SK-OV-3, A2780cis (higher or a little higher)
IC50 value for the monophosphorylated precursor wassmaller compared to the diphosphorylated precursor.
Zhou, J., Xu, B. et al. J. Am. Chem. Soc. 2016, 138, 3813–3823

HigherT98G

glioblas-toma
multiforma tumor
cellA little higher

Saos-2bone osteosarcoma
cell

Much higher

HS-5

immortalized
normal stromal cell(Standard)
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Short Summary of the number of enzyme recognition sites
Enzyme recognition sites

HeLa
SK-OV-3
A2780cis
Saos-2T98G

ü By increasing the number of enzyme recognition sites, peptides canavoid organizing in cells with moderately high ALP expression→ improving selectivity for targeting cells with very high ALP expression
Zhou, J., Xu, B. et al. J. Am. Chem. Soc. 2016, 138, 3813–3823

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Contents
Contents

l Introductionn What is the significance of peptide self-assembly?
n ALP-instructed self-assembling peptidesl Design of self-assembling peptides selectively targeting cancer cells
1. D-peptides vs. L-peptides
2. The number of the enzyme recognition sites
3. Addition of sites that react with other enzymesl Summary & Perspectives
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Lower selectivity of monophosphorylated peptides
Dual enzyme-instructed
HepG2
Hepatocellular carcinoma cells

How can we improve selectivity for cancer cells
over normal cells with highly ALP expression?
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Zhou, J., Xu, B. et al. J. Am. Chem. Soc. 2016, 138, 3813–3823

Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384

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Addition of CES-reacted sites
Dual enzyme-instructedReact with carboxy esterase (CES)Strategy
Carboxy esterase in somecancer cells are downregulated

OMe (Increasing hydrophobicityand neutralizing negative
charge of amino acid residues,
substrate of CES)vs. NHMe (Not react with CES)
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Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384

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Inhibitory activities against different cell lines
Dual enzyme-instructedü Peptides with much lower selfassembling ability were generated
by hydrolysis of the methyl ester
bond of 1p with CES.
Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384

Cell origin

ALP
expression
levelsCES
expression
levelsSaos-2bone
osteosarcoma
cellMuch higher

Down
regulated

MCF-7

Breastadenocarcinoma cell

T98G

glioblas-toma
multiforma
tumor cell

A little higher

HigherHS-5

immortalized
normal stromalcell(Standard)(Standard)

Cell
line

HigherFeng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 3950–3953

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Lower toxicity by introducing CES-reacted moiety
Dual enzyme-instructed

Cellline
HepG2

Cell origin

ALP
expression
levelsCESexpression
levelsHepatocellular
carcinoma cells

HigherMuch Higher

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Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384 Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 3950–3953

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Molecular transformation in cell
Dual enzyme-instructed

Cell origin

ALP
expression
levelsCES
expression
levelsSaos-2bone
osteosarcoma
cell

Much higher

Down
regulated

HepG2

Hepatocellular
carcinoma cells

HigherMuch Higher

HS-5

immortalized
normal stromalcell(Standard)(Standard)

Cellline

ALP acUviUes: Saos-2 > HepG2 > HS-5
↔ HepG2: high expression level of CES results in hydrolyzing 26% of carboxyl methyl ester
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Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384

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Short Summary of addition of other enzymes-reacted sites
Dual enzyme-instructed

Saos-2

ü Dual enzyme-instructed self-assembling peptides can selectivelyinhibit the osteosarcoma cells without harming liver cells.
Next : Activity of dual enzyme-instructed self-assembling peptides in vivo
Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384

Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 3950–3953

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Activity of self-assembling peptides in vivo
Dual enzyme-instructed

Orthotopic mouse model of osteosarcoma
n luciferase-labeled Saos-2 cells (Saos2-luc)n highly metastatic subtype (Saos2-lung) cells

ü 1P eﬃciently inhibited the tumor growth.
ü 1P exhibited higher eﬃcacy against themetastaWc osteosarcoma.

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Feng, Z., Xu, B. et al. Chem. 2019, 5, 2442-2449

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Effect of CES-reacted sites in vivo
Dual enzyme-instructedü 2P and 3P which did not have CES-reacted
moiety inhibited the growth of tumor lesseffectively than 1P.
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Feng, Z., Xu, B. et al. Chem. 2019, 5, 2442-2449

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Contents
Contents

l Introductionn What is the significance of peptide self-assembly?
n ALP-instructed self-assembling peptidesl Design of self-assembling peptides selectively targeting cancer cells
1. D-peptides vs. L-peptides
2. The number of the enzyme recognition sites
3. Addition of sites that react with other enzymesl Summary & Perspectives
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Summary
How can we design self-assembling peptides that selectively target cancer cells?
1Summary3

2

1. To maintain bio-stability, D-peptides were utilized.
2. Adjusting the number of enzyme recognition sites increased target cell selectivity.
3. Adding sites recognized by other downregulated enzymes improved cancer cell selectivity.
Li, J., Xu, B. et al. J. Am. Chem. Soc. 2013, 135, 9907–9914 Zhou, J., Xu, B. et al. J. Am. Chem. Soc. 2016, 138, 3813–3823
Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384

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Perspectives
Perspectives

l Multiple enzymes can control the EISA process and can precise regulateof the formation of the assemblies in different cellular environments.
← Need to design peptides with information of expression level of enzymes
l EISA has possibility to inhibit the multiple pathways of cells
simultaneously in space and time.
← Not rely on ligand-receptor binding

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Liu, Z., Xu, B. et al. Acc. Chem. Res. 2023, 56, 3076–3088

Feng, Z., Xu, B. et al. J. Am. Chem. Soc. 2017, 139, 15377–15384

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Thank you for your kind a.en0on!

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