A carboxylated Zn-phthalocyanine inhibits the fibril formation of Alzheimer’s amyloid β peptide

Atsushi Nagai

Dept. Laboratory Medicine

Shimane University Faculty of Medicine

Background and Purpose

3

Alzheimer’s Disease

Alzheimer’s disease (AD) is a common dementia disease of the elderly

Histopathological features of AD1. Degenerative and dystrophic neurons2. Reactive glial cells3. Extracellular Aβ peptide deposition4. Intracellular neurofibrillary tangles

Genetic and animal studies demonstrated the vital role of Aβ peptide in AD pathology

4

Current Management of Alzheimer’s Disease

• Diagnosis– Mainly depends on clinical assessment– Neuro-imaging is valuable, but either

expensive or nonspecific– Definitive diagnosis can be done by autopsy

examination of patient’s brain.• Treatment

– Currently no disease modifying therapy is available

5

Near-infrared Imaging

• Diagnostic tools– Near-infrared imaging is a technique to

detect target molecules with near-infrared probe.

Mol. Pharmaceutics 2013, 10, 3946−3958

• Example– NIR fluorophore conjugated folic

acid is used as a probe to visualize folate receptor

– Phthalocyanine-LHRH is used to visualize tumor in vivo

6

Phthalocyanine

• Water insoluble or tends to aggregate in water, resulting quenching of fluorescence properties

• Water soluble phthalocyanine substituted with sodium carboxylate group was prepared.

• Phthalocyanine is a large macrocyclic compound having optical properties in near-infrared region

UV-vis Spectra of ZnPc(COONa)8 in Buffer Solutions and MeOH at R.T.

Wavelength / nm

ε /

105

M-1c

m-1

673673

pH9.2

pH6.9

MeOH

642pH4.0

yield 46%

Syntheses and Properties of ZnPc Complex

Perspective

• Digagnostic strategy• Can phthalocyanine bind to A be detected

with NIRS?

• Therapeutic strategy• Potential of phthalocyanine for inhibition of

amyloidogenesis

Aim of this study

To investigate the interaction of

phthalocyanine with Aβ peptide

during fibril formation process

Methods and Results

1. Inhibition of Aβ fibril formation

2. Destabilization of Aβ fibril

3. Binding to Aβ

4. Secondary structure and hydrophobicity

5. Inhibition of oligomer formation

6. Cell toxicity

Phthalocyanines:

Phthalocyanines used in the study

(A) (B)

(C) (D)

ZnPc(COONa)8

(water soluble)

ZnPc(COONa)16

(water soluble)

ZnPc(OOC5H11)8

(water insoluble)

PdPc dimer(water insoluble)

0

40

80

0.1 1 5 10 20 µM

ThT

fluor

esce

nce

(% c

ontr

ol)

† † ††

0

40

80

120

0.1 1 5 10 20 µM

ThT

fluor

esce

nce

(% c

ontr

ol)

‡ ‡✴

ThT

fluor

esce

nce

(% c

ontr

ol)

0.1 1 5 10 20 µM

✴

‡

✴

0

100

200

300

400

0.1 1 5 10 20 µM

ThT

fluor

esce

nce

(% c

ontr

ol)

0

40

80

120

† †

ZnPc(COONa)8: ZnPc(COONa)16:

ZnPc(OOC6H13)8: PdPc dimer:

Effects of phthalocyanines on Aβ1-40 fibril formation

A1-40 (50 M)

*48 h incubation

Effects of phthalocyanines on Aβ1-42 fibril formation

0

40

80

0.1 1 5 10 20ZnPc(COONa)8: µM

ThT

fluor

esce

nce

(% c

ontr

ol)

††

††

0.1 1 5 10 20

ThT

fluor

esce

nce

(% c

ontr

ol)

ZnPc(COONa)16: µM0

40

80

120

✴

✴

0.1 1 5 10 20

ThT

fluor

esce

nce

(% c

ontr

ol)

µMZnPc(OOC6H13)8:0

100

200

300

400

0

40

80

120

ThT

fluor

esce

nce

(% c

ontr

ol)

0.1 1 5 10 20PdPc dimer: µM

A1-42 (12.5 M)

*24 h incubation

Aβ fibril formation kineticsTh

T flu

ores

cenc

e

0 20 40 60 800

20

40

60

h

h0 20 40 60 800

4

8

12

16

ThT

fluor

esce

nce

+ ZnPc(COONa)8

A1-40 (50 M)

A1-40

0 20 40 60 800

10

20

ThT

fluor

esce

nce

0

2

4

6

0 20 40 60 80

ThT

fluor

esce

nce

h

h

+ ZnPc(COONa)8

A1-42 (12.5 M)

A1-42

Aβ fibril morphology

+ ZnPc(COONa)8

A1-40 A1-42

A1-40

Bar=200 nm

+ ZnPc(COONa)8A1-42

0

10

20

30

0 0.1 1 10 50 M

ThT

fluor

esce

nce

✴

†

‡

†

0

20

40

0 0.1 1 10 50

ThT

fluor

esce

nce

M

†

††

‡

0 0.1 1 50 10 M 0 0.1 1 50 10 M

28

17

10

KDaKDa

Aβ fibril destabilization

28

17

10

A1-40 fibril (50 M) + ZnPc(COONa)8 A1-42 fibril (25 M) + ZnPc(COONa)8

buffer

Aβ1-42

Aβ1-42 fibril

Aβ1-42

0 1 5 10

ZnPc(COONa)8 (μM)

+ -

++

Aβ antibody

Binding of phthalocyanine to Aβ1-42 peptide or fibril

Incubation (24 h)

ZnPc(COONa)8＋ Immunoprecipitation with Aβ Ab

buffer

Aβ1-42

Aβ1-42 fibril

Aβ1-42

NIR scan

0

1

2

3

4

5

6

7

ANS

fluor

esce

nce

400 450 500 550 600 nm

random

turn sheet

α helix

ZnPc(COONa)8

0%

20%

40%

60%

80%

100%

- + - +0 h 2 h

Secondary structure and hydrophobicity of Aβ peptide or fibril

CD spectroscopyCD spectroscopy ANS fluorescence assay ANS fluorescence assay

emission spectra

A1-40 A1-42

Molecular species of Aβ1-42 during fibril formation

+ ZnPc(COONa)8

0 h 2 h 4 h 6 h 8 h 16h 24 h

ZnPc(COONa)8: - - - - - -+ + + + + +0 h 1 h 2 h 4 h 8 h 24 h

352817

10

KDa

delayed oligomer formation

Coomassie blue staining (SDS PAGE)

Coomassie blue staining (SDS PAGE)

Dot blot immunoassay with oligomer specific AbDot blot immunoassay

with oligomer specific Ab

A1-42

A1-42

A1-42 (50 μM)

0 0.1 1 5 10

MTT

- % c

ontr

ol

ZnPc(COONa)8

µM

MTT

- % c

ontr

ol

0 0.1 1 5 10 µM

✳ ✳

40

60

80

100

120

40

60

80

100

120

Cell viability assay of neuronal cell

ZnPc(COONa)8

ZnPc(COONa)8 protects A1 neuronal cells from Aβ-induced toxicity

A1-42

A1-42 (5 M)

22

Summary of the results

• Water soluble ZnPc(COONa)8

– has near-infrared properties

– binds to Aβ peptide

– inhibits its oligomer and subsequent fibril formation process

– destabilizes preformed Aβ fibrils

– decreases β-sheet conformation and hydrophobicity

– possesses neuroprotective feature against Aβ peptide

23

Conclusion

Phthalocyanine could be used as a safe diagnostic

probe as well as a therapeutic tool for AD.

24

Acknowledgements

• Shimane University• Faculty of Medicine,

• Dept. Laboratory Medicine• Shatera Tabassum• Abdullah Md. Sheikh• Shozo Yano

• Interdisciplinary Faculty of Science and Engineering, • Dept. Material Science• Takahisa Ikeue• Makoto Handa

Laboratory Medicine

• Funded by JSPS KAKENHI Grant Number 24310102