1. A carboxylated Zn-phthalocyanine inhibits the fibril formation of Alzheimer’s amyloid β peptide Atsushi Nagai Dept. Laboratory Medicine Shimane University Faculty of Medicine

2. Background and Purpose

3. 3 Alzheimer’s Disease Alzheimer’s disease (AD) is a common dementia disease of the elderly Histopathological features of AD 1. Degenerative and dystrophic neurons 2. Reactive glial cells 3. Extracellular Aβ peptide deposition 4. Intracellular neurofibrillary tangles Genetic and animal studies demonstrated the vital role of Aβ peptide in AD pathology

4. 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. 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. 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

7. UV-vis Spectra of ZnPc(COONa) 8 in Buffer Solutions and MeOH at R.T. Wavelength / nm ε / 10 5 M -1 cm -1 673 pH9.2 pH6.9 MeOH 642 pH4.0 yield 46% Syntheses and Properties of ZnPc Complex

8. Perspective Digagnostic strategy Can phthalocyanine bind to A  be detected with NIRS? Therapeutic strategy Potential of phthalocyanine for inhibition of amyloidogenesis

9. Aim of this study To investigate the interaction of phthalocyanine with Aβ peptide during fibril formation process

10. Methods and Results

11. 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:

12. Phthalocyanines used in the study (A)(B) (C) (D) ZnPc(COONa) 8 (water soluble) ZnPc(COONa) 16 (water soluble) ZnPc(OOC 5 H 11 ) 8 (water insoluble) PdPc dimer (water insoluble)

13. 0 40 80 0.1151020 µM ThT fluorescence (% control) † † † † 0 40 80 120 0.1151020 µM ThT fluorescence (% control) ‡ ‡ ✴ ThT fluorescence (% control) 0.1151020 µM ✴ ‡ ✴ 0 100 200 300 400 0.1151020 µM ThT fluorescence (% control) 0 40 80 120 † † ZnPc(COONa) 8 : ZnPc(COONa) 16 : ZnPc(OOC 6 H 13 ) 8 : PdPc dimer: Effects of phthalocyanines on Aβ 1-40 fibril formation A  1-40 (50  M) *48 h incubation

14. Effects of phthalocyanines on Aβ 1-42 fibril formation 0 40 80 0.1151020 ZnPc(COONa) 8 : µM ThT fluorescence (% control) † † † † 0.1151020 ThT fluorescence (% control) ZnPc(COONa) 16 : µM 0 40 80 120 ✴ ✴ 0.1151020 ThT fluorescence (% control) µM ZnPc(OOC 6 H 13 ) 8 : 0 100 200 300 400 0 40 80 120 ThT fluorescence (% control) 0.1151020 PdPc dimer: µM A  1-42 (12.5  M) *24 h incubation

15. Aβ fibril formation kinetics ThT fluorescence 020406080 0 20 40 60 h h 0204060 80 0 4 8 12 16 ThT fluorescence + ZnPc(COONa) 8 A  1-40 (50  M) A  1-40 020406080 0 10 20 ThT fluorescence 0 2 4 6 020406080 ThT fluorescence h h + ZnPc(COONa) 8 A  1-42 (12.5  M) A  1-42

16. Aβ fibril morphology + ZnPc(COONa) 8 A  1-40 A  1-42 A  1-40 Bar=200 nm + ZnPc(COONa) 8 A  1-42

17. 0 10 20 30 00.1110 50 MM ThT fluorescence ✴ † ‡ † 0 20 40 00.1110 50 ThT fluorescence MM † † † ‡ 0 0.1 1 50 10 MM 0 0.1 1 50 10 MM 28 17 10 KDa Aβ fibril destabilization 28 17 10 A  1-40 fibril (50  M) + ZnPc(COONa) 8 A  1-42 fibril (25  M) + ZnPc(COONa) 8

18. buffer Aβ 1-42 Aβ 1-42 fibril Aβ 1-42 0 1510 ZnPc(COONa) 8 (μM) + - + + Aβ antibody Binding of phthalocyanine to Aβ 1-42 peptide or fibril Incubation (24 h) ZnPc(COONa) 8 ＋ Immunoprecipitation with Aβ Ab buffer Aβ 1-42 Aβ 1-42 fibril Aβ 1-42 NIR scan

19. Aβ 1-42 -0h Aβ 1-42 -4h Aβ 1-42 +ZnPc-0h Aβ 1-42 +ZnPc-4h 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 spectroscopy ANS fluorescence assay emission spectra A  1-40 A  1-42

20. Molecular species of Aβ 1-42 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 h24 h 35 28 17 10 KDa delayed oligomer formation Coomassie blue staining (SDS PAGE) Coomassie blue staining (SDS PAGE) Dot blot immunoassay with oligomer specific Ab Dot blot immunoassay with oligomer specific Ab A  1-42 A  1-42 (50 μM)

21. 00.11510 MTT- % control ZnPc(COONa) 8 µM MTT- % control 00.11510 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 A  1-42 A  1-42 (5  M)

22. 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. 23 Conclusion Phthalocyanine could be used as a safe diagnostic probe as well as a therapeutic tool for AD.

24. 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