Chernii S. Investigation of phthalocyanine complexes with axially-coordinated ligands as inhibitors of amyloid aggregation of proteins

Українська версія

Thesis for the degree of Candidate of Sciences (CSc)

State registration number

0419U002188

Applicant for

Specialization

  • 03.00.03 - Молекулярна біологія

16-04-2019

Specialized Academic Board

Д 26.237.01

Institute of Molecular Biology and Genetics of NAS of Ukraine

Essay

In the pathogenesis of many conformational diseases, such as neurodegeneration and amyloidoses, proteins forming β-pleated stable aggregates called amyloid fibrils are involved. One of the important approaches against amyloid aggregation is a search for molecules able to inhibit or redirect the formation of amyloid fibrils. The goal of this work is to find potential inhibitors of amyloid aggregation among macrocyclic metal complexes (phthalocyanines, porphyrosines) with different geometry and investigate how their interaction with proteins affects protein aggregation and morphology of the aggregation products. A fluorescent dye-based assay was used to study the kinetics of protein aggregation, atomic force and scanning electron microscopies (SEM) – to characterize the morphology of single aggregates, dynamic light scattering (DLS) – to estimate the hydrodynamic parameters of the aggregates. Also, UV-VIS absorption and Raman spectroscopy were used to study the phthalocyanine aggregation and binding ability of phthalocyanines. Hf and Zr phthalocyanine complexes with axially coordinated ligands were strongly able to change the kinetics of the aggregation reaction and structure of the aggregates. It was shown that the intensity of the kinetics of amyloid fibril formation could be significantly suppressed by axially coordinated phthalocyanines (up to 96%). Zr and Hf phthalocyanines with quinolinium styryl ligand demonstrated the same high inhibitory activity of about 90% and similar values of IC50 equal to 0.160.08 mkM for PcZrQStr and 0.110.04 mkM for PcHfQStr. Similar values of inhibitory activity and IC50 suggest that the nature of the «closed» central metal atom does not affect the inhibitory activity. Depending on the structure of axially coordinated ligands, binding of phthalocyanines to insulin during its aggregation reaction could redirect fibrillization towards the formation of products of different morphology: single β­pleated protofilaments, oligomeric aggregates or large amorphous species. Insulin itself forms elongated filamentous species – amyloid fibrils with a diameter about 6-8 nm and length about 1-5 m with branched morphology. The presence of Hf phthalocyanine (PcHfCl2) with small-size ligands induces the appearance of large size aggregates with a diameter about 70-100 nm, that are identified as amorphous species. The presence of Zr phthalocyanine (PcZrDbm2) with bulky ligands redirects insulin fibril reaction to the formation of oligomeric aggregates with the diameter up to 10 nm as the main product. In the presence of Hf phthalocyanine (PcHfQStr) containing the quinolinium styryl fragment, the number, and diameter of formed fibrils is significantly reduced, only single filaments with a diameter about 1-2 nm and fibrils with diameter about 6 nm were observed. The inhibitory activity of Zn and Mg phthalocyanines and porphyrazines of planar structure on the kinetics of insulin fibrillization was also shown. Interaction of Mg­containing complexes with insulin more strongly suppresses the protein aggregation comparing to their Zn­containing analogs. Binding of Zn and Mg porphyrazines to insulin during fibrillization led to the formation of shorter fibrils comparing to free protein. Hydrodynamic diameters of fibrils population estimated by DLS generally correlate with the size of single fibrils obtained by SEM. UV-VIS absorption spectroscopy study revealed that axially coordinated phthalocyanines with a higher tendency to self-association demonstrate higher inhibitory activity in fibrillization reaction compared to poorly associated complexes. It was shown the destruction of phthalocyanine self-associates in the presence of insulin amyloid fibrils, which is evidence of complex formation between them. The formation of the complex between amyloid fibrils and phthalocyanine was also demonstrated by surface-enhanced Raman spectroscopy. The biggest increase in the intensity of the phthalocyanine peaks in the Raman spectrum in the presence of insulin fibrils was ten times. According to literature, the structural basis for the effect of the macrocyclic compound on amyloid fibril formation relies on specific π-π interactions between the aromatic ring system of molecules and aromatic residues of proteins. We have suggested that ligands of axially coordinated phthalocyanines, dependently on structure and geometry, could provide additional fixation upon the binding to the protein or prevent this binding. In the case of phthalocyanines and porphyrazines, «opened» central metal atom can contribute to the interaction of the macrocyclic compound to protein due to additional coordination bonding. The difference between Zn- and Mg-containing complexes could be explained by the ability of Zn atom of phthalocyanine to coordinate to the histidine residues of the protein.

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