Activity of Zn and Mg phthalocyanines and porphyrazines in amyloid aggregation of insulin

Abstract Formation of the deposits of protein aggregates—amyloid fibrils in an intracellular and intercellular space—is common to a large group of amyloid‐associated disorders. Among the approaches to develop of therapy of such disorders is the use of agents preventing protein fibrillization. Polyaromatic complexes—porphyrins and phthalocyanines—are known as compounds possessing anti‐fibrillogenic activity. Here, we explore the impact of related macrocyclic complexes—phthalocyanines (Pc) and octaphenyl porphyrazines (Pz) of Mg and Zn—on aggregation of amyloidogenic protein insulin. Pz complexes are firstly reported as compounds able to affect protein fibrillization. The effect of Pc and Pz complexes on the kinetics and intensity of insulin aggregation was studied by the fluorescent assay using amyloid sensitive cyanine dye. This has shown the impact of metal ion on the anti‐fibrillogenic properties of macrocyclic complexes—the effect on the fibrillization kinetics of Mg‐containing compounds is much more pronounced comparing to that of Zn analogues. Scanning electron microscopy experiments have demonstrated that filamentous fibrils are the main product of aggregation both for free insulin and in the presence of macrocyclic complexes. However, those fibrils are distinct by their length and proneness to lateral aggregation. The Pc complexes cause the increase in variation of fibrils length 0.9 to 2.7 nm in opposite to 1.4 to 2.0 nm for free insulin, whereas Pz complexes cause certain shortening of the fibrils to 0.8 to 1.6 nm. The averaged size of the fibrils population was estimated by dynamic light scattering; it correlates with the size of single fibrils detected by scanning electron microscopy.

the central metal atom, the most pronounced effect was observed for Ni-containing phthalocyanine with formation of small, amorphous aggregates and flat fibrils (in comparison with Al-containing and Zn-containing phthalocyanines as well as metal-free ones ( Figure S2 in Supporting Information).
Previously, we have discovered the anti-fibrillogenic activity of axially coordinated phthalocyanines ( Figure S3 in Supporting Information) that are the complexes with "spatial geometry" of the molecule. [7][8][9] It has been shown that these phthalocyanine complexes are able to affect amyloid aggregation of proteins redirecting their fibrillization toward the formation of oligomeric aggregates or substantially inhibiting the formation of the fibrils. 7,8 The impact of such phthalocyanine on morphology of the formed amyloid aggregates largely depends on the nature of the out-of-plane ligand.
In the present work, we examine the anti-fibrillogenic activity of the structure-related macrocyclic complexes Mg and Zn phthalocya-

| Materials
Mg and Zn phthalocyanines were prepared by metal template synthesis based on the corresponding metal alkanoates by methods described in Chernii et al 10 ; octaphenylporphyrazines were synthesized using general methods described in Cook and Linstead. 11

| Insulin fibril formation
Human insulin was dissolved at 340 μM (2 mg/mL) concentration in 0.1 M water solution of HCl (pH 2) either in the absence (control solution) or in the presence of one of the inhibiting compounds under study (concentration of compounds was 100 μМ). Fibrils were formed by incubating the mentioned protein solutions in a thermo mixer incubator at 65°C for 5 hours. The fluorescence assay based on the cyanine dye 7519 12 was used to monitor the amyloid aggregation degree. The presence and form of fibrillar aggregates were confirmed by SEM measurements.

| 7519 fluorescence assay
To investigate the kinetics of insulin fibril formation in the presence and in the absence of the studied compounds, 10-μL aliquots were removed from the incubated mixture at various time points during the reaction and

| Scanning electron microscopy studies
SEM studies of the products of fibrillization reaction of insulin in the absence and in the presence of the studied compounds were carried out using FEI NovaNano 450 scanning electron microscope. For layer deposition, the samples of insulin amyloid aggregates at the concentration of 340 μM were diluted 15 times with distilled water. Then, a drop of the solution was applied to the special surface (ITO-tin-doped indium oxide coated glass); the sample was studied after water evaporation. We use such parameters as 5 kV (useful for biological objects) and 5-mm distance. The length of the fibrils was determined using Gwyddion program.

| Dynamic light scattering
Buffers were filtered through a 0.2-μM filter prior to use. Following cessation of fibrils, the intensity of light (λ = 632.8 nm) scattered from samples at the concentration of 27.2 μM of insulin fibrils was measured at 6 angles between 40°and 160°(40°, 60°, 90°, 120°, 140°f or insulin amyloid fibrils in the presence of PcZn, PcMg, and PzMg; 40°, 60°, 90°, 120°, and 160°for free insulin amyloid fibrils and those obtained in the presence of PzZn). The sample was maintained at 25 ± 0.1°C using a circulating water bath to remove air bubbles.
Experiments were performed using the Brookhaven Instruments analyzer. In DLS experiments, the normalized intensity time correlation function G 2 (q, τ) was obtained, q defined as q = (4πn 0 /λ) × sin(θ/2) being thus a function of the angle where n 0 is a refraction index, and τ is a correlation time. For the obtained G 2 (q, τ) function, monoexponential fitting was performed by the dependence FIGURE 1 Structures of Mg and Zn phthalocyanines (PcMg, PcZn), octaphenylporphyrazines (PzMg, PzZn), and amyloid-sensitive dye 7519 (from left to right) G 2 (q, τ) = A + B × e −2Γτ , Γ = Dq 2 is the decay rate with D being the apparent translational diffusion coefficient. Results are presented as the dependence Γ/q 2 on q 2 . 13 Basing on D values, apparent hydrodynamic diameter of the fibrils can be estimated as d = kT/3πηD.

| Kinetics of insulin fibril formation monitored by amyloid sensitive dye
The influence of macrocyclic compounds on the kinetics of insulin fibrillization and degree of their inhibitory activity have been studied using fluorescent assay based on the amyloid-sensitive dye 7519 ( Figure 2). The dye specifically binds to the grooves of amyloid fibrils formed by the beta-pleats and this way increases its emission intensity The aggregation of amyloidogenic protein typically begins with a lag phase, when the formation and accumulation of the fibrillar seeds mainly occur and no significant increase in the fluorescence signal intensity is observed. 14 Studied complexes do not change the duration of the lag-phase of insulin fibrillization.
Mg-containing compounds PcMg and PzMg essentially suppress the intensity of insulin fibrillization (up to 54% and 40% correspondingly at the end of the reaction).
Zn complexes have weaker effect on the insulin fibrillization as compared with Mg ones. PzZn suppresses insulin fibril formation by approximately 11% and slightly (by 17%) increases the reaction intensity. It is observed that the nature of metal ion affects the insulin fibril formation intensity stronger than the periphery arrangement of the macrocycle does.
We have examined whether the interaction of the reference dye 7519 with metal complexes could be responsible for the changes in the dye fluorescence intensity. It is shown that the addition of macrocyclic complexes slightly affects the fluorescence characteristics of the free dye 7519 or its complexes with insulin fibrils. We thus consider that the changes of the dye fluorescent response along the fibrillization reaction pathway are not determined by probable phthalocyanine-dye interactions. Hence, the difference between the activities of Zn and Mg complexes shown by fluorescent assay is associated with their different behavior (supramolecular binding) in insulin aggregation reaction caused by the central metal atom effect.

| Scanning electron microscopy study
We study the impact of Mg and Zn phthalocyanines and porphyrazines on the morphology of amyloid aggregates of insulin by SEM (Figure 3).
Free insulin forms elongated filamentous species of the length 1.4 to 2 μm (Table 1) often with the branched structure. In addition, insulin fibrils have a proneness to lateral aggregation and thus form the so-called bundles 15 (Figure 3A,B). The high density of the fibrils and their bundles on the image may point on high intensity of the fibrillization reaction.
Previously, we have reported the ability of phthalocyanines with out-of-plane ligands to redirect the insulin fibrillization toward the formation of different kinds of aggregates (oligomeric or amorphous species) or cause changes of morphology of fibrils. 7,8 In the presence of earlier studied Hf phthalocyanine bearing quinolinium styryl ligand, the thinning and elongation of the fibrils are observed. 16 (Table 1) of the strongly branched structure and proneness to lateral aggregation.  (Table 1); moreover, a huge rod-like aggregate with the size of 4.3 μm is formed ( Figure 3J). Pc and Pz complexes differently affect the morphology of insulin aggregates, ie, they induce the formation of fibrils with different average length, "thickness," and tendency to sticking together. This could be explained by different "geometry" of the complexes. Phthalocyanines are planar molecules, while porphyrazines have planar macrocyclic core with non-planar arrangement on periphery provided by phenyl moieties (that are able to twist relatively to the core plane).
Thus, steric hindrances are caused by phthalocyanines and porphyrazines upon their supramolecular binding to polypeptide chains, and they change the growth of the fibrils and affect their morphology in different ways.

| Dynamic light scattering study
Dynamic light scattering measurements point on distribution of hydrodynamic parameters of aggregates formed in the presence of the studied compounds. The data obtained at different values of the scattering   17 It should be noted that fibrillar structures are not trivial to study by light scattering even in stationary solutions. 18 In our case, the solutions of fibrils with different shapes (separate fibrils, branched fibrils, or mature fibrils clusters) and sizes   Figure 3 and not presented ones); d, apparent hydrodynamic diameter. are characterized by estimated apparent hydrodynamic radius based on the sphere approximation. The values of apparent hydrodynamic radius for the fibrils can reach up to several micrometers due to a rod-like fibrils morphology. 19 Because we have shown by SEM the formation of fibrils of similar morphology in the presence of all complexes, we also compare the apparent hydrodynamic diameter of these fibrils obtained by DLS.
For this purpose, the apparent diffusion coefficients are estimated from the plots of Γ/q 2 on q 2 and are then converted to an apparent hydrodynamic diameter (

| Discussion of the impact of central metal atom on anti-fibrillogenic activity of macrocycles
The fluorescent dye-based assay has shown the noticeable difference In insulin, the histidine residue H10 ( Figure 5) is involved in hexamer formation by Zn +2 coordination. 21 This H10 is located near the amyloidogenic sequence (11)LVEALYL (17) in insulin B-chain, which is responsible for the formation of fibril core. 22 Generally, the structural basis for the effect of macrocyclic compound on amyloid fibril formation relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues of protein. 4 However, we suppose that due to the men-

ACKNOWLEDGEMENT
The research leading to these results has received funding from the H2020-MSCA-RISE-2014-RISE-645628 -METCOPH project.