Conformational-Switch Based Strategy Triggered by [18] Ï

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Conformational-switch based Strategy Triggered by [18] Heteroannulenes towards Reduction of Alpha Synuclein (#-Syn) Oligomer Toxicity Ritobrita Chakraborty, Sumit Sahoo, Nyancy Halder, Harapriya Rath, and Krishnananda Chattopadhyay ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00436 • Publication Date (Web): 08 Oct 2018 Downloaded from http://pubs.acs.org on October 9, 2018

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ACS Chemical Neuroscience

Conformational-switch based Strategy Triggered by [18]  Heteroannulenes towards Reduction of Alpha Synuclein (α-Syn) Oligomer Toxicity

Ritobrita Chakraborty,† Sumit Sahoo,Ϯ Nyancy Halder,Ϯ Harapriya Rath,*,Ϯ Krishnananda Chattopadhyay*,†

†Structural

Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4,

Raja S. C. Mullick Road, Kolkata 700032, INDIA; [email protected]; Tel: +913324995843 Ϯ School

of Chemical Sciences, Indian Association for the Cultivation of Science (IACS), 2A/ 2B

Raja S. C. Mullick Road, Kolkata 700032, INDIA; [email protected], Tel: +913324734971 * Corresponding

authors

1 ACS Paragon Plus Environment

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ABSTRACT

A water soluble meso-carboxy aryl substituted [18] heteroannulene (porphyrin) and its Zncomplex have been found to be viable in targeting α-Syn aggregation at all its key micro-events, namely, primary nucleation, fibril elongation and secondary nucleation, by converting the highly heterogeneous and cytotoxic aggresome into a homogeneous population of minimally toxic offpathway oligomers, that remained hitherto unexplored till date. With the EC50 and dissociation constants in the low micromolar range, these heteroannulenes induce a switch in the secondary structure of toxic prefibrillar on-pathway oligomers of α-Syn converting them into minimally toxic non-seeding off-pathway oligomers. The inhibition of the aggregation and the reduction of toxicity have been studied in vitro as well as inside neuroblastoma cells.

KEYWORDS: Heteroannulenes, α-Syn, conformational switch, on-pathway oligomer, off-pathway oligomer, neuroblastoma cell


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For Table of Contents Use Only

Conformational-switch Heteroannulenes

based

towards

Strategy

Reduction

of

Triggered Alpha

by

[18]

Synuclein

π

(α-Syn)

Oligomer Toxicity

Ritobrita

Chakraborty,

Sumit

Sahoo,

Nyancy

Halder,

Harapriya

Rath, Krishnananda Chattopadhyay



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INTRODUCTION

Alpha Synuclein (-Syn) is a 140 amino acid residue long, natively unfolded protein associated with incurable synucleopathies such as Parkinson’s disease (PD), dementia with Lewy bodies and multiple system atrophy.1, 2 PD is characterized by neuronal loss accompanied with motor and cognitive deficits in patients.3 The molecular interpretation of PD entails a conformational alteration of the soluble protein -Syn into misfolded monomers that self-coalesce into intermediate structures ultimately forming wellorganized amyloid fibrils. The deposition of these cross-β sheet-rich amyloid fibrils in insoluble cytoplasmic inclusions (Lewy bodies) within the dopaminergic neurons of the substantia nigra pars compacta of the brain is the physiological hallmark of PD.1 α-Syn consists of an amphipathic N-terminal domain (residues 1-60), a hydrophobic core domain, also known as the NAC (non-amyloid β component) region (residues 61–95), and an acidic, proline-rich C-terminal domain (residues 96–140).4 The amino acid sequence of the NAC also occurs individually as a 35 residue-long peptide fragment 4 ACS Paragon Plus Environment

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(non-amyloid ) along with the amyloid  peptide in the cytotoxic deposits of the related neurodegenerative Alzheimer’s disease (AD). 5, 6 Interestingly, this NAC sequence is also found as a domain within the α-Syn protein. As a domain within the α-Syn protein, the NAC due to its hydrophobicity, acts as the initiator of aggregation. Previous studies using paramagnetic relaxation enhancement and NMR dipolar coupling have reported that the native monomeric α-Syn forms an ensemble of fluctuating conformations that are stabilized by a network of long-range intra-molecular interactions involving the N and C termini.7, 8 These conformations are ring-like, compact and relatively stable, and thought to prevent oligomerization and fibrillogenesis by shielding the highly amyloidogenic NAC from the surrounding solvent. A promising approach to hinder aggregation resulting from protein misfolding is to use small molecules or chaperones to bind and stabilize these native auto-inhibitory states of the protein.8, 9 The development of such compounds has been successful in the case of globular proteins, such as transthyretin,10 a protein implicated in systemic amyloidosis, but the structural heterogeneity and transient nature of the early stage structural elements11 of intrinsically disordered proteins like α-Syn pose a major challenge in the discovery and design of small molecule inhibitors. Experimental evidence has also suggested that amyloid fibril formation is not linear but rather a competitive multi-pathway process that involves stable as well as metastable polymers, which show diverse degrees of cellular toxicity.12 The formation of amyloid fibrils of α-Syn typically follows the nucleation-conversion-polymerization model.13,

14

This process initiates through an event called primary nucleation in which the misfolded monomers of α-Syn associate via diverse pathways to form toxic oligomers and protofilaments, which elongate to form fibrils.15

Although the fibrils are minimally



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toxic,16 they have been shown to dissociate, acting as reservoirs of toxic misfolded monomers and oligomeric species.17 These misfolded species can nucleate again or seed collateral fibrillization in a process known as secondary nucleation.18-20 Several studies have suggested the existence of a variety of intermediate oligomeric species that transiently populate the conformational landscape of α-Syn. The ‘on-pathway’ oligomers consist of anti-parallel β sheet conformation and eventually form parallel β sheet fibrils. These oligomers contain a high percentage of exposed hydrophobic surfaces and are cytotoxic.21 In contrast, dead-end ‘off-pathway’ oligomers are those that contain parallel β sheet conformation with less exposed hydrophobic surfaces and do not form fibrils. These are not cytotoxic.12,

22-24

The precise molecular underpinnings of the

conformational switch between the on and off-pathway oligomers remain unclear. Although several small molecules have been tested as therapeutics for PD, none of them have cleared clinical trials.25 There is an upsurge of demand for small molecules that can inhibit and/ or terminate amyloid formation, and decrease PD-associated mitochondrial stress and cellular toxicity. In this manuscript, a novel mechanism of action of amyloidinhibition involving a ‘conformational switch’ in the oligomers of α-Syn has been explained, that might aid in developing neuroprotection against PD and other neurodegenerative diseases. We have explored the ability of two porphyrins (among a set of six porphyrins): TCPP (tetrakis (4-carboxyphenyl)porphyrin tetraSodium, 8) and ZnTCPP (Zinc-tetrakis (4-carboxyphenyl)porphyrin tetraSodium, 9), to interfere with the on-pathway intermediates (containing anti-parallel -sheet, formed via primary nucleation) of α-Syn leading to the formation of off-pathway species, with parallel β sheet content, that do not self-adhere to form amyloid fibrils. In contrast to the on-pathway


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oligomers, these off-pathway aggregates show reduced cytotoxicity to mammalian cells and are not capable of disrupting the integrity of synthetic liposomal membranes. We have also reported that 8 and 9 can block secondary processes such as the seeding capacity of α-Syn preformed aggregates. Furthermore, these porphyrins can also disintegrate preformed fibrils into smaller-sized oligomers which are rich in parallel β sheets, and more importantly, prevent these oligomers from seeding new aggregation reactions. 8 and 9 can also interrupt N-Methyl-4-phenylpyridinium Iodide (MPP+) induced aggregation inside SH-SY5Y neuroblastoma cells. Additionally, according to our observations, sub-micromolar doses of the porphyrins are sufficient to interfere with the production of toxic oligomers of α-Syn.

Scheme 1 Synthesis of macrocycles under study.

Results



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Synthesis and Reaction of the Porphryrin Macrocycles with α-Syn: Macrocycles 8 and 9 are the most effective inhibitors of α-Syn aggregation The macrocycles under biological investigation described in the present manuscript are shown in Scheme 1. We have taken into consideration, the parent basic porphyrin i.e. tetraphenyl porphyrin, and variations in its periphery with other meso-substituents. Synthesis of porphyrins 1, 4, and 7 were carried out following the Adler method.26 Since these macrocycles (1, 4 and 7) were not water-soluble, they were made soluble following literature method.27-29 Subsequently, these water soluble macrocycles were metallated with zinc salt. The UV-vis absorption spectra, NMR spectra and chemical analysis of all the macrocycles (both in free base forms and zinc-complexes) are well consistent with the structures. The viability of macrocycles under study towards inhibition of -Syn aggregation is based on the fact that tetrakis(4-carboxyphenyl)porphyrin tetraSodium) (8), Zn-

tetrakis(4-carboxyphenyl)porphyrin

sulfonatophenyl)porphyrin

tetrasodium

tetraSodium) (2)

and

(9), Zn-

Tetrakis

(4-

Tetrakis

(4-

sulfonatophenyl)porphyrin tetrasodium (3) are negatively charged at the periphery when water soluble. On the other hand, Tetra-(N-methyl pyridyl porphyrin) (5) and the Zinc complex of Tetra-(N-methylpyridyl porphyrin) (6) are positively charged at the periphery in the pH range in which they are water soluble. It is a well-known fact that the anionic porphyrins aggregate in water while the cationic porphyrins do not. It may be surmised that the positive centers on the periphery cause the delocalized -electron cloud to be more diffuse over the surface of the molecule, while negative centers lead to a partial localization of electron density near the centre. This localized electron density would make the centre a more attractive site for protons thereby increasing the basicity and


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decreasing the acidity of the species, and would lead to stronger van der Waal’s interactions for a stacking-type dimer. In other words, the extent of self aggregation properties of all the water soluble macrocycles under study has been considered as the key parameter in binding to the protein and inhibiting its aggregation. Initially, the binding between the six porphyrin molecules and monomeric α-Syn was investigated by titrating 0.1 μM of the porphyrins with α-Syn concentration ranging between 0.1 μM and 4 μM. The values of the dissociation constant Kd (Kd=1/Ka) are stated in Table 1. The dissociation constant (Kd) for TCPP, sodium salt (porphyrin 8) and ZnTCPP, sodium salt (porphyrin 9) was found to be 8.92 x 10-7 M and 4.69 x 10-7 M, respectively, and was lower by one order of magnitude than the Kd values of the remaining porphyrin molecules (Table 1 and Figure S1, Supporting Information). Porphyrin

Dissociation Constant (Kd), M

2

6.47 x 10-6

3

4.36 x 10-6

5

2.8 x 10-6

6

1 x 10-6

8

8.92 x 10-7

9

4.69 x 10-7

Table 1: Binding data derived for the binding of the six water-soluble porphyrins under study, with α-Syn.

Subsequently, we investigated the amyloid fibril formation of the protein by incubating 200 M α-Syn dissolved in sodium phosphate buffer, pH 7.4, at 37 ⁰C with constant agitation at 180 rpm for 96 hours in the absence and presence of 2 M porphyrins (protein: porphyrin 100: 1). Similar concentration of protein and experimental conditions has been shown by us to result in consistent and reproducible aggregation kinetics with 9 ACS Paragon Plus Environment


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defined lag time and saturation behaviour.30, 31 The extent of amyloid formation after 96 hours of incubation with or without the porphyrins was measured by steady state fluorescence emission of thioflavin T (ThT), and the results are shown in Figure 1. ThT is a

benzothiazole

dye

that

exhibits

enhanced

fluorescence

upon

binding

to amyloid fibrils.32 Figure 1 clearly shows that the molecules 8 and 9 offered maximum inhibition of amyloid fibril formation. Because of their better binding (Table 1) and more potent aggregation inhibition (Figure 1), these two molecules (8 and 9) were chosen for further detailed investigations as discussed below.

Figure 1 ThT fluorescence intensity of 200 μM α-Syn after 96 hours of incubation in the absence, and presence of 2 μM porphyrin macrocycles added from the beginning of the incubation period. Porphyrins 8 and 9 show maximum inhibition of amyloid fibrillization.

Figure 2a shows that the amyloid-formation kinetics of α-Syn follows a sigmoidal behaviour. In the absence of porphyrin, the kinetics was characterized by a lag phase of ~16 hours, in which a negligible change in ThT fluorescence was observed. This was followed by rapid exponential growth, until a saturation plateau phase of fibril maturation 10 ACS Paragon Plus Environment

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was achieved at ~75 hours (Figure 2a). This behaviour is typical for the nucleationconversion-polymerization13, 14 model of protein aggregation as outlined in Scheme 2. In the presence of both 8 and 9 (protein: porphyrin 100: 1), a large increase in the lag time accompanied by minimal increase in ThT fluorescence intensity was observed. (Figure 2a).

Scheme 2 The nucleation-conversion-polymerization model of α-Syn fibrillogenesis. Under pathogenic conditions, the monomeric protein is converted into and remains in equilibrium with an ensemble of misfolded conformations, which further self-associate via primary nucleation to form on-pathway oligomeric states and amyloid fibrils. Secondary processes of fibril fragmentation and secondary nucleation of these fragmented polymers onto the mature fibrils form a feedback loop that maintains the pathogenic amyloidogenesis cycle. Addition of porphyrins 8 or 9 to α-Syn leads to the formation offpathway olgomers which do not lead to mitochondrial dysfunction or membrane perforation. The micro-events that are inhibited by porphyrins 8 and 9 are represented by dotted lines.



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The half maximal effective concentration (EC50) was calculated by plotting on the y- axis, the ThT fluorescence intensity values of 200 μM α-Syn incubated (for 96 hours with constant agitation at 37 ⁰C) with various concentrations of 8 or 9. The log values of the porphyrin concentrations have been plotted on the x-axis. Figure 2b-c illustrate the representative sigmoidal semi log plots for the porphyrin 8 and 9 obtained by fitting the data using the Dose Response fitting function. Along with the EC50, the values of EC20 and EC80, which are the effective concentrations of the porphyrins at which 20% and 80% response are achieved, were also calculated. Doses of 8 and 9 as low as 1.22 μM and 1.49 μM, respectively, were found adequate to cause 50% reduction in aggregation of 200 uM α-Syn. Although both macrocycles were found effective in inhibiting the formation of fibrils, macrocycle 8 was found to be more potent than 9. Subsequently, we investigated if macrocycles 8 or 9 could show any ability to dissociate mature fibrils. When added at a molar ratio of 10: 1 (α-Syn: porphyrin) to mature fibrils (at the stationary phase, after 96 hours of aggregation), 8 and 9 caused disaggregation of the mature fibrils manifested by a sharp and permanent decrease in ThT fluorescence (Figure 2d). It was found that at a ratio lesser than 10: 1, the defibrillization by 8 or 9 was significantly slower. Thus, the 10: 1 ratio was used as the optimal concentration in these studies. In another experiment, ‘oligomer seeds’ were prepared by incubating 200 M monomeric α-Syn for 24 hours in the absence or in presence of 2 μM 8 or 9. These oligomer seeds were then added at the beginning of the aggregation assay consisting of 200 μM monomeric α-Syn. Figure 2e shows the aggregation profile of α-Syn as control (in the absence of seeds or porphyrins), which followed the typical sigmoidal behavior.


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Figure 2 ThT fluorescence of (a) 200 μM α-Syn incubated for 96 hours in the absence (blue) and presence of 2 μM 8 (black) or 2 μM 9 (red). Figures (b) and (c) illustrate the dose response curves and the calculated EC20, EC50 and EC80 (in μM) values of 8 and 9 when incubated with 200 μM α-Syn for 96 hours. (d) Fragmentation of 200 μM mature α-Syn fibrils (saturation phase) upon addition of 20 μM 8 (black) and 9 (red). (e) Comparison of the seeding effect of porphyrin-treated (α-Syn: Porphyrin 25: 1; 8: black; 9: red) and untreated (green) oligomers on α-Syn; the aggregation profile of 200 μM α-Syn (blue) has been added to this figure for comparison. TEM and AFM micrographs of 200 μM α-Syn incubated for



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96 hours in the absence (f-h) or presence of 2μM 8 (i-k) or 9 (l-n). TEM and AFM images of 200 μM saturation phase fibrils fragmented by 20 μM 8 (o-q) or 9 (r-t). The representative AFM height distribution profile is shown below each micrograph. Oligomers formed as a result of fibril disintegration are marked with black arrows in the TEM images. The white bar corresponds to 200 nm in the TEM and AFM images.

In the presence of porphyrin-untreated oligomer seeds, the kinetics became hyperbolic, a behaviour observed by other research groups12,

24

in seeded aggregation. The oligomer

seeds that had been incubated for 24 hours in presence of 8 and 9 (in the ratio of α-Syn: porphyrin 100: 1) showed complete inhibition of seeding-induced aggregation, indicating that porphyrin-generated oligomers do not function as templates for the conversion into amyloid and are therefore off-pathway structures. The ThT fluorescence measurements were substantiated by negative stain TEM and AFM. According to the TEM micrographs, the diameter of an individual mature fibril after 96 hours of incubation in the absence of porphyrins measured between 6-7 nm (Figure 2f), while its AFM height ranged between 2.8 nm and 6 nm (Figure 2g-h). The length of each fibril measured between 0.5- 2 μm. In contrast, the TEM images of 200 μM α-Syn coincubated from the lag phase with 2 μM 8 (Figure 2i) or 9 (Figure 2l) intimate the absence of fibrillar networks even after 96 hours. Instead, small-length fibrils clumped laterally along their longitudinal axes, with diameters ranging between 10 and 15 nm and lengths of approximately 0.1- 0.3 μm were observed. Spherical oligomers (depicted in the TEM micrographs using arrows) with diameters ranging between 15 nm and 30 nm were also present. AFM images (Figure 2j-k: 8 and Figure 2m-n: 9) report the height of individual


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‘broken’ fibrils to be ~ 4 nm and those of oligomers to be between 2.5 nm and 15 nm. Figures 2o-q and 2r-t show the disaggregation of 200 μM mature α-Syn fibrils (after 96 hours of aggregation) into shorter length clumped fibrils and oligomers upon the addition of 20 μM 8 or 9, respectively. Under these circumstances as well, the TEM images (Figure 2o: 8 and 2r: 9) showed broken fibrils evidently clustered lengthwise, with the clumped fibrils having diameters ranging between 10 nm and 15 nm and length between 0.1 μm and 0.3 μm. In addition, numerous oligomers with diameters varying between 10 nm and 20 nm were also present. AFM (Figure 2p-q: 8 and Figure 2s-t: 9) images quantified the diameters of the clumped fibrils to be in the range between 7 nm and 20 nm and length between 0.3 μm and 1 μm. The diameters of the oligomers were found to be between 3 nm and 9 nm. The macrocycles 8 and 9 inhibit aggregation of α-Syn inside live neuroblastoma cells Further, we examined α-Syn aggregation inside SH-SY5Y neuroblastoma cells in the absence and presence of 8 or 9. Figure 3a represents the confocal image of cells that were transiently transfected using an EGFP-α-Syn construct, and the aggregation of α-Syn was induced by treating these cells with 5 μM N-Methyl-4-phenylpyridinium Iodide (MPP+) dissolved in DMEM for a period of 6 hours. The intense green dots (marked by white arrows) within the cytoplasm represented α-Syn aggregates as has been shown in previous literatures.33,

34

The incubation for 2 hours with 5 μM of either 8 or 9 of the

transfected MPP+-treated cells, represented by figures 3b and 3c, respectively, prevented the formation of the aggregates marked by the absence of intense green dots in the cell cytoplasm (only the outlines of the cells can be ascertained from the images). The fluorescence from these cells (Figure 3b and 3c) were comparable to the basal



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fluorescence of the control SH-SY5Y cells that contained only minute green aggregates showing diminished fluorescence, because these cells were transfected with the protein construct, but not treated with MPP+ (Figure 3d). The corresponding intensity profiles along the yellow lines drawn across the confocal images are presented in Figure 3 and show that the fluorescence emission of the MPP+-treated cells in Figure 3a is about threefold greater than that of those cells that were treated 8 or 9 (Figures 3b and 3c, respectively).

Figure 3 Confocal microscopy images of SH-SY5Y cells: (a) The white arrows indicate the intense punctate cytoplasmic aggregates of GFP-tagged α-Syn induced by the exposure to 5 μM MPP+ for 6 hours. Further incubation for 2 hours of the MPP+-treated cells with 5 μM (b) 8 or (c) 9 reduces the 16 ACS Paragon Plus Environment

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presence of the high intensity cytoplasmic aggregates. (d) Control cells transfected with α-Syn-GFP, but without the addition of MPP+. The white scale bars denote 20 μm. The intensity profiles of the fluorescence signals along the yellow lines in the confocal images are shown on the right for comparison.

We studied the co-localization with other cellular organelles of porphyrin 9, because of its higher fluorescence compared to porphyrin 8 inside SH-SY5Y cells. The difference in fluorescence between 8 and 9 inside SH-SY5Y cells (Figure 4a-b) may have a direct correlation with the self-association propensity of these porphyrins when dissolved in buffer/ culture medium. Tetrapyrrolic compounds are known to remain aggregated in aqueous solution, thus emitting lesser fluorescence than their monomeric counterparts in organic solvents such as DMSO.35-37 When compared, we observed a higher stacking propensity of 8 than 9 (Figure S2, Supporting Information). For the colocalization studies, SH-SY5Y cells incubated for 2 hours with 9 were further coincubated with MitoTracker Green FM or LysoTracker Green DND-26, which are specific dyes for the mitochondria and lysosomes, respectively, and then subjected to confocal imaging.



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Figure 4 Uptake of porphyrins (a) 8 and (b) 9 inside SH-SY5Y cells. The superior fluorescence of 9 inside cells is presumably due to its lesser propensity to self-associate in aqueous medium. (c) Colocalization of 9 with LysoTracker Green DND-26 within lysosomes. (d) Porphyrin 9 does not colocalize within mitochondria with MitoTracker Green FM.


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The concentration of 9 was kept at 5 μM. It was seen that porphyrin 9 colocalized only in the lysosomes with LysoTracker (Figure 4c). A lesser extent of 9 was also detected in the cell cytoplasm, but not at all in the mitochondria or the nucleus (Figure 4d). This is in accordance with previous studies that have reported that cationic porphyrins localize in mitochondria, whereas those with a more anionic character tend to localize in lysosomes.37 Porphyrins 8 and 9 alleviate membrane perturbation propensity and cytotoxicity of -Syn oligomers The aggregation pathway of α-Syn is extremely heterogeneous and comprises different microscopic events that may individually or collectively contribute to the generation of toxic oligomers and overall the cytotoxicity. The inhibition of primary nucleation by 8 or 9 strongly delayed the fibril formation but increased the amount of oligomers formed as evidenced by the ThT measurements, AFM and TEM results. Moreover, suppression of fibril and seed elongation and disaggregation of mature fibrils lead to an increase in the amount of oligomers, as has also been reported earlier.38 As a result, the treatment of porphyrins at any stage in the aggregation pathway of α-Syn leads to an overall increase in oligomer content. Since on-pathway oligomers are known to generate significant toxicity, while their off-pathway counterparts do not,22-24 determination of the overall cytotoxicity would further validate the on- vs off-pathway nature of the oligomers formed by the addition of 8 and 9. Alternatively, these measurements would provide direct information of whether the oligomers formed due to the addition of 8 or 9 at either the lag phase or at the saturation phase were as toxic as the oligomers formed in the absence of the porphyrins. 19 ACS Paragon Plus Environment


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We compared the membrane permeation and cellular toxicity of the oligomers, which formed in the absence and presence of porphyrins. For this purpose, we synthesized calcein-loaded small unilamellar vesicles (SUVs) composed of 3: 7 POPC: DOPS and added them to 2 μM oligomeric α-Syn (either treated or untreated with porphyrins) at a protein: lipid ratio of 1:10. Triton X-100 was used to determine 100% calcein release and all results were normalized to this value. Upon addition of oligomers (formed in the absence of porphyrins) to the SUVs, a 62% increase in calcein fluorescence was observed (Figure 5a). In contrast, oligomers formed when 2 μM 8 or 9 were added at the beginning of aggregation kinetics (lag phase) showed a much lesser calcein release of 35% and 41%, respectively. Moreover, the oligomers obtained because of the fragmentation of saturation phase fibrils with 20 μM 8 or 9 also led to a decreased calcein release of 32% and 22%, respectively. Addition of the porphyrins alone as controls (at a concentration of 20 μM) to the SUVs caused a minor calcein release of

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