Conformational-Switch Based Strategy Triggered by [18] π Heteroannulenes toward Reduction of Alpha Synuclein Oligomer Toxicity

Article abstract of DOI:10.1021/acschemneuro.8b00436

A water-soluble meso-carboxy aryl substituted [18] heteroannulene (porphyrin) and its Zn-complex have been found to be viable in targeting α-Syn aggregation at all its key microevents, namely, primary nucleation, fibril elongation, and secondary nucleation, by converting the highly heterogeneous and cytotoxic aggresome into a homogeneous population of minimally toxic off-pathway oligomers, that remained unexplored until recently. 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 nonseeding off-pathway oligomers. The inhibition of the aggregation and the reduction of toxicity have been studied in vitro as well as inside neuroblastoma cells.

Full text of DOI:10.1021/acschemneuro.8b00436

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Article
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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Conformational-switch based Strategy Triggered by [18] 
Heteroannulenes towards Reduction of Alpha Synuclein (α-Syn)
Oligomer Toxicity
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†
Ϯ
Ϯ
*,Ϯ
Ritobrita Chakraborty, Sumit Sahoo, Nyancy Halder, Harapriya Rath, Krishnananda
Chattopadhyay
*,†
†Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4,
Raja S. C. Mullick Road, Kolkata 700032, INDIA; krish@iicb.res.in; 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; ichr@iacs.res.in, Tel:
+
913324734971
*
Corresponding authors
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ABSTRACT
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A water soluble meso-carboxy aryl substituted [18] heteroannulene (porphyrin) and its Zn-
complex 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 off-
pathway oligomers, that remained hitherto unexplored till date. With the EC and dissociation
50
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 based Strategy Triggered by [18]
π
Heteroannulenes towards Reduction of Alpha Synuclein (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
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accompanied with motor and cognitive deficits in patients. 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 well-
organized 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),
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and an acidic, proline-rich C-terminal domain (residues 96–140). The amino acid
sequence of the NAC also occurs individually as a 35 residue-long peptide fragment
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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
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has been successful in the case of globular proteins, such as transthyretin, a protein
implicated in systemic amyloidosis, but the structural heterogeneity and transient nature
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of the early stage structural elements 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
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polymers, which show diverse degrees of cellular toxicity. 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, they have been shown to dissociate, acting as reservoirs of toxic misfolded
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monomers and oligomeric species. 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
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cytotoxic. 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
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have cleared clinical trials. 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 amyloid-
inhibition 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
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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.
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Synthesis of porphyrins 1, 4, and 7 were carried out following the Adler method. 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
tetrasodium
tetraSodium)
(2) and
(9),
Zn-
Tetrakis
Tetrakis
(4-
(4-
sulfonatophenyl)porphyrin
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.
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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 K (K =1/K ) are
d
d
a
stated in Table 1. The dissociation constant (K ) for TCPP, sodium salt (porphyrin 8) and
d
-7
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ZnTCPP, sodium salt (porphyrin 9) was found to be 8.92 x 10 M and 4.69 x 10 M,
respectively, and was lower by one order of magnitude than the K values of the
d
remaining porphyrin molecules (Table 1 and Figure S1, Supporting Information).
Porphyrin
Dissociation Constant (K ), M
d
-
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8
9
6.47 x 10
-6
4.36 x 10
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2.8 x 10
-6
1 x 10
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8.92 x 10
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
2
00 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
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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
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a
benzothiazole dye that exhibits enhanced fluorescence upon binding
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2
to amyloid fibrils. 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
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was achieved at ~75 hours (Figure 2a). This behaviour is typical for the nucleation-
conversion-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
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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 off-
pathway 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 (EC ) was calculated by plotting on the y- axis,
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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 EC , the values of EC
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and EC , which are the effective concentrations of the porphyrins at which 20% and 80%
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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 EC , EC and EC (in μM) values of 8 and 9 when incubated with 200 μM α-Syn for 96
20
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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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6 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.
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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 co-
incubated 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
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(
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
.1 μm and 0.3 μm. In addition, numerous oligomers with diameters varying between 10
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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
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construct, but not treated with MPP (Figure 3d). The corresponding intensity profiles
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along the yellow lines drawn across the confocal images are presented in Figure 3 and
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show that the fluorescence emission of the MPP -treated cells in Figure 3a is about three-
fold 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
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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
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presence of the high intensity cytoplasmic aggregates. (d) Control cells transfected with α-Syn-GFP, but
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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.
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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 co-
localization studies, SH-SY5Y cells incubated for 2 hours with 9 were further co-
incubated 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
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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
38
amount of oligomers, as has also been reported earlier. 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.
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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
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(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 <2%.
Figure 5 (a) Calcein-loaded SUV membrane permeation, (b) % of early apoptotic SH-SY5Y cells and (c)
%
of SH-SY5Y cells showing mitochondrial membrane depolarization in presence of following
conditions: (1)- 20 mM sodium phosphate buffer; (2)- 20 μM 8; (3)- 20 μM 9; (4)- α-Syn oligomers
incubated for 28 hours at 37 ⁰C with constant agitation; (5 and 6)- porphyrin-treated oligomers formed
when 200 μM α-Syn monomers were incubated from the lag phase with (5) 2 μM 8 or (6) 2 μM 9 for 28
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hours; (7 and 8)- porphyrin-treated oligomers formed when 200 μM α-Syn fibrils (agitated for 96 hours)
were further incubated for 48 hours with (7) 20 μM 8 or (8) 20 μM 9.
A subsequent flow cytometry study was performed to investigate the pro- versus anti-
apoptotic effect of the oligomeric species on the neuroblastoma cell line SH-SY5Y.
Figure 5b (also Figure S3, Supporting Information) illustrates that the oligomers formed
in the absence of porphyrins lead to a higher percentage of cells in the early apoptotic
phase (Q4 of Figure S3, Supporting Information) compared to their porphyrin-treated
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(both at the lag phase and saturation phase) counterparts. Since mitochondrial dysfunction
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plays a central role in the pathogenesis of PD, we measured the effect of the oligomers
on the mitochondrial transmembrane potential using the dye JC-1. Figure 5c (also Figure
S4, Supporting Information) shows that the porphyrin-stabilized (both at the lag phase
and saturation phase) oligomers are substantially capable of reducing the percentage of
cells demonstrating mitochondrial membrane depolarization as compared to that of the
untreated oligomers. Altogether, these studies confirm an overall decrease of cytotoxicity
of the oligomers formed as a result of porphyrin treatment.
Porphyrins 8 and 9 alter the secondary structure and hydrophobic surface exposure of the
oligomeric species of α-Syn
In order to identify the underlying mechanism of the prevention of α-Syn aggregation by
8
and 9 and the reduction of toxicity, we compared the structure of α-Syn oligomers
formed in the absence and presence of the porphyrins. The secondary structure of the
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oligomers was estimated using the deconvolution of the FTIR spectra. In the absence of
porphyrins, the oligomers formed after 28 hours of incubation under aggregation-
inducing conditions of constant agitation at 180 rpm at 37 ⁰C, were largely composed of
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anti-parallel β sheets as characterized by the presence of a band at ~1624 cm and
another band at ~1683 cm-1 (Figure 6a). The same conclusion was also obtained from the
second derivative analysis of the FTIR spectra (Figure S5, black trace). The band at 1683
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cm was approximately four-fold weaker than the band at ~1624 cm-1 (Figure 6a, and
Figure S5, black trace, for the second derivative plot) which is suggestive of anti-parallel
β sheet content.24 On the other hand, oligomers with parallel β structure
Figure 6 Deconvoluted FTIR spectra of (a) α-Syn oligomers formed after 28 hours of aggregation; (b and
c) α-Syn oligomers formed when the monomeric protein was incubated with porphyrins (b) 8 and (c) 9
(
α-Syn: Porphyrin 100: 1) for 28 hours; (d and e) α-Syn oligomers formed when α-Syn mature fibrils
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were treated for 48 hours with (d) 8 or (e) 9 (α-Syn: Porphyrin 10: 1). The representative FTIR peaks
denoting the secondary structures are colour-coded and the percentage of secondary structural content is
mentioned underneath each curve; (f and g) Fluorescence emission from ANS binding experiments for α-
Syn oligomers aggregated for 28 hours (black), and porphyrin treated oligomers (red: 8 added at the lag
phase; blue: 8 added at the saturation phase; magenta: 9 added at the lag phase; green: 9 added at the
saturation phase).
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were formed when 200 μM α-Syn monomers were incubated from the lag phase with 2
μM 8 (Figure 6b) or 9 (Figure 6c) for 28 hours. Additionally, the oligomers formed when
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in parallel β sheet structures as identified by the presence of a band at 1620-1630 cm and
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the absence of the band at ~1683 cm (Figure 6d for 8 or Figure 6e for 9). ANS binding
experiments showed an increase in ANS fluorescence intensity for oligomers generated in
the absence of porphyrins (Figure 6f-g) indicating that these oligomers contained large
solvent exposed hydrophobic surfaces. In case of porphyrin-stabilized oligomers
(prepared by the addition of 8 or 9 either at the lag phase or at the saturation phase of
aggregation), the ANS intensity was comparatively lower (Figure 6f: 8 and Figure 6g: 9).
Discussion
Till date, several studies have attempted to characterize the large diversity of the
prefibrillar α-Syn oligomers that differ in molecular weight, morphology and toxicity.17
These intermediates may or may not culminate into fibrils and have been described as
spheres (2–6 nm in size), chains of spheres (protofibrils) and rings (annular
protofibrils).41-43 The transient unstable and metastable oligomers may also vary in their
seeding capacity.44 Recent reports have attempted to characterize the oligomeric
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population depending on whether or not they lead to fibril formation. The oligomers that
eventually polymerize to form parallel β sheet-containing fibrils have been named ‘on-
pathway’. These oligomers consist of anti-parallel β sheet conformation and a higher
percentage of exposed hydrophobic surfaces. These have been found to be more
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cytotoxic. In contrast, dead-end ‘off-pathway’ oligomers do not form fibrils and contain
parallel β sheet conformation with less exposed hydrophobic surfaces. These are not
cytotoxic.12, 22-24 In our study, these diverse-sized polymers formed in the presence and in
absence of porphyrins 8 or 9, have been visualized by ThT fluorescence measurements,
electron microscopy and atomic force microscopy (Figure 2).
In the absence of porphyrins, the primary nucleation process of α-Syn aggregation begins
when the monomers misfold to form nuclei, which self-adhere to form toxic early
oligomers (Scheme 2). Early oligomers have a nominal amount of amyloid structure and
hence correspond to the low ThT intensity lag phase (Figure 2a). The oligomers further
combine to form amyloid fibrils in the exponential phase which terminates into the
saturation phase (Scheme 2 and Figure 2a). The high ThT fluorescence intensity of the
saturation phase is maintained even without the addition of fresh monomers due to the
secondary processes of fibril fragmentation into monomers, and secondary nucleation of
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these fragmented monomers on fibril surfaces. When porphyrins 8 or 9 were added to α-
Syn monomers at the lag phase, the primary nucleation stage appeared to be inhibited as
evidenced by the reduced ThT fluorescence value and absence of the subsequent
exponential phase. Thus, 8 and 9 can bind to monomeric α-Syn and retard the
oligomerization process from a very early step at a very low porphyrin: protein molar
ratio of 1: 100 (Figure 2a). It may be suggested that 8 and 9 can inhibit amyloid
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fibrillogenesis because they stabilize the unstructured state of the natively unfolded
protein and reinforce the auto-inhibitory intra-molecular interactions in the protein that
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causes the shielding of the NAC region from the surrounding solvent. Additionally, the
sharp decrease in ThT fluorescence when 8 or 9 is added to mature fibrils in the saturation
phase suggests that these porphyrins can inhibit the secondary processes as well (Figure
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d and Scheme 2). Templated seeding experiments by preformed oligomers enable the
aggregation reaction to proceed faster and lead to the growth of more fibrils by monomer
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addition, without primary nucleation. The seeding experiments performed by us (Figure
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e) have suggested that the oligomers formed in presence of 8 or 9 do not elongate to
form amyloid fibrils.
In addition, the oligomers formed in the presence of 8 and 9 did not possess significant
toxicity. Our in cell toxicity findings have revealed that while the extracellular addition of
oligomers formed in the absence of porphyrins to neuroblastoma cells increases
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cytotoxicity, the porphyrin-remodelled off-pathway species show reduced apoptotic
susceptibility and mitochondrial dysfunction in these cells (Figure 5b-c). It has been
reported in literature that the toxic species of α-Syn can be transmitted from diseased cells
to nearby healthy neurons where they seed/ induce the conversion of native α-Syn into
misfolded monomers that assemble to form toxic oligomeric species, following a seeding-
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nucleation mechanism. Since porphyrin stabilized oligomers are non-toxic, it can be
interpreted that even if they spread to healthy cells they would not result in cell death. We
have also reported that the oligomers formed in the absence of porphyrins increase the
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permeation of synthetic lipid vesicles in vitro, similar to as reported previously. This
phenomenon has been explained as the propensity of α-syn annular oligomers to integrate
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into membranes resulting in the formation of pores or ion channel-like structures that may
cause uncontrolled membrane permeability.50, 51 In compliance to the cytotoxicity studies,
we found that the porphyrin-treated oligomers show a much reduced pore formation
tendency, thus confirming their off-pathway nature (Figure 5a). In vitro aggregation
kinetics measurements combined with the toxicity studies clearly suggest that the
porphyrins convert the fibril-forming toxic ‘on-pathway’ oligomers into dead-end and
non-toxic ‘off-pathway’ oligomers.
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In order to gain a deeper insight into the underlying mechanism behind the porphyrin-
induced conversion between on-pathway and off-pathway oligomers, we examined their
secondary structure and hydrophobic content using FTIR and ANS fluorescence,
respectively. It was found that a switch in the β-sheet content (from anti-parallel to
parallel) and an expansion in the hydrophobic surface exposure of the oligomers caused
the inhibition of oligomer-association into amyloid fibrils (Figure 6). Similar β sheet
orientation and hydrophobic surface exposure in the oligomeric species of α-Syn have
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been reported previously, and oligomer populations with differing characteristics have
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been shown to cause difference in the membrane perturbation and cell death via the
apoptotic pathway.54-56
This study proposes a strategy to limit the cytotoxic progression in PD by inducing the
conversion of on-pathway anti-parallel β sheet oligomers into off-pathway parallel β sheet
oligomers using meso-substituted porphyrin molecules. Aromatic molecules have been
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shown to inhibit α-Syn amyloid formation by forming π-π stacking interactions with
aromatic residues within the polypeptide chain of the protein. We propose a mechanism
in which the porphyrins 8 and 9 when added to monomeric α-Syn, form stacking
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interactions with the aromatic residues of the protein, which in turn compromise the
formation of the early inter- and intra-molecular contacts stabilizing the cross β amyloid
assembly. Similarly, when added to preformed aggregates, the aromatic groups of 8 and 9
stack in between individual strands thus breaking the hydrogen bonds that hold the cross
β structure in place. We have observed that both the meso-substituted groups at the
periphery of the macrocycle, as well as the central metal ion influence protein binding
and thus the aggregation-deterring activity. Self-association properties of the metal
coordinated porphyrins are well known, and it has been established for the lower order
stacked porphyrins, that the tendency towards self-association is directly proportional to
their anti-aggregatory effect.58 We found higher self-association (Figure S2), higher
binding affinity to α-Syn (Table 1) and higher anti-amyloid activity (Figure 2) of the free
base porphyrin 8 over the metal-coordinated porphyrin 9. Since porphyrins with cationic
peripheral groups show lesser extent of self-association, a lesser amount of anti-amyloid
activity of the free base and zinc-coordinated Tetra-(N-methylpyridyl porphyrins
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(Macrocycles 5 and 6, Figure 1) was observed in our results. We also observed that the
meso-carboxy aryl substituted porphyrins were more potent amyloid inhibitors than the
corresponding meso-aryl sulfonated porphyrins (Figure 1). Thus, the efficiency of these
molecules as anti-amyloid agents could also be triggered by the nature of meso-
substituents. The π-π stacking of aromatic rings can be enhanced when one of the
aromatic rings involved in the process is electron-poor and the other is electron-rich,
limiting possible repulsion between the electrons of both rings. In case of compounds 8
and 9, the presence of strong electron-withdrawing groups such as COOH as the meso-
substituents results in better inhibition, possibly due to the formation of a strong electron
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acceptor- donor complex between the inhibitor (porphyrin) and α-Syn. The conversion of
toxic anti-parallel β sheet-containing oligomers into comparatively non-toxic parallel β
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sheet-containing fibrils is believed by several research groups to be a physiological
salvaging mechanism to reduce cytotoxicity. However, the processes of disintegration of
the fibrils into oligomers and monomers that seed collateral aggregation through
secondary nucleation compromises the ability of the aforementioned cellular quality
control mechanism to minimize cytotoxicity.18, 19 Here, we propose a therapeutic
approach, in which the added porphyrin molecules (8 and 9) compete with the monomeric
protein to bind to the toxic on-pathway oligomers. As an efficient mimic of the natural
system (in which the toxic antiparallel oligomers generate non-toxic parallel fibrils), the
presence of porphyrins eventually lead to the form minimally toxic end-products, which
are stable and non-seeding. Investigations are currently underway in our laboratories to
develop a residue specific understanding of the binding process.
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Another aspect of the competence of porphyrins 8 and 9 as anti-PD therapeutics lies in
their ability to limit the aggregation of α-Syn at any stage in its pathogenesis. After the
initiation/ lag phase in the aggregation pathway of α-Syn is over, approximately all the
secondary processes begin to occur simultaneously. Most small molecules designed and
studied till date can target the late-stage micro-processes only when used at relatively
high concentrations. For example, the poplyphenolic compound EGCG ((–)-
epigallochatechin gallate)12 and the benzoic acid derivative gallic acid (3, 4, 5-
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trihydroxybenzoic acid) can effectively hinder fibrillogenesis only when added at a ratio
of 1:1 (inhibitor: α-Syn) or higher. A recent study on the use of the antibiotic
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the commencement of aggregation at a ratio higher than 1:1 (doxycycline: α-Syn), but
was inefficient in salvaging the already formed higher oligomers and fibrils. This
suggests that doxycyline can target only the primary nucleation micro-event of the
aggregation pathway. However, we find that porphyrins 8 and 9 can limit primary
nucleation at a ratio as low as 1:100 (porphyrin: α-Syn) and secondary processes at a ratio
of 1:10. These observations are imperative for potential drug-treatment studies because
they indicate that these porphyrins might be able to antagonize amyloid formation even
after substantial amounts of fibrils have been formed in PD patients.
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An additional obstruction faced while designing small molecules against PD is that the
drug candidates that can block the aggregation process in vitro do not work suitably in
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vivo due to their toxicity or low bioavailability in the brain. The class of porphyrins in
our study can be easily functionalized and modified using available synthetic chemistry
methods in order to increase their affinity to a particular protein conformation, or
decrease their self-stacking behaviour or to increase their lipophilicity to enable efficient
crossing of the blood-brain barrier.
Conclusion
In conclusion, our results suggest that the presence of porphyrins 8 and 9 at a low
effective micromolar dose can assuage the pathology of PD by targeting several processes
that occur during the α-Syn amyloid amplification cycle. These processes include de novo
as well as seed-induced aggregation, secondary nucleation after fibril fragmentation,
aggregate build-up inside live cells, mitochondrial dysfunction and cell death by
apoptosis. The meso-carboxy aryl-substituted porphyrins showed higher binding to α-Syn
and maximum inhibition of its aggregation, which could be attributed to the formation of
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