A fluorescent molecular imaging probe with selectivity for soluble tau aggregated protein

Article abstract of DOI:10.1039/c9sc05620c

Soluble forms of aggregated tau misfolded protein, generally termed oligomers, are considered to be the most toxic species of the different assembly states that are the pathological components of neurodegenerative disorders. Therefore, a critical biomedical need exists for imaging probes that can identify and quantify them. We have designed and synthesized a novel fluorescent probe, pTP-TFE for which binding and selectivity profiles towards aggregated tau and Aβ proteins were assessed. Our results have shown pTP-TFE to be selective for early forms of soluble tau aggregates, with high affinity of dissociation constants (K_d) = 66 nM, and tenfold selectivity over mature tau fibrils. Furthermore, we found that pTP-TFE is selective for tau over Aβ aggregates and had good cell permeability. This selectivity of pTP-TFE towards early forms of aggregated tau protein ex vivo was also supported with studies on human brain tissue containing tau and Aβ pathology. To the best of our knowledge, this is the first fluorescent molecule to be reported to have this form of selectivity profile, which suggests that pTP-TFE is a unique probe candidate for imaging-based detection of early stages of Alzheimer's disease and other tauopathies.

Full text of DOI:10.1039/c9sc05620c

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DOI: 10.1039/C9SC05620C
ARTICLE
A Fluorescent Molecular Imaging Probe with Selectivity for
Soluble Tau Aggregated Protein
Yanyan Zhao,a† Ole Tietz,a† Wei-Li Kuan,b Abdul K. Haji-Dheere,a Stephen Thompson, a Benjamin
Received 00th January 20xx,
Accepted 00th January 20xx
Vallin,b Elisabetta Ronchi,a Gergely Tóth,c David Klenerman,d,e and Franklin I. Aigbirhio.a*
DOI: 10.1039/x0xx00000x
Soluble forms of aggregated tau misfolded protein, generally termed oligomers, are considered to be the most toxic species
of the different assembly states that are the pathological components of neurodegenerative disorders. Therefore, a critical
biomedical need exists for imaging probes that can identify and quantify them. We have designed and synthesized a novel
fluorescent probe, pTP-TFE for which binding and selectivity profiles towards aggregated tau and A proteins were assessed.
Our results have shown pTP-TFE to be selective for early forms of soluble tau aggregates, with high affinity of dissociation
constants (Kd) = 66 nM, and tenfold selectivity over mature tau fibrils. Furthermore, we found that pTP-TFE is selective for
tau over A aggregates and had good cell permeability. This selectivity of pTP-TFE towards early forms of aggregated tau
protein ex vivo was also supported with studies on human brain tissue containing tau and A pathology. To the best of our
knowledge, this is the first fluorescent molecule to be reported to have this form of selectivity profile, which suggest that
pTP-TFE is a unique probe candidate for imaging-based detection of early stages of Alzhiemer’s disease and other
tauopathies.
likely to be more correlated with disease progression than the
levels of mature fibrils.7 Given that early diagnosis of
Introduction
neurodegenerative disease has become
an important
The accumulation of misfolded protein aggregates is a
pathological hallmark of neurodegenerative diseases.1 In the
case of Alzheimer’s disease (AD), the most prevalent pathology
consists of extracellular -amyloid protein (A) plaques and
intracellular tau protein-containing neurofibrillary tangles.2
Protein aggregates have high beta-sheet structural
characteristic,3 which led to the identification of small
molecules with specific binding affinity for them,4 several of
which have been used successfully for in vivo imaging with
positron emission tomography (PET).5 However, it is becoming
increasingly apparent that proteins associated with
neurodegenerative diseases can adopt different assembly
states, which can impact disease on-set and their progression to
different extent.6 Research on tau protein has shown that the
earlier forming smaller soluble aggregates, generally termed
oligomers, are the most toxic species and their in vivo levels are
objective, a critical need exists for the development of novel
imaging probes that can identify and quantify soluble
aggregated protein species in vivo. Moreover, such probes
could greatly enhance our understanding of disease progression
and be used for assessing the efficacy of new therapeutic
candidates.
Efforts to design a facile imaging agent that can be used for
investigation of soluble protein aggregates have thus far
focused on A-protein, leading to the development of a
monoclonal antibody based probe ([125I]8D3-F(ab’)2-h158)8
and a modest number of small molecules, most notably BD-
Oligo based on the boron-dipyrromethene (BODIPY)
fluorescence structure9 and AN-SP based on a spiropyran
scaffold.10 In addition, luminescent conjugated oligothiophenes
(LCOs) have been shown to bind to soluble aggregates of both
A and tau protein.11 Despite the success in recent years of
developing PET probes for imaging tau neurofibrillary tangles12
presently there are no probes that have selective binding
toward tau soluble aggregated species. With tau protein
a. Molecular Imaging Chemistry Laboratory, Wolfson Brain Imaging Centre,
Department of Clinical Neurosciences, University of Cambridge, Cambridge,
United Kingdom.
pathology being
a
constituent feature of several
b. John van Geest Centre for Brain Repair, Department of Clinical Neuroscience,
University of Cambridge, Cambridge, United Kingdom.
c. TTK-NAP B - Drug Discovery Research Group – Neurodegenerative Diseases,
Institute of Organic Chemistry, Research Center for Natural Sciences, Budapest,
Hungary.
neurodegenerative disorders, collectively termed tauopathies,
the development of such probes, represents a major biomedical
need.
The scaffold diversity of recently reported tau binding com-
pounds highlights the fact that, despite recent progress, there
is currently no clear structure activity relationship consensus.
Nonetheless, flat -conjugated scaffolds with length limited to
13 Å and polarizable ligands are thought to be essential design
d. Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge,
United Kingdom.
e. UK Dementia Research Institute, University of Cambridge, Cambridge, United
Kingdom.
† These authors contributed equally.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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elements for tau probes.13 Previously reported LCO, pFTAA and (iii) may enhance in vivo pharmacokinetic properties, such
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DOI: 10.1039/C9SC05620C
as metabolic instability and reduce blood plasma retention.
(Scheme 1), fulfils these requirements by including
a
pentathiophene backbone and four carboxylic acid (CA) Herein we report the synthesis of the novel fluorescent probe
functional groups and has shown affinity for the soluble pTP-TFE as well as the characterization of its binding to tau and
aggregates of tau and A However, with no apparent selectivity A aggregated proteins in comparison to pFTAA. This includes
between protein types (i.e. tau or A) or their various forms (i.e. the application of a sucrose gradient method to obtain known
soluble and fibril).14-16
sizes of aggregate proteins and thereby quantify binding
Towards developing molecular imaging probes with selectivity affinities of pTP-TFE towards specific soluble aggregated
for soluble aggregates we noted an important structural feature species. Finally, we assessed if pTP-TFE can penetrate intact live
of mature tau fibrils is
a positively charged flexible cell membranes and evaluated its binding to tau pathology in
polyelectrolyte brush, also known as a fuzzy coat.17 On the basis human brain tissue.
that the positive charges of the coat may interact strongly with
the negative charges present in anionic dyes such as pFTAA, we
Results and Discussion
Synthesis of pTP-TFE
reasoned that we may enhance selectivity toward soluble
aggregate forms by reducing this chemical property.18 To impart
selectivity but retain the CA binding properties toward
aggregated proteins, we chose to replace the CAs with
bioisosteric 2,2,2-trifluoroethan-1-ol (TFE) functional groups, to
yield the compound pTP-TFE (pentathiophene-trifluoroethanol
– Scheme 1). TFE functional groups have been shown to act as
excellent replacements for CA groups, while imparting superior
pharmacokinetic properties on molecules.19 Furthermore, the
inclusion of fluorine into the molecule introduces several other
advantages; (i) provides a natural position for labelling with
The strategy for the synthesis of pTP-TFE relied on the
trifluoromethylation of individual thiophene building blocks and
their subsequent assembly to the pentameric target compound
through Suzuki cross-coupling reactions (Scheme 1). Commercially
available starting material (1) was brominated to yield 2 and
subsequently treated with (trifluoromethyl)trimethylsilane and
caesium fluoride to yield silyl protected compound 3, which was
purified, deprotected (4) and reduced to give 2,2,2-trifluoroethan-1-
ol bearing thiophene 5. 5 was cross-coupled to commercially
available 2,5-Bis-thiopheneboronic acid pinacol ester to yield 6,
which was brominated to give 7. The terminal 2,2,2-trifluoroethan-
1-ol bearing thiophene (10) was synthesized from commercially
available starting material 2-bromo-5-trifluoroacetylthiophene (8),
which was reduced (9) and subsequently boronated using
bis(pinacolato)diboron (10). Finally, pTP-TFE was synthesized by
Suzuki cross-coupling of substrates 7 and 10. Purification of pTP-TFE
by preparative thin-layer chromatography (TLC) afforded the
compound in acceptable purity for in vitro evaluation. The overall
yield for the synthesis of pTP-TFE from starting material (1) is 0.7 %.
pFTAA was synthesized from starting material (1), analogues to the
previously reported methodology.14 The fluorescence properties of
pTP-TFE were obtained (Figure S1. pTP-TFE) including its quantum
yield value, which at 0.27 is similar to pFTAA.14
fluorine-18 radioisotope so that
a PET probe can be
developed;20 (ii) allows 19F magnetic resonance imaging (MRI)
and magnetic resonance spectroscopy (MRS) measurements;
Binding to A and tau proteins in aggregation assay
We started our investigation into pTP-TFE’s binding and selectivity
properties by performing an A protein aggregation assay. In this
assay monomeric Aβ40 is aggregated in the presence of pTP-TFE or
pFTAA and the fluorescence intensities of the compounds monitored
over time. An increase in fluorescence intensity is driven by
realignment of -orbitals due to conformation changes in the
thiophene backbone and it is indicative of binding to protein
aggregates.14 Initial results were encouraging as pTP-TFE showed an
earlier response to Aβ40 aggregates in comparison to pFTAA (Figure
1A). Transmission electron microscopy (TEM) images of Aβ40
aggregates at 90 min confirm the presence of small aggregate forms
(Figure 1B) and mature fibrils at later time points (700 min – Figure
1D). To quantitatively assess the binding affinity of pTP-TFE and
pFTAA to different types of aggregates, Aβ40 protein was collected
from an aggregation assay (not containing compounds) at time
points of 90 min, 200 min and 24 h. These aggregates were treated
Scheme 1. Synthesis of pTP-TFE; Synthesis of pFTAA (analogues to previously
report). 14
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with different concentrations of the probes to determine binding aggregates at different time points confirmed that initially small tau
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constants (Kd) (Figure 1E, F) by measuring their fluorescence aggregates were detectable as early as 2D4OhI: 1(0F.i1g0u3r9e/C29BSC),05w6h2i0cCh
intensity. These experiments confirmed that pTP-TFE displays a became more apparent at 96 h (Figure 2C) and then mature fibrils
higher binding affinity to the earlier species (90 min) of aggregates are found in the 240 h samples (Figure 2D). These results suggest
(Kd = 7.58 M), compared to pFTAA which showed no binding affinity preferential binding of pTP-TFE to early soluble tau aggregates in
for aggregates at this stage of maturation. Inversely, pFTAA showed comparison to pFTAA.
stronger binding affinity to later aggregates (Kd = 0.93 M {200 min},
Kd = 0.65 M {24 h}) compared to pTP-TFE (Kd = 3.27 M {200 min},
Kd = 3.79 M {24 h}). In addition, the binding profile of pTP-TFE to a
mixture of Aβ40:Aβ42 (9:1 ratio), which is considered a more
accurate representation of human cerebrospinal fluid A aggregates
in vivo, was also determined. On the basis of the analysis of
fluorescence intensities during the assays (Figure S6A) pTP-TFE
interacted with A aggregates at an earlier time compared to pFTAA
and ThT. Thus, we observe a similar relative interaction profile as
with just Aβ40 (Figure 1A). The binding affinities of pTP-TFE to
Aβ40:Aβ42 (9:1) mixture studied were Kd = 2.1 M at 200 min and
Kd = 0.75 M at 24 h, therefore higher binding affinity than just with
Figure 2. (A) Normalized fluorescence intensities of pTP-TFE (n=3), pFTAA (n=3)
and ThT (n=3) in tau fibril growing assay starting with tau monomers. (B-D)
Representative TEM image of tau aggregates 24 h (B), 96 h (C) and 240 h (D)
(White bar = 200 nm). (E, F) Binding affinity of pTP-TFE to tau aggregates
collected at 96 hours (E) and 240 hours (F) of the aggregation assay.
Interestingly, we observed a shift in the emission spectrum of pTP-
TFE over the course of the experiment. Emission spectra recorded
with tau aggregates at 24, 96 and 240 h show distinct shapes (Figure
S4A). An analysis of the ratio of normalized fluorescence intensities
at 495 nm and 560 nm revealed a gradual shift towards the blue until
96 h (ratio = 2.2 ± 0.20), followed by a shift back towards the red
(ratio = 1.5 ± 0.74) (Figure S4B). These results suggest the pTP-TFE
can bind to distinct oligomers of tau that are generated along the
Figure 1. (A) Normalized fluorescence intensities of pTP-TFE, pFTAA and ThT in
Aβ growing assay starting with Aβ40 monomers (n=3). (B-D) Representative
aggregation pathway of the protein. No emission spectrum shifts
were observed in experiments with pFTAA (Figure S5B).
The binding affinity for pTP-TFE towards aggregates collected from
the aggregation assay (not containing compounds) at 96 h and 240 h
were determined by treatment with different concentrations of
probe (Figure 2E, F). pTP-TFE displayed exceptional affinity for small
aggregates (96 h) with a Kd of 38 nM and, encouragingly, showed no
detectable affinity towards the 240h tau fibril sample (Kd < 10 μM).
These results indicate good selectivity towards soluble aggregated
species.
TEM image of Aβ aggregates 90 min (B), 200 min (C) and 700 min (D) (White
bar = 200 nm). (E, F) Fluorescence intensity based binding curve of pTP-TFE (E)
and pFTAA (F) with A aggregates collected at 90min (red), 200min (blue) and
24 hours (black) of the aggregation assay.
A40. This is consistent with presence of higher affinity binding to
A42 as previously observed.21
We then investigated the binding profile of pTP-TFE to tau protein.
To this end, tau monomers were subjected to fibril-forming
conditions using heparin and aliquots removed at 1, 5, 24, 48, 80, 96,
168 and 240 h. Treatment of these aliquots with pTP-TFE showed a
Binding to fractionated aggregates of A and tau proteins
gradual increase of fluorescence intensity, which reached
a
It has been well documented that protein samples collected at
different time points of an aggregation assay contain heterogeneous
compositions of different aggregated species, from small soluble
form to fibrils. Therefore, we recognized the need to purify these
maximum after 96 h and declined thereafter until the end point of
the experiment (Figure 2A). Experiments with pFTAA showed a curve
of similar shape and characteristic trajectory.16 TEM images of tau
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samples to obtain homogenous protein aggregate fractions of tau to fibrils. Encouragingly, these results furthermore show that the use of
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confirm the binding profile discussed above. To achieve this, we TFE bioisostere endows the compound witDhOsIe: 1le0c.1t0iv3i9ty/Cf9oSrC0so56lu2b0lCe
employed a sucrose gradient ultracentrifugation technique by means aggregates of tau over soluble aggregates of A
of which protein aggregates of different sizes can be separated into
homogenous fractions22 and then used these samples for affinity
measurement (Figure 3).
Aggregates
pTP-TFE
Kd (μM)
pTP-TFE
Binding
Ratioa
14
pFTAA
Kd (μM)
pFTAA
Binding
Ratiob
148
The binding affinity of pTP-TFE and pFTAA with 10% to 50% sucrose
gradient tau aggregates were then tested by a fluorescent binding
assay (Table 1). The data obtained confirms results from the previous
experiments (Figure S7) indicating pTP-TFE binds most strongly to
the 20 % sucrose gradient fraction (Kd = 66 nM), which consist of
soluble aggregated tau as confirmed by TEM (Figure 3). Furthermore,
selectivity of pTP-TFE for small aggregates over fibrils was confirmed
by a difference in Kd for 40 % and 50 % sucrose gradient tau
aggregates of approximately one order of magnitude. Comparisons
of pTP-TFE to tau binding affinity data with that to A aggregates at
200min (Table 1) show a 50-fold selectivity in favour of tau (Kd (Aβ40)
= 3.3 M vs. Kd (tau) = 66 nM). This indicates that pTP-TFE is also
selective for tau soluble aggregates over A soluble aggregates.
pFTAA, by comparison, displays highest affinity to tau aggregates in
the 50 % sucrose gradient fraction (Kd = 0.20 M) and A fibril
aggregates (24 h; Kd = 0.65 μM; Table 1). These results support our
hypothesis that the removal of negatively charged functional groups
imparts higher selectivity for early aggregates of tau over mature
10% sucrose
Tau
20% sucrose
Tau
30% sucrose
Tau
40% sucrose
Tau
50% sucrose
Tau
90 minute
Aβ
200 minute
Aβ
24 hour
Aβ
0.96±0.20
0.07±0.03
3.04±0.71
0.59±0.20
1.08±0.17
7.58±2.47
3.27±0.64
3.79±0.34
29.57±2.10
2.75±0.57
5.27±1.25
1.29±0.20
0.20±0.01
No affinity
0.93±0.30
0.65±0.19
1
49
9
14
26
6
15
108
47
54
1
N/A
5
3
Table 1. Binding affinity of pTP-TFE and pFTAA with 10% to 50% tau fraction
(n=3~6) after ultracentrifugation and Aβ40 aggregates at 90 min, 200 min and 24
hours (n=3), Kd values fitted using GraphPad Prism5. Binding ratio is based on
a
comparison of Kd values with the 20% sucrose tau Kd. b Binding ratio is based on
comparison of Kd values with the 50% sucrose tau Kd.
Cellular uptake of pTP-TFE into primary human fetal neurons
To determine whether pTP-TFE can penetrate live intact cell
membranes we then investigated the cellular uptake of pTP-TFE in
live primary human fetal neurons. This was performed by
adding pTP-TFE to a final concentration of 4 µM in a culture medium,
and incubating for 15 min, 30 min, 60 min and 120 min. pTP-
TFE uptake in the neurons at each time point was then determined
by acetonitrile extraction of PBS-washed cultures, and quantifying
using an LC-MS/MS assay23 (Figure S9). This showed 5 % of the total
applied pTP-TFE in the neuronal cells within 30 min and 10 % in 2
hours (Figure 4 and Table S1), which indicates it can cross the cell
membrane with rapid uptake. The quantity and kinetics of this
uptake are comparable to other compounds, including established
PET probes in oncology24 and agents delivered into neuronal cells.25
Figure 3. (A) Tau (0N4R) protein were collected 100 μL every 24 hours from the
incubation and then stored at -80◦C after flash frozen. After 240 hours of
incubation 1mL of tau aggregates was collected and loaded on the top of the
ultracentrifuge. (B-G) TEM images of tau aggregates structures at fractions from
50 % to 5 % sucrose gradient after 4 hours of ultracentrifugation (White bar = 500
nm).
Figure 4: Kinetics of pTP-TFE uptake in primary human fetal neurons quantified
by measuring the concentration of pTP-TFE in live neurons using LC-MS/MS; data
presented as % of total administered amount. Y values is calculated from table
S1(n=3).
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Ex vivo imaging of early tau aggregates in AD and PSP human brain for the study of tauopathy development and progressiVoienwaAsrtwicleelOl anlsinae
DOI: 10.1039/C9SC05620C
slices
proof of concept chemotype for the development of small molecule
therapeutics with enhanced affinity and selectivity. We aim to utilize
the potential displayed by pTP-TFE in vitro and ex vivo through in vivo
optical, fluorine-19 MRI/MRS and fluorine-18 PET imaging modalities
in the future.
Finally, we investigated whether the selectivity demonstrated in in
vitro assays could be replicated in ex vivo human brain tissue. To this
end we evaluated the binding of pTP-TFE to tau pathology in human
brain tissue of progressive supranuclear palsy (PSP), a pure
tauopathy, and Alzheimer’s disease, which contains both A and tau
pathology. Low-magnification epifluorescence microscopy showed
that pTP-TFE signals can be found colocalizing with
hyperphosphorylated, AT8-positive tau pathology in both PSP and
AD brains (Figure 5A). Closer inspection using confocal microscopy
revealed that pTP-TFE signals were predominantly observed within
the cell soma, co-localizing with AT8-positive tau pathology, although
the presence of pTP-TFE signals could also be found in the axon-
al/dendritic compartments (Figure 5B, arrows). Conversely, co-
localization with tau fibril antibody AT100 is less well established;
AT100-positive immunoreactivity has been shown to selectively label
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
The authors thank Dr. Gabriele Schierle and Dr. Na Yu (Chemical
Engineering Department, University of Cambridge), Dr. Karin
Muller (Cambridge Advanced Imaging Center) for technical
support, Dr. David Williamson and Cambridge Brain Bank for the
post-mortem brain samples, EPSRC Mass Spectrometry Service
(University of Swansea). Cambridge Brain Bank is supported by
the NIHR Cambridge Biomedical Research Centre. This study
was funded by the National Institute for Health Research (NIHR)
Cambridge Biomedical Research Centre, Medical Research
Council grant (MR/K02308X/1), the Engineering and Physical
Sciences Research Council (ST, EP/P008224/1), Royal Society
(DK), and Amgen Foundation Scholarship (ER). WLK and BV are
supported by the Medical Research Council (MR/S005528/1).
GT was supported by the Hungarian Brain Research Program
(2017-1.2.1-NKP-2017-00002).
Figure 5. pTP-TFE staining with human PSP (n=5) and AD brain slides (n=4). Left
are fluorescent imaging of pTP-TFE in AD and PSP human brain slides with scale
bar = 50μm and on right is confocal imaging of pTP-TFE in AD and PSP with scale
bar = 10μm. Arrows indicate axonal/dendritic compartment.
Notes and references
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Conclusions
We have designed and synthesized a novel fluorescent probe, pTP-
TFE for which binding and selectivity profiles towards aggregated tau
and A proteins were assessed. This included use of a sucrose
gradient ultracentrifugation method for more accurate
quantification of binding affinities to specific tau aggregated protein
of various sizes. Our results show pTP-TFE to be selective for soluble
tau aggregates, with a high affinity of Kd = 66 nM, and ten-fold
selectivity over mature fibrils. Furthermore, we found that pTP-TFE
is tau selective over A, the other major misfolded protein aggregate
of neurodegenerative disorders. To the best of our knowledge, pTP-
TFE is the first fluorescent molecule to have this form of selectivity
for soluble aggregates of tau over tau fibrils. In addition, we
established that pTP-TFE could penetrate intact live cell membranes
rapidly, while its selectivity towards early forms of aggregated tau
protein was also supported by studies on human brain tissue
containing tau and A pathology.
5
6
7
8
9
The high affinity and selectivity for early soluble aggregates of tau
and its cell permeability make pTP-TFE a best in class molecular tool
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137, 13503; L. P. Jameson, S. V. Dzyuba, Bioorg. Med. Chem.
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Chemical Science
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DOI: 10.1039/C9SC05620C
A table of contents entry
pTP-TFE imaging probe can distinguish soluble tau aggregated proteins from other aggregated proteins
enabling earlier detection of neurodegenerative diseases