A novel small molecule screening platform for disrupting toxic tau oligomers in cells
Chih Hung Lo
1
, Colin Kin-Wye Lim
1
, Zhipeng Ding
1
, Sanjula Wickramasinghe
2
, Anthony R.
Braun
1
,
Elizabeth Rhoades
2
, David D. Thomas
3,4
and Jonathan N. Sachs
1*
1
Department of Biomedical Engineering, University of Minnesota, Minneapolis, MN 55455
2
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104
3
Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota,
Minneapolis, MN 55455
4
Photonic Pharma LLC, Minneapolis, MN 55410
*To whom correspondence should be addressed: jnsachs@umn.edu
KEY WORDS
Tau oligomerization
Toxic tau oligomers
Fibrillation kinetics
Small-molecule inhibitors
Time-resolved FRET
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Abstract
Tauopathies, including Alzheimer’s disease, are a group of neurodegenerative disorders
characterized by pathological aggregation of the microtubule binding protein tau. Recent studies
suggest that toxic tau oligomers, which are soluble and distinct from insoluble beta-sheet fibrils,
are central players in neuronal cell death. To exploit this new therapeutic window, we engineered
two first-in-class FRET based biosensors that monitor tau conformations in cells. Because this new
technology platform operates in cells, it enables high-throughput screening of small molecules that
target tau oligomers while avoiding the uncertainties of idiosyncratic in vitro preparations of tau
assemblies from purified protein. We found a small molecule, MK-886, that disrupts tau oligomers
and reduces tau-induced cell cytotoxicity with nanomolar potency. Using SPR and an advanced
single-molecule FRET technique, we show that MK-886 directly binds to tau and specifically
perturbs the folding of tau monomer in the proline-rich and microtubule-binding regions.
Furthermore, we show that MK-886 accelerates the tau aggregation lag phase using a thioflavin-
T assay, implying that the compound stabilizes a non-toxic, on-pathway oligomer. The technology
described here should generalize to the study and targeting of conformational ensembles within
the aggregation pathways of most intrinsically disordered proteins.
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted January 3, 2019. ; https://doi.org/10.1101/510412doi: bioRxiv preprint
Introduction
Tauopathies, including Alzheimer’s disease (AD), are a group of neurodegenerative disorders
characterized by the presence of tau inclusions (neurofibrillary tangles, NFTs) in affected brain
regions
1
. Despite the past few decades of rigorous and focused research, there are currently no
cures or significant disease modifying therapies for AD and related tauopathies
2
. There is a
particular dearth of compounds that target tau, as out of the 105 small molecules that are currently
in clinical trials for AD, only five are tau-related disease modifying small molecules
3-4
. Hence,
there is desperate need for technologies that enable the identification of more effective disease-
modifying and tau-focused treatments
4-6
.
Tau is an intrinsically disordered protein that plays an important role in the regulation of
microtubule stability and axonal transport
7
. Under pathological conditions, tau is
hyperphosphorylated and detaches from microtubules, accumulating in the cytosol
8
. Unbound tau
has a tendency to misfold, undergoing conformational changes that initiate the tau amyloidgenesis
cascade, with an initial formation of tau oligomers that subsequently nucleate into paired helical
filaments (PHFs), and eventually intracellular NFTs
9
. NFTs have been the primary
histopathological hallmark of tauopathies, with their presence in the brain showing significant
correlation with the degree of cognitive impairment
10
. However, recent studies suggest that these
large insoluble NFTs are not the principle toxic species, implicating soluble oligomeric tau—
intermediate tau assemblies formed prior to PHFs and NFTs—in the induction of
neurodegeneration
11-12
. Tau oligomers promote cytotoxicity in vitro and are linked to
neurodegeneration and cognitive phenotypes in vivo
12-18
. As a result, the focus in therapeutic
development has begun to shift from targeting large fibrillar aggregates to inhibiting or disrupting
the formation of toxic soluble tau oligomers
11, 19-21
.
Several recent efforts to discover small molecules that target toxic tau oligomers have yielded
efficacious, cytoprotective compounds
22-28
. However, these studies were done in vitro with
purified proteins, and the compounds were protective only in the low micromolar range
22-28
. Tau
oligomers exist as an ensemble of distinct assemblies which include both toxic and non-toxic, on-
and off-pathway species along the fibrillogenesis cascade
29-35
. The formation of these toxic tau
oligomers has been associated with mutations and overexpression of numerous chaperone
proteins
36-37
, highlighting the importance of other components of the tau-protein interactome in the
pathogenesis of tauopathies. Capturing this complexity in an in vitro setting is extremely difficult,
if even possible, and established protocols for aggregating purified tau protein into oligomers,
PHFs, and NFTs have, not surprisingly, been shown to produce different tau assemblies depending
on aggregation conditions
17, 31, 34, 38-41
. Critically, no specific toxic tau species has been isolated or
identified to date
21, 42-43
.
Here, we shift the tau oligomerization process into the cellular environment, where the ensemble
of tau conformations should more closely recapitulate the distribution of oligomeric assemblies.
In addition, cellular oligomers, unlike those produced from purified proteins, may include non-tau
components and thus tau oligomer structures only accessible via interaction with chaperone
proteins. Building on the groundbreaking biosensor developed by the Diamond group (which is
focused on the detection of pathogenic species in biofluids as a biomarker for AD diagnosis)
44
, we
have developed a technology platform that directly monitors tau oligomerization in cells, enabling
the therapeutic targeting of early-stage tau pathology. Furthermore, with this platform, we have
established a robust assay that can be easily adjusted in the future for additional cell lines and new
pathological conditions that more closely mimic disease conditions as they become known. Such
a platform increases the likelihood of targeting the true toxic species, and has the added advantage
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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of identifying compounds that act both directly (by binding tau) and indirectly (through orthogonal
biochemical pathways) to modify toxic oligomers.
To discover small molecules that disrupt toxic tau oligomers in cells, we engineered two distinct
fluorescence resonance energy transfer (FRET) biosensors to monitor tau oligomerization. We
used these biosensors in conjunction with a state-of-the-art fluorescence lifetime plate reader as a
high-throughput screening (HTS) platform for drug discovery
45
, using the NIH Clinical Collection
(NCC) library. Fluorescence lifetime detection increases the precision of FRET-based screening
by a factor of 30 compared with conventional fluorescence intensity detection
46
, and provides
exquisite sensitivity to resolve minute structural changes within protein ensembles. This sensitivity
allows direct detection of conformation changes within an ensemble of oligomers (e.g. conversion
from toxic to non-toxic oligomer conformation), the dissociation of oligomers, or even changes in
the ensemble of monomer conformations
47-49
. The FRET biosensors were engineered by
expressing full-length wild-type (WT) 2N4R tau and fluorescent protein fusion constructs in living
cells, allowing us to directly detect and monitor either inter-molecular or intra-molecular tau
interactions. While FRET techniques have been employed to investigate tau aggregation with
aggregation-prone constructs, such as the caspase-cleaved form of tau
50
or the K18 isoform with
familial mutants including P301L
44
, the use of full-length WT 2N4R tau to study tau
oligomerization has not been reported. The novel use of full-length WT 2N4R tau in this study
enables the specific detection of ensembles of tau oligomers, not fibrils, as the 2N4R isoform does
not spontaneously fibrillize, even at supersaturating concentrations, and therefore forms mostly
oligomers
51-53
.
After first establishing that the new technology platform specifically targets oligomers (using
known tau aggregators), we identified a small molecule, MK-886, that directly binds tau and
strongly attenuates FRET with an EC
50
of 1.40 μM. The compound rescues tau-induced cell
cytotoxicity with an IC
50
of 435 nM. To elucidate the mechanism of action, we used an advanced
single-molecule FRET (smFRET) technique to show that MK-886 perturbs the folding of purified,
monomeric tau in the proline-rich and microtubule-binding regions. This effect is recapitulated in
the cellular biosensor that monitors intra-molecular FRET and indicates an unfolding of the two
termini of tau. In addition, we used a thioflavin-T assay to show that MK-886 shortens the lag
phase in tau fibrillization, implying that the compound stabilizes a non-toxic, on-pathway tau
oligomer, hence defining a new therapeutic approach to targeting toxic tau oligomers.
Results
Inter-molecular FRET biosensor directly monitors structural changes in tau oligomers in cells
To develop an in-cell HTS platform that can detect small-molecule modulation of tau
oligomerization and/or perturbation of tau conformational states, we engineered a tau FRET
biosensor expressed in living cells. We used HEK293 cells expressing full-length WT 2N4R tau
fused to GFP or RFP (tau-GFP/RFP or tau FRET biosensor) (Fig. 1A). Full-length WT 2N4R
isoform was used because it does not spontaneously fibrillize, even at supersaturating
concentrations, forming mostly oligomers
51-53
. Expression and homogeneity of the FRET
biosensor were determined by fluorescence microscopy and immunoblotting. Fluorescence
microscopy images showed that the tau proteins were evenly distributed in the cytosol of the cells,
with no discernable puncta (which would have indicated more progressive aggregation, e.g. fibril
formation) or other non-uniformities (Fig. 1B). Western blot analysis of the tau biosensor cell
lysates confirmed the expression of fluorophore-tagged tau (Supplementary Fig. 1A).
certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint (which was notthis version posted January 3, 2019. ; https://doi.org/10.1101/510412doi: bioRxiv preprint
We next tested the functionality of the tau FRET biosensor by measuring FRET efficiency using
the fluorescence lifetime plate reader
45
. FRET between tau-GFP (donor) and tau-RFP (acceptor)
in live cells showed hyperbolic dependence on acceptor concentration (Fig. 1C), with a maximum
energy transfer efficiency (E) of 0.086±0.002, illustrated through a substantial decrease in the
donor fluorescence lifetime in the presence of the acceptor (Fig. 1D), indicating the formation of
tau oligomers in cells. As a negative control, we expressed free soluble GFP and RFP in cells using
the same DNA concentrations as the tau biosensor to ensure that the FRET we observed was tau-
specific and not caused by nonspecific interactions between the fluorophores in the cytosol
(Supplementary Fig. 1B). The low FRET efficiency (E=0.019±0.004 between free soluble GFP
and RFP) indicates that FRET observed from cells expressing tau biosensor arises from specific
tau-tau interactions and not from nonspecific interactions between the free fluorophores
(Supplementary Fig. 1C).
We further tested the sensitivity of the tau FRET biosensor with the addition of forskolin, a small
molecule known to induce tau hyperphosphorylation and self-association
54
. Cell treatment with
forskolin (20 µM) showed a significant increase in FRET, illustrating increased self-association of
tau (Supplementary Fig. 1D). Interestingly, cell treatment with gossypetin (10 µM), a known
inhibitor of fibril formation
27
, did not show any significant change in FRET, suggesting that under
our cellular conditions only oligomeric tau species were contributing to the observed FRET
(Supplementary Fig. 1D).
To further confirm that only oligomeric species of tau, but not fibrils, were present in the tau
biosensor cells, we performed a thioflavin-S (ThS) assay in cells expressing tau-RFP at the same
concentration of tau-GFP/RFP dual transfected cells, with treatment of tau pre-formed fibrils (PFF)
as positive controls. Tau-RFP was used instead of tau-GFP/RFP as tau-GFP emission interferes
with the ThS signal. Results from the ThS assay illustrate that the cells treated with PFF show a
positive ThS signal, but not the cells expressing the biosensors (Fig. 1E), confirming that no fibrils
(specifically β-sheet tau assemblies) are present in the biosensor cells, and more importantly that
the observed FRET is mainly the result of tau oligomerization. The combination of our biosensor’s
basal FRET signal and response to positive control tool compounds demonstrates that time-
resolved FRET detection in cellular tau FRET biosensors is sensitive to conformationally distinct
tau assemblies, providing a powerful platform to identify novel compounds that modulate the
ensemble of tau oligomers.
Identification of novel small molecules from HTS of the NCC library that perturb the
conformational ensembles of tau oligomers
Using our cellular tau FRET biosensor, we performed a HTS of the NIH Clinical Collection (NCC:
727 bioactive compounds) to identify compounds that perturb the conformational ensembles of
tau oligomers. The NCC library is a collection of small molecules that have been previously tested
in clinical trials, and therefore have known safety profiles and details on potential mechanisms of
action. These compounds can provide excellent starting points for medicinal chemistry
optimization and may even be appropriate for direct human use in new disease areas.
After an initial quality control check of the cells expressing the tau FRET biosensor on each day
of screening (fluorescent waveform signal level and coefficient of variance), the cells were
dispensed into drug plates and incubated with the compounds (10 µM) or DMSO control wells for
2 hours. Lifetime measurements were acquired with the fluorescence lifetime plate reader. A
single-exponential fit was used to determine the lifetime from cells expressing the tau FRET
biosensor (τ
DA
) or expressing a tau-GFP donor-only control (τ
D
) to determine FRET efficiency
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