Alpha Synuclein Fibrils Contain Multiple Binding Sites for Small Molecules

Article abstract of DOI:10.1021/acschemneuro.8b00177

The fibrillary aggregation of the protein alpha synuclein (Asyn) is a hallmark of Parkinson's disease, and the identification of small molecule binding sites on fibrils is essential to the development of diagnostic imaging probes. A series of molecular mo

Full text of DOI:10.1021/acschemneuro.8b00177

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Letter
Alpha Synuclein Fibrils Contain Multiple Binding Sites for Small Molecules
Chia-Ju Hsieh, John J. Ferrie, Kuiying Xu, Iljung Lee, Thomas J. A. Graham, Zhude Tu,
Jennifer Yu, Dhruva Dhavale, Paul Kotzbauer, E. James Petersson, and Robert H. Mach
ACS Chem. Neurosci., Just Accepted Manuscript • DOI: 10.1021/acschemneuro.8b00177 • Publication Date (Web): 11 May 2018
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ACS Chemical Neuroscience
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Alpha Synuclein Fibrils Contain Multiple Binding Sites for Small Molecules
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Chia-Ju Hsieh, John J. Ferrie, Kuiying Xu, Iljung Lee, Thomas J. A. Graham, Zhude Tu, Jennifer Yu, Dhruva Dhavale, Paul
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Kotzbauer, E. James Petersson, and Robert H. Mach
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Department of Radiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104,
USA, ; Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA; Mallinckrodt Insti-
tute of Radiology, Washington University School of Medicine, St. Louis, MO, 63110; Department of Neurology, Washing-
ton University School of Medicine, St. Louis, MO, 63110.
2
3
4
Supporting Information Placeholder
ABSTRACT: The fibrillary aggregation of the protein alpha synuclein (Asyn) is a hallmark of Parkinson’s disease, and the identi-
fication of small molecule binding sites on fibrils is essential to the development of diagnostic imaging probes. A series of molecu-
lar modelling, photoaffinity labeling, mass spectrometry, and radioligand binding studies were conducted on Asyn fibrils. The re-
sults of these studies revealed the presence of three different binding sites within fibrillar Asyn capable of binding small molecules
with moderate to high affinity. A knowledge of the amino acid residues in these binding sites will be important in the design of high
affinity probes capable of imaging fibrillary species of Asyn.
KEYWORDS: Alpha synuclein, Lewy Bodies, Lewy Neurites, Parkinson’s disease
INTRODUCTION
syndromes do not include the pathologic accumulation of
Asyn.
Alpha synuclein (Asyn) is a small (140 amino acid), intrinsi-
cally disordered protein that is localized in presynaptic nerve
terminals in the central nervous system (CNS). Although its
biological function is poorly understood, Asyn is currently
believed to play a key role in docking of neurotransmitter ves-
icles to active zones of nerve terminals to facilitate the release
of neurotransmitters into the synaptic cleft. In Parkinson’s
disease (PD), Asyn forms fibrillar protein aggregates contain-
ing a beta pleated sheet structure. These fibrillar protein ag-
Positron Emission Tomography (PET) is an in vivo imaging
technique that is capable of quantifying a change in protein
levels, rates of cellular metabolism, or protein-protein interac-
tions that serve as the foundation of the molecular basis of
disease. Perhaps the most significant advance in the past dec-
ade in PET imaging studies is the development of radiotracers
1
-2
3
1
0-
capable of imaging the presence of amyloid β (Aβ) plaques
and tau-based neurofibrillary tangles (NFTs)
13
14-16
4
in patients
diagnosed with Alzheimer’s disease (AD). Because of the
availability of Aβ- and tau-specific probes, it is now clear that
Aβ plaques represent an “antecedent” marker for identifying
patients at risk for developing AD, whereas NFTs occur rela-
tively late in the disease process when the neuronal loss lead-
ing to cognitive impairment has already occurred.
gregates accumulate to the level where they form insoluble
intraneuronal inclusions termed Lewy bodies (LBs) and Lewy
neurites (LNs). The accumulation of LBs and LNs in the sub-
stantia nigra and other brain regions represents a hallmark
5
feature of PD. A second form of fibrillary Asyn, termed glia
cell inclusions (GCIs), is found in the oligodendrocytes of
white matter tracts in patients diagnosed with Multiple System
Atrophy (MSA). The identification of dominant mutations in
SNCA, the gene encoding Asyn, in familial PD, including
point mutations that promote fibril formation and triplication
of the SNCA gene provides a strong link between fibrillary
The success of Aβ plaque and tau imaging in AD has led to
a similar effort towards developing a PET radiotracer for im-
aging insoluble Asyn that occurs in LBs and LNs in PD, and
6
9, 17
GCIs in MSA.
Since Aβ and tau are found in both AD and
18
PD postmortem brain samples, a suitable PET probe for im-
aging insoluble Asyn aggregates must have a high affinity for
Asyn and low affinity for fibrillary Aβ and tau. Unfortunately,
most ligands developed to date have not shown this desired
selectivity for Asyn versus Aβ and tau for PET imaging stud-
ies in PD.
7-8
Asyn and the pathogenesis of PD.
The diagnosis of PD has traditionally relied on the clinical
presentation of a number of motoric deficits including brady-
kinesia, rigidity, rest tremor and postural instability. Although
accuracy of the clinical diagnosis of PD is very good in later
stages of the disease, the early diagnosis of PD is problematic
since not all features are present in early PD. Furthermore, the
same clinical features can be present in other Parkinsonian
syndromes such as vascular parkinsonism, MSA, corticobasal
Our group has recently reported a number of structurally-
diverse compounds that are capable of binding to Asyn fi-
19-22
brils.
Although structure-activity relationship studies have
identified compounds displaying a higher affinity for Asyn
versus Aβ and tau, the properties of the ligand that possess this
property are not ideal for functioning as a PET radiotracer for
degeneration (CBD), and progressive supranuclear palsy
PSP). With the exception of MSA, these other Parkinsonian
9
(
21, 23
imaging Asyn.
In this regard, a greater understanding of
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the location of the binding sites for small molecules on Asyn, zine analog (4) showed a preference for sites 2 and 9, whereas
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and the amino acid residues that comprise the binding sites,
would be useful in guiding the design of a PET radiotracer
having the affinity, selectivity, and physicochemical properties
required for imaging LBs and LNs. The publication of the
solid-state NMR structure of Asyn fibrils by Rienstra and col-
the tricyclic compounds (5-7) and the indolinone-diene analog
(8) showed a near exclusive preference for site 3/13 (Table
S2). The identification of sites 2, 9 and 3/13 as potential bind-
ing sites is also supported by the high degree of hydration and
stability of these sites using the molecular dynamics program
NAMD with the VMD visualization and analysis module.
The results of these studies can be found in Figure S2 and
Table S3.
24
29
30
leagues served as the starting point for the series of studies
described in this communication.
RESULTS AND DISCUSSION
The structure of Asyn was imported from the Protein Data
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Bank (PDB ID: 2N0A) and visualized with the 3-dimension
visualization program PyMol (pymol.org). Docking studies
26
were conducted using the AutoDock 4.2 plugin on PyMol.
The test set for docking studies included four different classes
of compounds shown in Figure 1. A grid box that covered the
major beta-sheet structure of residue K34 to K97 was applied
to the Asyn fibril structure for molecular blind docking to
search the potential binding sites. The blind docking studies
allow for predicting potential sites of interaction with the pro-
tein by calculating a % probability of interaction and binding
energy for each putative site. The values for each of these
measurements for the eight test compounds are shown in Ta-
ble S1 and S2.
Figure 2. Solid-state NMR structure of Asyn showing the three
putative binding sites for small molecules. Color labeled cyan,
site 2; purple, Site 3/13; orange, site 9.
O
N
HN
N
HN
N
S
OCH3
S
S
OCH
3
N
3
In order to confirm the location of these binding sites, pho-
toaffinity labeling of Asyn fibrils was conducted using the
azide-containing styrene analog, 3. In addition to sites 2 and 9,
this compound also has a modest probability of interacting
with site 3/13. Disaggregation of the photoaffinity-labeled
Asyn fibrils into their corresponding monomers followed by
positive ion and negative ion matrix-assisted laser desorption
ionization mass spectrometry (MALDI-MS) revealed the la-
beling of peptide fragments corresponding to residues T44-
K58 or E46-K60 (Figure 3 and S3, and Table S4 and S5).
While we favor assignment of the site of crosslinking to T44-
K58 due to the absence of the E46-K58 photo-adduct, we were
unable to definitively assign this by MS-MS sequencing. The-
se results indicate that azido analog 3 binds to either site 2 or
3/13, and site 9 can be excluded as a potential binding site for
this compound. Since 3 had a modest binding affinity for dis-
N
N
N
F
1
2
3
F
H
N
H
N
O
OCH
3
N
N
NH
O
S
NO
2
O
2
N
S
O
I
N
4
5
6
NO
2
H
MeO
N
N
O
N
O
NO
2
F
7
8
O
Figure 1. Structures of the compounds used in the docking and
in vitro binding assays.
The results of the docking studies revealed thirteen putative
binding sites in Asyn for small molecules (Figure S1 and Ta-
ble S1). However, it was possible to exclude a number of these
sites based on their low % probability of interaction. This re-
duced the number of putative binding sites to six. Further-
more, site 1 was excluded as a credible binding site since it is
located in the N-terminal region that is highly disordered in
the NMR structure. We note that some evidence indicates that
these regions are more ordered in fibrils formed under differ-
ent conditions, so sites in terminal regions could be investigat-
3
placing [ H]1 from Asyn fibrils (Figure S4), and given their
structural similarity, we surmised that 1 and 2, also bind to site
2
or 3/13.
2
7
ed in subsequent studies. Because the side chains of site 6
2
4
were reported disordered in the fibril structure , and the inner
core of the Asyn fibril was also reported as a relatively inac-
cessible region for small molecule binding, site 6 was also
2
4, 28
excluded.
The remaining binding sites included: 1) site 2,
which contains Y39-S42-T44; 2) site 9, which contains G86-
F94-K96; 3) site 3, which contains L45-V48-H50; and 4) site
13, which contains L43-L45-V48-H50. Since site 3 is con-
tained within site 13, these two sites can collectively be
termed site 3/13 (Figure 2).
Figure 3. Site of the peptide fragments T44-K58 labeled (blue)
by the photoaffinity probe, 3. This peptide fragment contains
sites 2 and 3/13. Inset: MALDI-MS data showing a photoad-
The docking studies also revealed that the different classes
of compounds displayed selectivity among the binding sites.
For example, the styrene-based analogs (1-3) and the pipera-
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7) in in vitro binding studies using [ I]5 as the radioligand.
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duct of 3 with a peptide corresponding to residues 44-58 or
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46-60 of Asyn (red). No peak at this mass is observed when
Asyn fibrils are irradiated in the absence of 3 (blue).
However, neither compound was able to displace the binding
3
of [ H]1 from Asyn fibrils (K > 1,000 nM). Furthermore,
i
18
[ F]8 was shown to bind to Asyn fibrils with a K value of 9
d
21
3
As a means of further characterizing the binding of small
molecules to the three putative binding sites, a series of in
vitro binding studies in Asyn fibrils was conducted using two
nM, but it also did not displace [ H]1 from Asyn fibrils (Fig-
ure S5). These data support the binding of the styrene-based
analogs (1-3) to site 2 and not site 3/13.
3
3
different tritiated ligands, [ H]1 and [ H]4. These radioligands
were synthesized for in vitro binding studies aimed at screen-
ing potential ligands for PET imaging studies, and in vitro
autoradiography studies for imaging LBs and LNs in postmor-
tem samples of PD brain. The in vitro characterization of these
To confirm that compounds predicted to bind to site 3/13 do
not bind to site 9, in vitro binding assays were conducted
3
measuring the ability of compounds 7 and 8 to displace [ H]4
0
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from Asyn fibrils. The K values under both assays conditions
i
radioligands will be published separately. However, homolo-
were >1,000 nM showing that they indeed bind to distinct sites
(Figure S5). Although direct site-specificity data are only
available for compound 3, taken together, our competition and
photo-crosslinking studies allow us to generate a self-
consistent model for binding site selectivity that is in agree-
ment with the predictions from our docking studies. While
further studies would be necessary to definitively assign the
compound classes to each site, the current model is as follows:
styrene-based analogs (1-3) bind to site 2 (with weaker affinity
for site 9), the piperazine analog (4) binds to site 9 (with
weaker affinity for site 2), and the tricyclic compounds (5-7)
and indolinone-diene analog (8) bind to site 3/13. The interac-
tions of representative compounds with each of the binding
sites is shown in Figure 5.
3
gous competition studies revealed that [ H]1 has a K value of
d
3
4
7 nM and [ H]4 has a K value of 6.5 nM for recombinant
d
Asyn fibrils, respectively. In vitro competition curves measur-
3
3
ing the displacement of [ H]1 and [ H]4 from Asyn fibrils
revealed different K values for compound 2. That is, 2 had a
i
3
K value of 71 nM for displacing [ H]1, and a K value of 488
i
i
3
nM for displacing [ H]4 from Asyn fibrils (Figure 4). These
data are consistent with the in silico prediction that the styrene
analogs (1-3) bind with higher affinity to site 2 than site 9,
while 4 binds with highest affinity to site 9.
The importance in understanding where small molecules are
predicted to bind to Asyn in designing an in vitro binding as-
say to measure their affinity to Asyn fibrils is evident from the
data shown in Figure 4 and Figure S5. The K value of 2 for
i
3
displacing [ H]1 from Asyn fibrils was 71 nM, and would be
considered to be a good lead compound for radiotracer devel-
opment (Figure 4). In contrast, the K value for displacing
i
3
3
Figure 4. In vitro binding studies of 2 using either [ H]1 or
[ H]4 from Asyn fibrils was 488 nM, an affinity that would
3
[
H]4 as the radioligand. Since 2 is predicted to bind to site 2,
exclude its consideration from further radiotracer develop-
ment. Similarly, one would conclude that the indolinone-diene
analog 8 has no affinity for Asyn fibrils based on the competi-
its affinity for Asyn fibrils was higher when a site 2 preferring
3
ligand (i.e., [ H]1) was used in the binding assay.
3
3
tion data using [ H]1 and [ H]4 as the radioligands (Figure
18
The next set of experiments was designed to confirm that the
styrene-based analogs 1-3 bind to site 2 and not site 3/13.
Since analogs 6, 7 and 8 were predicted to bind near exclu-
sively to site 3/13, in vitro competition studies were conducted
S5), whereas direct binding assays of the F-labeled com-
pound reveals that it has a K value of 9 nM for Asyn fibrils.
d
For protein structures such as fibrils, with many shallow sur-
faces, having a set of compounds with varied site selectivity is
essential to profiling how changes to candidate molecules af-
fects not only affinity, but binding site preference.
3
measuring the potency of these compounds to displace [ H]1
from Asyn fibrils. Previous studies have shown 6 and 7 bind to
Asyn fibrils with an affinity of 16 nM (for 6) and ~70 nM (for
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Figure 5. Docking studies of 1 to site 2 (a and b) and 9 (c and d), 4 to sites 2 (e and f) and 9 (g and h), and 8 to site 3/13 (i and j). (a,
c, e, g, and i) are viewed from the fibril axis, and (b, d, f, h, and j) are viewed from the fibril side view.
In summary, we have utilized a combination of in silico
docking, photoaffinity labeling, and radioligand binding stud-
ies to identify three putative binding sites for small molecules
in Asyn fibrils. The location of these binding sites is critical in
designing radioligand binding studies aimed at determining
the affinity of small molecules for Asyn fibrils. The identifica-
tion of high-affinity compounds for Asyn fibrils represents the
first step in developing a radiotracer for PET imaging studies
of LBs and LNs in PD. We are currently using the strategy
described in this study in the screening of chemical libraries as
a means of identifying suitable lead compounds for the devel-
opment of a radiotracer for imaging insoluble Asyn aggregates
with PET.
144.40, 154.39, 160.45. 162.73, 163.68. MS (ESI) m/z 285
+
(M+H) .
O
O
O
HO
Cl
HO
N
3
N
3
NO
2
IL-IV-037
IL-IV-039
H
N
O
OH
N
N
N
S
S
N3
N
3
IL-IV-040
3
(E)-3-(4-Azidophenyl)acrylic acid (IL-IV-037)
Metal zinc (1.95 g, 29.9 mmol) was added to AcOH (45.0 mL)
and water (5.0 mL) followed by the addition of (E)-3-(4-
nitrophenyl)acrylic acid (2.89 g, 14.9 mmol). The reaction
mixture was stirred at 25 °C for 0.5 h and filtered. The filtrate
was cooled to 0 °C for 10 min, and a solution of sodium nitrite
METHODS
Chemistry. Reagents were purchased from Sigma-Aldrich
and Fisher Scientific. Silica gel chromatography was carried
TM
out on a Biotage Isolera Spektra One chromatograph sys-
(1.24 g, 17.9 mmol) in water (10 ml) was added dropwise at 0
tem. All synthesized compounds were analyzed and confirmed
to have purity over 95% with a Waters Alliance LC-MS sys-
tem. NMR spectra were measured on a Brucker 500 or 360
MHz spectrometer as indicated. Chemical shifts (δ values)
were reported in ppm relative to TMS. For multiplicity, s =
singlet, d = doublet, t = triplet, m = multiplet. H NMR spectra
data were presented as follows: Chemical shifts (multiplicity,
coupling constants, integration).
°
C. The reaction mixture was stirred at 0 °C for 10 min. Then
sodium azide (1.16 g, 17.9 mmol) in water (10.00 ml) was
added dropwise to the reaction mixture. The mixture was kept
stirred at 0 °C for another 15 min. After the reaction, the sol-
vent was filtered, and washed with water. The solid was dried
over via high vacuum, recrystallized with ethyl ace-
tate/hexane, washed with hexane, and dried over to yield (E)-
1
3
-(4-azidophenyl) acrylic acid (738.6 mg, 3.90 mmol, 26.1 %)
1
Synthesis
of
(E)-3-(4-methoxystyryl)-5-(thiazol-2-
as a light yellow solid. H NMR (500 MHz, DMSO-d ) δ ppm
6
yl)isoxazole (1)
To a solution of (2Z,4E)-3-hydroxy-5-(4-methoxyphenyl)-1-
6.48 (d, J = 16.0 Hz, 1H), 7.14 (d, J = 8.5 Hz, 2H), 7.56 (d, J =
16.0 Hz, 1H), 7.73 (d, J = 8.5 Hz, 2H), 12.35 (s, 1H).
(E)-3-(4-azidophenyl)acryloyl chloride (IL-IV-039)
22
(thiazol-2-yl)penta-2,4-dien-1-one (250 mg, 0.87 mmol) in
EtOH (5.00 mL) was added NH OH-HCl (121 mg, 1.74
Sulfurous dichloride (0.30 ml, 4.19 mmol) was added to a
solution of (E)-3-(4-azidophenyl)acrylic acid (720 mg, 3.81
mmol) in dichloromethane (10 ml). The reaction mixture was
stirred at 80 °C for 2.5 h. The solvent was then evaporated and
the solid formed was dried via high vacuum. The solid was
2
mmol) and stirred at 80 °C for 3 h. The reaction mixture was
cooled down to the ambient temperature and removed EtOH in
vacuo. The crude compound was purified by Biotage purifica-
tion system (Hex: EtOAc = 3:1) which gave a 1:1 mixture of
two isomers. Further purification with dichloromethane yield
1
used for the next step without further purification. H NMR
1
compound 1 (40 mg, 16.0%) as a colorless solid. H NMR
(500 MHz, CDCl ) δ ppm 6.60 (d, J = 15.5 Hz, 1H), 7.09 (d, J
3
(
2
1
500 MHz, CDCl ) δ ppm δ 3.84 (s, 3H), 6.93 (d, J = 8.7 Hz,
H), 7.03 (d, J = 16.5 Hz, 1H), 7.07 (s, 1H), 7.20 (d, J = 16.5,
= 8.5 Hz, 2H), 7.58 (d, J = 8.5 Hz, 2H), 7.79 (d, J = 15.5 Hz,
1H).
(2Z,4E)-5-(4-Azidophenyl)-3-hydroxy-1-(thiazol-2-
yl)penta-2,4-dien-1-one (IL-IV-040)
3
H), 7.49 (d, J = 8.7 Hz, 2H), 7.53 (d, J = 3.1 Hz, 1H), 7.99
13
(d, J = 3.1 Hz, 1H). C NMR (126 MHz, CDCl ) δ ppm
3
5
5.33, 98.88, 113.02, 114.30, 121.28, 128.32, 128.51, 136.35,
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volume at 310 K for 100 ps with a time step of 1 fs. The sys-
LiHMDS (2.65 mL, 2.65 mmol) was added dropwise to a so-
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
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lution of 1-(thiazol-2-yl)ethan-1-one (306 mg, 2.41 mmol) at -
78 °C, and the mixture was stirred for 1 h at the same tempera-
ture. (E)-3-(4-Azidophenyl)acryloyl chloride (IL-IV-039, 600
mg, 2.89 mmol) in THF (2.145 mL) was added to the reaction
mixture dropwise at -78 °C, and then the mixture was warmed
up to 25 °C and stirred for 3 h at room temperature. After the
tem was then equilibrated in NPT ensemble at 310 K and 1
atm for 200 ps with 1 fs time step. The Langevin dynamics
and Langevin piston method were used to maintain the pres-
sure at 1 atm and temperature at 310 K. A final molecular
dynamics simulation was run in a NPT ensemble for 10 ns
with a time step of 2 fs, and saved the trajectory every 1 ps.
Photocrosslinking. Asyn was expressed recombinantly in E.
Coli from a pT-T7 plasmid and was purified by size exclusion
chromatography followed by ion exchange chromatography as
reaction, saturated NH Cl aqueous (100 mL) was added to a
4
reaction mixture, which was then extracted with ethyl acetate
(50 mL) twice. The combined organic layer was dried over
31
Na SO and concentrated. The residue was recrystallized with
previously described. Following purification purified protein
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MeOH giving the product as an orange solid (350 mg, 48.4%).
was dialyzed against 20 mM tris, 100 mM NaCl pH7.5 at 4 °C
overnight. Subsequently, samples were prepared containing
100 µM Asyn which were shaken for 48 hours at 1500 rpm
and 37 °C producing fibrils. To remove residual monomers
post-aggregation, fibrils were pelleted at 13,500 rpm for 1.5
hours in a bench top centrifuge and resuspended in the same
volume and buffer that was removed. Compound 3 was dis-
solved in DMSO producing a 136 mM stock. Samples contain-
ing ~100 µM Asyn fibrils with and without 900 µM Com-
pound 3 were prepared in glass vials which were irradiated
with a standard TLC lamp for 5 hours. Following irradiation,
samples were disaggregated by adding sodium dodecyl sulfate
(SDS) to a final concentration of 15 mM and boiling for 1
hour. After disaggregating the Asyn fibrils, SDS was removed
1
H NMR (500 MHz, CDCl ) δ ppm 6.59 (d, J = 15.8 Hz, 1H),
3
6.76 (s, 1H), 7.06 (d, J = 8.5 Hz, 2H), 7.55 (d, J = 8.5 Hz, 1H),
7.65 (d, J = 15.8 Hz, 1H), 7.67 (d, J = 3.0 Hz, 1H), 8.02 (d, J =
3
.0 Hz, 1H).
(E)-2-(3-(4-Azidostyryl)-1H-pyrazol-5-yl)thiazole (3)
Hydrazine hydrate (171mg, 3.42 mmol) was added to a solu-
tion of (2Z,4E)-5-(4-azidophenyl)-3-hydroxy-1-(thiazol-2-
yl)penta-2,4-dien-1-one (500 mg, 1.676 mmol) in Ethanol (4
ml), stirred at 80 °C for 3 h. After the reaction, EtOH was re-
moved in vacuo, purufied by Biotage. The purification gave
(
E)-2-(5-(4-azidostyryl)-1H-pyrazol-3-yl)thiazole (150 mg,
1
0.510 mmol, 30.4 % yield) as a light yellow solid. H NMR
(360 MHz, DMSO-d ) δ ppm 6.95 (s, 1H), 7.08-7.16 (m, 3H),
6
3
2
7.29 (d, J = 7.2 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H). 7.67 (d, J =
via protein precipitation using choloform and methanol.
3
.4 Hz, 1H), 7.86 (d, J = 3.4 Hz, 1H).
Trypsin was added to monomeric protein samples at a final
concentration of 0.05 mg/mL and samples were incubated
overnight at 37 °C overnight. Mass spectra of each digested
sample were acquired via matrix assisted laser desorption ion-
ization (MALDI-MS) on a Bruker µLtraflex III (Billerica,
MA, USA). Samples were shot using two methods, one meas-
uring positive ions with a reflection (RP) and the other meas-
uring negative ions via a linear method (LN).
Molecular Docking. All the structures of compound were
drew on ChemDraw Profession 15.1 (PerkinElmer Informat-
ics, Inc.), then imported to Chem3D Ultra 15.1 (PerkinElmer
Informatics, Inc.) to minimize individual structures by
MMFF94 force field for preparation of molecular docking.
Molecular blind docking studies were performed via Auto-
26
Dock 4.2 plugin on PyMOL (pymol.org). The solid-state
NMR structure of full-length Asyn fibril (PDB ID: 2N0A) was
Binding Assay. The radioligand competition binding assays
used Asyn fibrils as binding substrate. Asyn fibrils were pre-
pared from recombinant human Asyn protein as previously
obtained
from
RCSB
protein
data
bank
(https://www.rcsb.org/) as a target protein for blind docking.
Non-polar hydrogens were removed from both compounds
33
described. Binding mixtures contained Asyn fibrils at a con-
3
and protein structures. A grid box with a dimension of 95 × 50
centration of 25 nM for assays with [ H]4 and 150 nM for
3
3
×
95 Å was applied to Asyn fibril structure, majorly including
assays with [ H]1. At radioligand concentrations of 2 nM in
beta-sheet section (residue K34-K97) with parts of N- and C-
terminus disorder residues that close to the beta-sheet area.
The Lamarckian Genetic Algorithm with a maximum of
the binding assays, these fibril concentrations resulted in 5% -
10% of the radioligand bound to fibrils in the absence of com-
petitor. Different concentrations of competitor compounds,
ranging from 1 nM to 1000 nM were combined with Asyn
fibrils and 2 nM radioligand in 30 mM Tris-HCL pH 7.4, 0.1%
BSA, in a total volume of 150 ꢀl. The binding mixtures were
incubated at 37 °C for 2 hrs in 96 well plates. Bound and free
radioligand were then separated by vacuum filtration through
1.0 ꢀm Glass Fiber filters in 96-well filter plates (Millipore),
followed by three 200 ꢀl/well washes with cold assay buffer.
Filters containing the bound ligand were mixed with 150 ꢀL
of Optiphase Supermix scintillation cocktail (PerkinElmer)
and counted after overnight incubation. All data points were
performed in triplicate. Data were analyzed using Graphpad
2
,500,000 energy evaluations was used to calculate 1,000 pro-
tein-ligand binding poses for each compound. The % probabil-
ity, predicted best binding energy, and average binding energy
of each binding site that determined from blind docking were
reported.
Molecular Dynamics Simulation. In order to investigate
the stability and accessibility of the Asyn fibril structure in
solution, molecular dynamics simulation was performed via
29
NAMD 2.12 with CHARMM22 force field, and analyzed by
30
using visualization program VMD . The protein was solvated
in a TIP3P water box with a minimized volume of 160 × 168 ×
3
1
20 Å , and then 90 sodium ions were randomly added to the
Prism 7 software to obtain EC50 values by fitting the data to
(x-logEC50)
solvated system for charge neutralization. Periodic boundary
conditions were used for the molecular dynamics simulation.
A cut-off of 12 Å was set to non-bonded interaction, and long-
range electrostatics interaction was calculated by using Parti-
cle Mesh Ewald (PME) method. SHAKE algorithm was used
to constrain bonds involving hydrogen atoms. A 50,000 steps
of energy minimization was implemented. Then, the mini-
mized system was heated in NVT ensemble with constant
the equation Y=bottom+(top-bottom)/(1+10
). K values
i
were calculated from EC50 values using the equation
K =EC /(1+[radioligand]/K ).
i
50
d
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website.
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McCormack, A.; Chegeni, N.; Chegini, F.; Colella,
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Details for the docking studies, photocrosslinking, and repre-
sentative in vitro binding data. (PDF)
A.; Power, J.; Keating, D.; Chataway, T., Purification of
alpha-synuclein containing inclusions from human post
mortem brain tissue. Journal of neuroscience methods 2016,
AUTHOR INFORMATION
2
7
66, 141-50.
. Chartier-Harlin, M. C.; Kachergus, J.; Roumier, C.;
Corresponding Author
*RHM: Phone: (215) 746-8233
E-mail: rmach@pennmedicine.upenn.edu.
ORCID
Robert H. Mach: 0000-0002-7645-2869
Author Contribution
C.-J.H. performed molecular docking and molecular dynamics
simulation. J.J.F. and E.J.P. performed photocrosslinking.
K.X., I.L., and T.J.A.G. performed organic synthesis. Z.T,
J.Y., D.D., and P.K. performed binding assays. R.H.M. di-
rected the project.
Mouroux, V.; Douay, X.; Lincoln, S.; Levecque, C.; Larvor,
L.; Andrieux, J.; Hulihan, M.; Waucquier, N.; Defebvre, L.;
Amouyel, P.; Farrer, M.; Destee, A., Alpha-synuclein locus
duplication as a cause of familial Parkinson's disease. Lancet
2
8
004, 364 (9440), 1167-1169.
. Singleton, A. B.; Farrer, M.; Johnson, J.; Singleton,
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A.; Hague, S.; Kachergus, J.; Hulihan, M.; Peuralinna, T.;
Dutra, A.; Nussbaum, R.; Lincoln, S.; Crawley, A.; Hanson,
M.; Maraganore, D.; Adler, C.; Cookson, M. R.; Muenter, M.;
Baptista, M.; Miller, D.; Blancato, J.; Hardy, J.; Gwinn-Hardy,
K., alpha-Synuclein locus triplication causes Parkinson's
disease. Science 2003, 302 (5646), 841.
Funding
This research was supported by the Michael J. Fox Foundation
and the National Institutes of Health (NIH NS081033 to
E.J.P.). J.J.F. thanks NSF(DGE-1321851) and the Parkin-
son’s Disease Foundation (PF-RVSA-SFW-1754) for
fellowship support. The other members of the Michael J. Fox
Alpha Synuclein Imaging Consortium include Jamie Eberling,
Eugene Johnson, and Kalpana M. Merchant from the Michael
J. Fox Foundation; Chester A. Mathis, William E. Klunk, and
N. Scott Mason from University of Pittsburgh Medical Center;
Dale Mitchell, Wolfgang Schmidt, and David Hardick from
BioFocus-Charles River; Edilio Borroni, Luca Golbi, Michael
Horner, and Kevin Nash from Roche; and Joel Mercier from
UCB.
9
.
Kotzbauer, P. T.; Tu, Z.; Mach, R. H., Current status
of the development of PET radiotracers for imaging alpha
synuclein aggregates in Lewy bodies and Lewy neurites.
Clinical and Translational Imaging 2017, 5 (1), 3-14.
1
0.
Klunk, W. E.; Engler, H.; Nordberg, A.; Wang, Y.;
Blomqvist, G.; Holt, D. P.; Bergström, M.; Savitcheva, I.;
Huang, G. F.; Estrada, S., Imaging brain amyloid in
Alzheimer's disease with Pittsburgh CompoundꢀB. Annals of
neurology 2004, 55 (3), 306-319.
1
1.
Wong, D. F.; Rosenberg, P. B.; Zhou, Y.; Kumar, A.;
Raymont, V.; Ravert, H. T.; Dannals, R. F.; Nandi, A.; Brašić,
J. R.; Ye, W., In vivo imaging of amyloid deposition in
Alzheimer disease using the radioligand 18F-AV-45
(flobetapir F 18). Journal of Nuclear Medicine 2010, 51 (6),
9
1
R.; Pike, K. L.; O'Keefe, G.; Tochon-Danguy, H.; Chan, G.;
Berlangieri, S. U.; Jones, G., Imaging of amyloid β in
Alzheimer's disease with 18F-BAY94-9172, a novel PET
tracer: proof of mechanism. The Lancet Neurology 2008, 7 (2),
1
1
†
These authors contributed equally to this study.
13-920.
2.
Rowe, C. C.; Ackerman, U.; Browne, W.; Mulligan,
Notes
The authors declare no competing financial interests.
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Figure 1. Structures of the compounds used in the docking and in vitro binding assays.
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Figure 2. Solid-state NMR structure of Asyn showing the three putative binding sites for small molecules.
Color labeled cyan, site 2; purple, Site 3/13; orange, site 9.
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Figure 3. Site of the peptide fragments T44-K58 labeled (blue) by the photoaffinity probe, 3. This peptide
fragment contains sites 2 and 3/13. Inset: MALDI-MS data showing a photoadduct of 3 with a peptide
corresponding to resi-dues 44-58 or 46-60 of Asyn (red). No peak at this mass is observed when Asyn fibrils
are irradiated in the absence of 3 (blue).
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In vitro binding studies of 2 using either [ H]1 or [ H]4 as the radioligand. Since 2 is predicted to bind to
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site 2, its affinity for Asyn fibrils was higher when a site 2 preferring ligand (i.e., [ H]1) was used in the
binding assay.
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