Aminopyrimidine Class Aggregation Inhibitor Effectively Blocks Aβ-Fibrinogen Interaction and Aβ-Induced Contact System Activation

Article abstract of DOI:10.1021/acs.biochem.7b01214

Accumulating evidence suggests that fibrinogen, a key protein in the coagulation cascade, plays an important role in circulatory dysfunction in Alzheimer's disease (AD). Previous work has shown that the interaction between fibrinogen and β-amyloid (Aβ), a hallmark pathological protein in AD, induces plasmin-resistant abnormal blood clots, delays fibrinolysis, increases inflammation, and aggravates cognitive function in mouse models of AD. Since Aβ oligomers have a much stronger affinity for fibrinogen than Aβ monomers, we tested whether amyloid aggregation inhibitors could block the Aβ-fibrinogen interaction and found that some Aβ aggregation inhibitors showed moderate inhibitory efficacy against this interaction. We then modified a hit compound so that it not only showed a strong inhibitory efficacy toward the Aβ-fibrinogen interaction but also retained its potency toward the Aβ42 aggregation inhibition process. Furthermore, our best hit compound, TDI-2760, modulated Aβ42-induced contact system activation, a pathological condition observed in some AD patients, in addition to inhibiting the Aβ-fibrinogen interaction and Aβ aggregation. Thus, TDI-2760 has the potential to lessen vascular abnormalities as well as Aβ aggregation-driven pathology in AD.

Full text of DOI:10.1021/acs.biochem.7b01214

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Article
Aminopyrimidine class aggregation inhibitor effectively blocks A#-
fibrinogen interaction and A#-induced contact system activation
Pradeep K. Singh, Masanori Kawasaki, Hanna E. Berk-Rauch, Goushi Nishida, Takeshi
Yamasaki, Michael A Foley, Erin H. Norris, Sidney Strickland, Kazuyoshi Aso, and Hyung Jin Ahn
Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.7b01214 • Publication Date (Web): 02 Feb 2018
Downloaded from http://pubs.acs.org on February 4, 2018
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Aminopyrimidine class aggregation inhibitor effectively blocks Aβꢀfibrinogen interaction and Aβꢀ
induced contact system activation
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Pradeep K. Singh‡1, Masanori Kawasaki§&1, Hanna E. BerkꢀRauch‡, Goushi Nishida§, Takeshi Yamasaki§,
Michael A. Foley§, Erin H. Norris‡, Sidney Strickland‡, Kazuyoshi Aso§&*, Hyung Jin Ahn‡*
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‡Patricia and John Rosenwald Laboratory of Neurobiology and Genetics, The Rockefeller University,
New York, NY 10065, USA; TriꢀInstitutional Therapeutics Discovery Institute, New York, NY 10021,
§
USA
*To whom correspondence should be addressed: Kazuyoshi Aso, 26ꢀ1 MuraokaꢀHigashi 2ꢀChome,
Fujisawa, Kanagawa 255ꢀ0012, Japan, Tel: 81ꢀ466ꢀ32ꢀ1121, Fax: 81ꢀ466ꢀ29ꢀ4457; Eꢀmail:
kazuyoshi.aso@takeda.com
*To whom correspondence should be addressed: Hyung Jin Ahn, 1230 York Avenue New York, NY
10065, USA, Tel: 1ꢀ212ꢀ327ꢀ8773, Fax: 1ꢀ212ꢀ327ꢀ8774; Eꢀmail: hahn@rockefeller.edu
1These authors contributed equally.
&Present address: Research Division, Chemistry, Axcelead Drug Discovery Partners, Inc., 26ꢀ1 Muraokaꢀ
Higashi 2ꢀChome, Fujisawa, Kanagawa 255ꢀ0012, Japan
Abstract
Accumulating evidence suggests that fibrinogen, a key protein in the coagulation cascade, plays an
important role in circulatory dysfunction in Alzheimer’s disease (AD). Previous work has shown that the
interaction between fibrinogen and βꢀamyloid (Aβ), a hallmark pathological protein in AD, induces
plasminꢀresistant abnormal blood clots, delays fibrinolysis, increases inflammation, and aggravates
cognitive function in mouse models of AD. Since Aβ oligomers have a much stronger affinity for
fibrinogen than Aβ monomers, we tested whether amyloid aggregation inhibitors could block the Aβꢀ
fibrinogen interaction and found that some Aβ aggregation inhibitors showed moderate inhibitory
efficacy against this interaction. We then modified a hit compound so that it not only showed a strong
inhibitory efficacy towards the Aβꢀfibrinogen interaction but also retained its potency towards the Aβ42
aggregation inhibition process. Furthermore, our best hit compound, TDIꢀ2760, modulated Aβ42ꢀinduced
contact system activation, a pathological condition observed in some AD patients, in addition to
inhibiting the Aβꢀfibrinogen interaction and Aβ aggregation. Thus, TDIꢀ2760 has the potential to lessen
vascular abnormalities as well as Aβ aggregationꢀdriven pathology in AD.
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Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder that leads to profound cognitive decline in the
elderly population 1, 2. AD is a complex, multifactorial disease, and it is likely that various disease
mechanisms contribute to pathology 3ꢀ5. Although the direct neuronal toxicity of the ADꢀrelated peptide,
βꢀamyloid (Aβ), is well recognized in vitro, the precise mechanism by which Aβ disrupts neuronal
function in AD patients is unclear 2, 6ꢀ8. Increasing evidence suggests that AD may be a neurological
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disorder in which circulatory deficiencies significantly contribute to the pathophysiology of the disease
14. Furthermore, it has been shown that Aβ plays an important role in this cerebrovascular dysfunction in
AD 15ꢀ20. Therefore, some of the neuronal loss observed in AD patients could be secondary to circulatory
problems. For example, many AD patients display altered cerebral blood flow, damaged cerebral
vasculature, neuroinflammation, and cerebral microinfarcts 5, 21ꢀ24
.
Components of the coagulation cascade could contribute to these vascular abnormalities as well as
cognitive decline and neurodegeneration in AD 17, 25. AD patients show increased coagulation factor XII
(FXII) activation, kallikrein activity, and high molecular weight kininogen (HK) cleavage in their plasma
17. Depletion of FXII inhibits HK cleavage as well as reduces neuroinflammation, neurodegeneration, and
fibrin(ogen) deposition in the brains of AD mice 25. Moreover, fibrinogen, the end product of the
coagulation cascade, interacts with Aβ with strong affinity, and this interaction increases the aggregation
kinetics of Aβ26. This interaction also induces abnormal clot structure and increases clot resistance
towards fibrinolysis 15, 18, 27, 28. In addition, fibrinogen is frequently localized to Aβ deposits around the
blood vessels of the brain (cerebral amyloid angiopathy, CAA) and the brain parenchyma in AD patients
as well as AD mouse models 15, 29ꢀ31. These findings suggest that the Aβꢀfibrinogen interaction could be
crucial to the onset and progression of neurovascular damage and cognitive impairment in AD. To further
support this hypothesis, administration of RUꢀ505, a small compound inhibitor of the Aβꢀfibrinogen
interaction, reduced cortical inflammation, vascular Aβ deposition, and lessened the severity of cognitive
impairment in AD mouse models 27.
The binding affinity of soluble Aβ oligomers to fibrinogen is nearly 10ꢀfold higher than that of Aβ
monomers 27. This finding suggests that minimizing the population of Aβ oligomers could indirectly
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affect the Aβꢀfibrinogen interaction. In addition, several Aβ aggregation inhibitors directly bind Aβ
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suggesting they may also inhibit the Aβꢀfibrinogen interaction directly. Therefore, we screened several
known Aβ aggregation inhibitors for the ability to block the Aβꢀfibrinogen interaction and found a subset
of hits with inhibitor capacity. We then improved the inhibitory efficacy of hit compounds using
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medicinal chemistry approaches and found that the best hit compound, TDIꢀ2760, not only showed a
strong inhibitory efficacy towards the Aβꢀfibrinogen interaction but also modulated Aβ42ꢀinduced contact
system activation, a pathological condition observed in some AD patients 17.
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Materials and Methods
Synthesis of Aβꢀaggregation inhibitors. The derivatives of SEN1269 were synthesized as shown in
Scheme 1.
Scheme 1. Synthetic scheme of TDIꢀ2760 derivatives.
As described in Scheme 1, 2ꢀchloroꢀ5ꢀfluoropyrimidine (1) was treated with 3ꢀdimethylaminophenol in
the presence of K2CO3 to afford chloro analog (2). Buchwald amination of (2) using palladium catalyst
with various amines worked smoothly to give corresponding amino analogs (3).
Synthesis of 3ꢀ(2ꢀChloropyrimidinꢀ5ꢀyl)oxyꢀN,Nꢀdimethylꢀaniline. A mixture of 3ꢀ(dimethylamino)
phenol (11.9 g, 87.1 mmol), 2ꢀchloroꢀ5ꢀfluoroꢀpyrimidine (15.0 g, 113 mmol, 14.0 mL), and K2CO3 (24.1
g, 174 mmol) in DMF (100 mL) was stirred at 60 °C for 48 h. The mixture was quenched with water and
diluted with EtOAc. The organic layer was separated, washed with brine, dried over MgSO4, filtered and
concentrated in vacuo. The residue was purified by column chromatography (silicaꢀgel, 10ꢀ30% EtOAc in
1
hexane) to give the title compound (3.1 g, 12.5 mmol, 14% yield) as an offꢀwhite solid; H NMR (500
MHz, DMSOꢀd6) δ 8.54 (s, 2H), 7.22 (t, J = 8.5 Hz, 1H), 6.58 (d, J = 8.6 Hz, 1H), 6.49 (s, 1H), 6.45 ꢀ
6.32 (m, 1H), 2.90 (s, 6H).
General procedure of palladiumꢀcatalyzed coupling reaction. A mixture of 3ꢀ((2ꢀchloropyrimidinꢀ5ꢀ
yl)oxy)ꢀN,Nꢀdimethylaniline (20.0 mg, 80.1 µmol), amine (160 µmol), BrettPhos Pd G3 (7.25 mg, 8.00
µmol), BrettPhos (4.29 mg, 8.00 µmol), KOtBu (27.0 mg, 240 µmol) and DME (1 ml) was heated 100°C
for 2 h under microwave irradiation (Synthos 3000, 4x48MC). The reaction mixture was diluted with
EtOAc (3 ml), quenched with H2O (1 ml) and stirred for 2 min. The organic layer was separated, and the
aqueous layer was then extracted with EtOAc (2 ml). The combined organic layer was evaporated by
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60°C. The residue was purified by preparative HPLC (Actus Triart C18, eluted with MeCN/10mM
NH4HCO3 aq 5:95 to 100:0). Pure fractions were combined and concentrated by blowing away with the
air at 60°C to afford product.
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Preparation of Aβ42 solutions. Biotinylated and nonꢀbiotinylated synthetic Aβ42 were purchased from
Anaspec (Fremont, CA). For AlphaLISA, Aβ42 was reconstituted in a minimal volume of 0.1% NH4OH
and then diluted with 20 mM HEPES buffer (pH 7.4) with 140 mM NaCl. Solubilized Aβ42 was spun at
20,000 x g for 10 min to remove aggregated material and the protein concentration was estimated by BCA
(Thermo Scientific). For Aβ42 aggregation studies, monomerized Aβ42 films (Hexafluoro isopropanol,
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HFIP treated) were prepared as in and diluted in 20 mM TrisꢀHCl (pH 7.4) or 20 mM HEPES buffer
(pH 7.4) with 140 mM NaCl. Aβ42 oligomers were prepared as described in 34. Briefly, monomerized
Aβ42 films were reconstituted in DMSO by sonication for 10 min and incubated overnight with slow
agitation (40 rpm) at 4°C either in 20 mM TrisꢀHCl (pH 7.4) or 20 mM HEPES buffer (pH 7.4) with 140
mM NaCl. After, overnight incubation, the solution was centrifuged at 20,000 x g for 10 min. The protein
concentration was estimated by Pierce BCA protein assay kit (Thermo Scientific).
AlphaLISA. Various concentrations (0. 1 – 100 µM) of compounds were plated in white 384ꢀwell plates
(Greiner) and were incubated with 10 nM biotinylated Aβ42 (Anaspec) and 1 nM fibrinogen for 30 min at
room temperature (RT) in a final volume of 10 µl assay buffer (25 mM TrisꢀHCl, pH 7.4, 150 mM NaCl,
0.05% Tweenꢀ20, and 0.1% BSA). The mixture was then incubated with anti–fibrinogen antibody (Dako),
20 µg/ml streptavidinꢀconjugated donor, and protein A–conjugated acceptor beads (PerkinElmer) for 90
min at RT. Samples were read by a PerkinElmer EnVision plate reader. RUꢀ505, which is a known
inhibitor of Aβ42ꢀfibrinogen interaction27, was used as a positive control and also for comparison of its
efficacy with other compounds. All the experiments were performed in triplicate. To determine IC50 of
compounds, the data were fitted to a sigmoidal doseꢀresponse equation (Y= Bottom + (Top – Bottom)/1 +
10(logIC50 ꢀ X)⋅ Hill coefficient)) using GraphPad Prism 4 to calculate IC50.
In vitro clotting (turbidity) assay. Turbidity assays were performed at RT in high binding 96ꢀwell plates
(Fisher Scientific) in triplicate using a Spectramax Plus384 reader (Molecular Devices). For in vitro fibrin
clot formation, fibrinogen (1.5 ꢁM) with or without Aβ42 oligomers (3 ꢁM) was mixed with thrombin
(0.5 U/mL) and CaCl2 (5 mM) in 20 mM HEPES buffer (pH 7.4) with 137 mM NaCl in a volume of
200ꢁl. To test the efficacy of SEN1269 and its analogs in restoring the Aβ42ꢀinduced reduction in clot
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turbidity, clot formation was also initiated in the presence of these compounds (20 ꢁM) with Aβ42
oligomers (3ꢁM). The effect of these compounds was also tested in normal fibrin clotting experiments
without adding Aβ42 oligomers. Clot formation was monitored by measuring the change in turbidity at
450 nm over time.
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Monitoring Aβ42 aggregation kinetics. Monomerized Aβ42 films (Hexafluoro isopropanol, HFIP,
treated) were reconstituted in DMSO by sonication as in 34. The solution was then diluted in 20 mM
HEPES buffer (pH 7.4) with 140 mM NaCl and centrifuged at 14,000 rpm at 4°C for 10 min to remove
any aggregated species. The supernatant was collected, and Aβ42 concentration was estimated using the
BCA method for protein quantification. The Aβ42 solution was kept on the ice and used for aggregation
study reaction setup within 30 min. For amyloid aggregation kinetics, we used 96ꢀwell flat bottom
microtiter black plates (Greiner), and the Aβ42 solution was added to the wells in the presence or absence
of compounds. The final concentration of Aβ42 was 12.5 ꢁM, and the final concentration of compounds
was 48 ꢁM in a total reaction mixture of 150 ꢁl. Because the stock solutions of these compounds were in
DMSO, the equivalent residual DMSO concentration was also added to Aβ42 alone solution. To monitor
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the time dependent Aβ amyloid assembly, ThT dye was added to each well to a final concentration of
33 ꢁM in the solution. Buffer alone in the presence and absence of compounds was also used with ThT as
controls. The plate was sealed with an adhesive film (Thin Seal) and incubated at 37°C with a shaking
speed of 25 rpm (Environ Orbital Shaker). Time dependent ThT fluorescence was recorded (once an
hour) by quickly taking out the microtiter plate from the shaker and directly measuring the emission
maxima at 480 nm with an excitation wavelength of 440 nm, using a Spectramax M2e plate reader
(Molecular Devices). The microtiter plate was sealed after every measurement and incubated at 37°C for
a total of 26 hrs. All the experiments were performed in triplicate.
Scanning electron microscopy (SEM). Fibrin clots were formed using purified human fibrinogen
(Calbiochem) on round glass coverslips at RT. Fibrin clots in the presence or absence of oligomeric Aβ42
were formed as described above for the turbidity assay. To test the efficacy of SEN1269 and its analogs,
these compounds (20 ꢁM) or DMSO were preincubated with Aβ42 oligomers (3.0 ꢁM) for 10 mins with
slow shaking at RT. Fibrinogen (1.5 ꢁM) was added into compoundꢀAβ42 mixtures, and the reaction was
initiated by adding thrombin (0.5 U/mL). Thirty mins after adding thrombin, clots were gently washed
twice with cold sodium cacodylate buffer (0.1M; 2 min each) and fixed with 2% glutaraldehyde (on ice
for 30 min). After serial dehydration by ice cold ethanol (20ꢀ100%), dried fibrin clots were sputterꢀcoated
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with gold palladium. Images were obtained using a LEO 1550 scanning electron microscope at the
Electron Microscopy Resource Center at The Rockefeller University.
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Transmission electron microscopy (TEM). For visualizing the morphology of Aβ42 aggregates in the
presence and absence of hit compounds, after 26 hrs of incubation, the undiluted Aβ42 (12.5 ꢁM) samples
(in the presence and absence of compounds) were applied to glowꢀdischarged CF200ꢀCu grids (Electron
Microscopy Sciences), washed three times with ultrapure water, negatively stained with 2% uranyl
acetate, and airꢀdried. Electron micrographs were acquired using the FEI TECNAI G2 Spirit BioTwin
transmission electron microscope (120 Kv) equipped with Gatan 4K x 4K digital camera in Electron
Microscopy Resource Center at The Rockefeller University.
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Kallikrein activity assay. To test the effect of TDIꢀ2760 on Aβ42 oligomerꢀinduced kallikrein
generation in vitro, Aβ42 oligomers in the presence or absence of TDIꢀ2760 were incubated in 20 mM
HEPES buffer (pH 7.4) with 140 mM NaCl in a 96ꢀwell microtiter plate (Fisher Scientific). The plate was
sealed with an adhesive sealing film (Thin Seal) and incubated at RT with a shaking speed of 25 rpm for
30 min. Buffer (in the presence or absence of TDIꢀ2760) without Aβ42 oligomers was also incubated.
After 30 min, 10 nM of purified human coagulation factor XII (FXII; Haematologic Technologies Inc)
and 10 nM of human plasma prekallikrein (PPK; Enzyme Research Laboratories) were added to each
well. Finally, chromogenic substrate Sꢀ2302TM (Chromogenix) was added to each well to a final
concentration of 0.67 mM in a 100 ꢁl reaction mixture. Time dependent change in the absorbance was
recorded by measuring the absorbance at 405 nm at RT with a Spectramax Plus 384 reader (Molecular
Devices). In the reaction mixture of 100 ꢁl, the final concentration of Aβ42 oligomer was 1ꢁM and TDIꢀ
2760 was 40 ꢁM. Experiments were performed in triplicate, and dextran sulfate (DXS) (0.1 ꢁg/ml) was
used as a positive control. DXS (0.1 ꢁg/ml) in the presence of TDIꢀ2760 (40 ꢁM) was also used to check
the specificity of TDIꢀ2760. The rate of substrate (Sꢀ2302TM) conversion over time (30 min) was also
calculated for each sample by Softmax 6.1 (Molecular Devices) and represented as milliꢀunits per min.
Statistical analysis. All numerical values presented in graphs are mean ± SEM. Statistical significance in
Figure 5 was determined using Student’s twoꢀtailed tꢀtest. For all the AlphaLISA IC50 analyses, 95%
confidence interval (95% CI) was used as the statistical parameter.
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Some Aβ aggregation inhibitors can block the Aβꢀfibrinogen interaction. Biochemical analysis using
AlphaLISA showed that the binding affinity of soluble Aβ oligomers to fibrinogen is nearly 10ꢀfold
higher than that of Aβ monomers 27. Therefore, we hypothesized that Aβ aggregation inhibitors may
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indirectly minimize the Aβꢀfibrinogen interaction by decreasing Aβ oligomerization. In addition, we
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hypothesized that some Aβ aggregation inhibitors may directly bind to Aβ
and disrupt the Aβꢀ
fibrinogen interaction directly. Based on these hypotheses, we synthesized and evaluated a series of
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known Aβ aggregation inhibitors/βꢀsheet breakers
for their efficacy against the Aβꢀfibrinogen
interaction using AlphaLISA (Table 1). Some of the βꢀsheet breakers possessed efficacy against the
fibrinogenꢀAβ interaction (IC50 values are shown in Table 1). However, the efficacy of these compounds
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(IC50) varied considerably. For example, RSꢀ0406, SEN1576, and tanshinone
showed almost no
efficacy (IC50 > 100 ꢁM), whereas SEN1269 32 showed the best efficacy in blocking the Aβ42ꢀfibrinogen
interaction (IC50 = 17.8 ꢁM). Since SEN1269 has the highest inhibitory efficacy, we selected this
compound for structure activity relationship (SAR) study.
Chemical modification of SEN1269 to improve its inhibitory efficacy against the Aβ42ꢀfibrinogen
interaction. To improve the potency of SEN1269, we modified the chemical structure of the SEN1269
scaffold. SAR of the rightꢀhand side (RHS) of SEN1269 is summarized in Table 2. The inhibitory activity
of SEN1269 analogs against the Aβ42ꢀfibrinogen interaction was determined using AlphaLISA, and IC50
values were calculated (Table 2). The methoxy analogs (Table 2; TDIꢀ2758 Entry 2, TDIꢀ3066 Entry 3,
and TDIꢀ3067 Entry 4) were evaluated, and it was found that the substituent in the meta position (TDIꢀ
2758) was most effective among the three analogs (IC50 = 6.42 µM for Entry 2 vs IC50 = 25.0 and 29.0
µM, for Entry 3 and 4, respectively). Compared to the parent molecule, the substituents in the ortho and
para positions actually led to decreased inhibitory activity against the Aβ42ꢀfibrinogen interaction.
Furthermore, the phenyl analog (TDIꢀ2760; Table 2 Entry 6 and Figure 1A) showed much higher efficacy
against this interaction (IC50 = 1.67 µM) in AlphaLISA (Table 2 and Figure 1B). In comparison to its
parent molecule, the inhibitory efficacy (IC50) of TDIꢀ2760 was increased ~ 10ꢀfold (17.8 µM vs 1.67
µM). RUꢀ505 is a known inhibitor of the Aβ42ꢀfibrinogen interaction27, and thus we included it in our
study as a positive control (Figure 1).
Next, a SAR study of the left hand side (LHS) was also conducted (Table S1). The dimethylamino
substituent on the phenyl group was crucial to disrupt the interaction (Table S1; Entry 1 and 2), and the
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pyrrolidine analog, TDIꢀ3222, led to enhanced activity (Table S1; Entry 5; IC50 = 3.12 µM). This result
would suggest that an electronꢀdonating amino group leads to enhanced potency. To test this hypothesis,
difluoropyrrolidine analog TDIꢀ3906 was evaluated and revealed a decreased potency (Table S1; Entry 6;
IC50 = 88.3 µM). In addition, we tested several other LHS analogs including morpholino and monoꢀNꢀ
methyl analog, but only the Nꢀmethyl analog led to improved potency effectively compared with the
original compound (Table S1; Entry 10, IC50 = 3.76). Based on these preliminary SAR results, we found
that TDIꢀ3222 and TDIꢀ3256, which were obtained by LHS modifications, have better inhibitory efficacy
against the Aβ42ꢀfibrinogen interaction (IC50 = 3.12 µM and 3.76 µM, respectively). Overall, the TDIꢀ
2760, which was obtained by RHS modifications exhibited the most potent efficacy against the Aβ42ꢀ
fibrinogen interaction as its IC50 value was 10ꢀfold lower than its parent molecule, SEN1269 (1.67 µM vs
17.8 µM, respectively).
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Figure 1. TDIꢀ2760 blocks the interaction
between Aβ42 and fibrinogen in AlphaLISA.
(A) The chemical structures of TDIꢀ2760 and
RUꢀ505. (B) Representative AlphaLISA
reading, showing the effect of TDIꢀ2760 on the
Aβ42ꢀfibrinogen interaction. IC50 of TDIꢀ2760
was determined, and RUꢀ505 was used as a
positive control since it inhibits the Aβ42ꢀ
fibrinogen interaction. The experiment was
performed in triplicate, and mean ± SEM values
were plotted. Blue = TDIꢀ2760; Red = RUꢀ505.
Validating the inhibitory efficacy of SEN1269 and its analogs against the Aβ42–fibrinogen
interaction using an in vitro clotting assay. As the interaction between fibrinogen and Aβ induces a
structurally abnormal fibrin clot 15, 18, 27, we tested whether SEN1269 and its analogs (TDIꢀ3222, TDIꢀ
3256, and TDIꢀ2760) can restore Aβꢀinduced altered thrombosis in vitro. We also included TDIꢀ2758
(Entry 2, Table 2) in the assays, since this analog also showed improved inhibitory efficacy against the
Aβꢀfibrinogen interaction by AlphaLISA.
During clot formation, fibrinogen molecules are cleaved by thrombin and associate into a fibrin network,
which scatters light and causes the solution to increase in turbidity. Thus, the kinetics of turbidity can be
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used as a readꢀout to examine fibrin clot formation27. Each compound (20 µM) or vehicle (0.4% DMSO)
was incubated with purified human fibrinogen in the presence or absence of oligomeric Aβ42. Fibrin clot
formation was analyzed by measuring the turbidity immediately after adding thrombin to the mixture. The
maximum turbidity of the fibrin clot decreased in the presence of Aβ42, since it alters fibrin clot structure
(Figure 2AꢀD). In the in vitro clotting assay, SEN1269 further decreased the turbidity of the clot formed
in the presence or absence of Aβ42 (Figure 2A). This result indicates that SEN1269 also altered normal
fibrin clot structure, which was undesirable. In contrast, TDIꢀ2758 and TDIꢀ3256 did not alter the
turbidity of the normal fibrin clot formed in the absence of Aβ42 (Figure 2B and 2C, green), but clot
turbidity was further decreased in the presence of both Aβ42 and TDIꢀ2758/TDIꢀ3256 (Figure 2B and 2C,
purple). This result was the opposite of what we expected, as these analogs showed an inhibitory effect on
the Aβ42ꢀfibrinogen interaction in AlphaLISA. TDIꢀ3222 did not show any effect in the turbidity assay
due to its color causing a high background (data not shown).
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In contrast to the other compounds, phenyl analog TDIꢀ2760 was very effective in the turbidity assay as it
partially restored the Aβ42ꢀinduced abnormal fibrin clot formation (Figure 2D, purple vs red).
Additionally, TDIꢀ2760 did not alter the turbidity of the normal clot in the absence of Aβ42 (Figure 2D,
green vs blue). This observation indicates that TDIꢀ2760 specifically restores the Aβꢀinduced decrease in
turbidity during fibrin clot formation without affecting normal thrombosis. In conclusion, TDIꢀ2760
effectively inhibits the Aβꢀfibrinogen interaction and partially restores Aβ42ꢀinduced abnormal fibrin clot
formation.
The results of the turbidity assay were further validated by analyzing in vitro fibrin clots using scanning
electron microscopy (SEM). Fibrin clots were prepared from purified fibrinogen in the presence of CaCl2
and thrombin, and SEM images of fibrin clots showed a typical threeꢀdimensional mesh of elongated
fibrin threads (Figure 3A). However, in the presence of Aβ42 oligomers, the fibrin threads were relatively
thinner (Figure 3B) and several clumped structures were observed, indicating that the architecture of the
fibrin clot was altered in the presence of Aβ42 (Figure 3B). Consistent with the turbidity assay, TDIꢀ2760
partially restored the Aβ42ꢀinduced abnormal fibrin clot structure as evidenced by fewer clumps and
relatively thicker fibrin threads under SEM (Figure 3C). However, the SEM analysis of fibrin clots in the
presence of Aβ42 and SEN1269 (Figure 3D) or TDIꢀ2758 (Figure 3E) suggested that SEN1269 and TDIꢀ
2758 failed to restore the Aβ42ꢀinduced clot abnormality. In fact, clots formed in the presence of Aβ42
with SEN1269 or TDIꢀ2758 showed more clumps and compromised clot architecture more severely than
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clots formed in the presence of Aβ42 oligomers only (Figure 3D and 3E). This finding was consistent
with the results obtained from the turbidity assay, in which SEN1269 and TDIꢀ2758 further decreased the
turbidity of the fibrin clots formed in the presence of Aβ42 (Figure 2A and 2B).
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Even though TDIꢀ2760 did not affect normal thrombosis in the turbidity assay (Figure 2D), we further
tested whether TDIꢀ2760 affects thrombin proteolytic activity or blood coagulation, using a chromogenic
substrate assay and activated partial thromboplastin time (aPTT) test, respectively. Our assay results
showed that TDIꢀ2760 did not alter the activity of thrombin in the chromogenic substrate assay (Figure
S1) and did not have any effect on the coagulation profile of human pooled normal plasma in a microtiter
plate basedꢀaPTT test (Figure S2).
Figure 2. TDIꢀ2760 restores the Aβ42ꢀinduced decrease in fibrin clot turbidity without affecting normal
clotting in an in vitro clotting assay. Individual test compounds or DMSO (vehicle) were incubated with
fibrinogen in the presence or absence of Aβ42 (3 µM), followed by addition of thrombin and CaCl2.
Fibrin clot formation was assessed by measuring timeꢀdependent turbidity changes at 450 nm (n = 3
experiments/compound). (A) SEN1269 decreased the clot turbidity of the normal clot formed in the
absence of Aβ42 (green vs blue) as well as the turbidity of the clot formed in the presence of Aβ42
(purple vs red). (B) In the presence of TDIꢀ2758, the turbidity of clots formed with Aβ42 was decreased
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(purple vs red). (C) In the presence of TDIꢀ3256, the turbidity of clots formed with Aβ42 was also
decreased (purple vs red). (D) TDIꢀ2760 did not alter the turbidity of the normal clot (blue vs green) but
restored the Aβ42ꢀinduced decrease in turbidity during fibrin clot formation (purple vs red).
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Figure 3. SEM analysis of the effect of hit compounds on Aβ42ꢀinduced abnormal fibrin clot structure.
Individual compounds (20 µM) or DMSO (vehicle) were incubated with fibrinogen (1.5 µM) in the
presence or absence of Aβ42 oligomers (3 µM), and clot formation was initiated by adding thrombin into
the mixture. Fibrin clot structures were analyzed by SEM. SEM images of clots formed by (A) Fibrinogen
alone, (B) Fibrinogen+Aβ42, (C) Fibrinogen+Aβ42+TDIꢀ2760, (D) Fibrinogen+Aβ42+SEN1269, (E)
Fibrinogen+Aβ42+TDIꢀ2758. SEM analysis revealed that Aβ42 oligomers altered the fibrin clot
structure, and TDIꢀ2760 partially restored the Aβ42ꢀinduced structural abnormality of fibrin clot while
SEN1269 and TDIꢀ2758 did not. Scale bar is 1 µm.
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TDIꢀ2760 effectively inhibits Aβ42 aggregation. SEN1269 was not effective in reversing the Aβ42ꢀ
induced decrease in clot turbidity and altered clot structure (Figure 2A and Figure 3D). However,
SEN1269 has been shown to be a potent Aβ aggregation inhibitor32, and it also effectively inhibited Aβ42
aggregation in our study (Figure S3). Therefore, we investigated if SEN1269 phenyl analog, TDIꢀ2760,
also retained this inhibitory efficacy towards Aβ42 aggregation in vitro. The timeꢀdependent Aβ42
aggregation kinetics were probed using an amyloidꢀspecific dye, thioflavin T (ThT)35. The typical
amyloid aggregation process (when probed using ThT) shows a sigmoidal growth curve with lag phase,
log phase, and stationary phase. The stationary phase (with a plateau), usually represents the completion
of the amyloid fibrillization process43. The ThT fluorescence of Aβ42 (12.5 ꢁM) in the presence and
absence of TDIꢀ2760 (48 ꢁM) was recorded at different time intervals. The ThT fluorescence data of
Aβ42 alone showed a typical sigmoidal curve, characteristic of the amyloid aggregation pathway (red in
Figure 4A).
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In contrast, Aβ42 in the presence of TDIꢀ2760 did not show such characteristics and the ThT fluorescence
was significantly lower (blue in Figure 4A). This result indicates minimal Aβ42 amyloid fibril formation
in the presence of TDIꢀ2760. ThT fluorescence of buffer in the presence and absence of TDIꢀ2760
without Aβ42 addition remained low during the incubation period (data not shown). To confirm this
result, we used transmission electron microscopy (TEM) to examine the end products of Aβ42
aggregation in the presence or absence of TDIꢀ2760 after 26 hrs incubation. TEM of Aβ42 showed
mainly fibrillar structures with few oligomers (Figure 4B), consistent with high ThT fluorescence (Figure
4A). However, TEM of Aβ42 in the presence of TDIꢀ2760 showed very few fibrillar structures (Figure
4C), consistent with low ThT fluorescence (Figure 4A). The ThT fluorescence data and TEM analysis
clearly revealed that TDIꢀ2760 effectively inhibits Aβ42 aggregation. This observation suggests that
removing the hydroxyl (ꢀOH) group improves the inhibitory efficacy of SEN1269 against the Aβ42ꢀ
fibrinogen interaction without diminishing its ability to inhibit Aβ42 aggregation.
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Figure 4. TDIꢀ2760 inhibits Aβ42 fibrillization. Aβ42 aggregation in the presence and absence of TDIꢀ
2760 was examined by monitoring amyloid aggregation kinetics by ThT fluorescence. (A) ThT
fluorescence was monitored over time by measuring the emission maxima at 480 nm, with an excitation
wavelength of 440 nm. ThT fluorescence of the Aβ42 alone (red) showed a timeꢀdependent increase and
displayed a sigmoidal growth curve, which is a typical characteristic of amyloid aggregation. In the
presence of TDIꢀ2760 (blue), the Aβ42ꢀinduced timeꢀdependent increase of ThT fluorescence was
significantly reduced, suggesting that TDIꢀ2760 strongly inhibits Aβ42 fibrillization. The experiment was
performed in triplicate, and the graph is presented as mean± SEM value. (B, C) After 26 hrs, these
samples were visualized by TEM. TEM images of Aβ42 alone (B) showed predominately fibrillar species
along with some oligomers. The fibrillar and oligomeric species of Aβ42, however, were substantially
decreased in the presence of TDIꢀ2760 (C), suggesting the inhibitory efficacy of this compound against
Aβ42 aggregation. Scale bar is 100 nm.
Effect of TDIꢀ2760 on Aβ42ꢀmediated initiation of contact activation system. Induction of the
coagulation factor XII (FXII)ꢀdriven contact activation pathway can be initiated by Aβ42 17, 44ꢀ47, leading
to cleavage of coagulation factor XI (FXI) and blood clotting and activation of plasma prekallikrein
(PPK) and release of bradykinin from high molecular weight kininogen (HK) 48. This activation could
play a role in the vascular and inflammatory aspects of AD pathology by launching both prothrombotic
and proinflammatory pathways 25. Since SEN1269 binds to Aβ42 in a surface plasmon resonance
experiment 32, TDIꢀ2760, the phenyl analog of SEN1269, may bind to Aβ42, since it showed strong
inhibition against the Aβ42 aggregation process (Figure 4). We speculated that if TDIꢀ2760 interacts with
Aβ42, it may also affect Aβ’s activation of FXII and subsequent cleavage of FXI to FXIa and/or PPK to
kallikrein. To test this hypothesis, we measured the in vitro kallikrein activity using purified human
coagulation components (FXII and PPK) and a chromogenic substrate, Sꢀ2302TM. Aβ42 oligomers in the
presence and absence of TDIꢀ2760 were tested for their potential to activate FXII and thus produce
kallikrein. Kallikrein then can cleave its chromogenic substrate Sꢀ2302TM which can be quantified by
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measuring absorbance at 405 nm 17. Aβ42 oligomers (1µM) showed high kallikrein activity in the reaction
mixture of purified coagulation components and the Sꢀ2302TM substrate, as indicated by high absorbance
at 405 nm (red in Figure 5A).
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However, Aβ42 oligomers incubated in the presence of TDIꢀ2760 (40 µM) showed significantly less
absorbance at 405 nm compared to Aβ42 oligomers alone (blue in Figure 5A). The buffer in the presence
and absence of TDIꢀ2760 (40 µM) without Aβ42 addition showed insignificant kallikrein activity (purple
and green in Figure 5A). This suggests that binding of TDIꢀ2760 to Aβ42 oligomers lessens the ability of
Aβ to induce FXII activation and its subsequent generation of kallikrein. Consistent with this result, the
rate of Sꢀ2302TM substrate conversion was also significantly lower in the Aβ42 oligomers incubated with
TDIꢀ2760 sample (22.06±0.73 vs 10.06±0.78 milliꢀunits per min, respectively) (Figure 5B). Dextran
sulfate (DXS, MW 500, KDa) 17 was used as a positive control and showed high kallikrein activity (red in
Figure 5C). Additionally, TDIꢀ2760 (40 µM) did not alter the kallikrein activity induced by DXS (0.1
µg/ml) (blue in Figure 5C), indicating the specificity of TDIꢀ2760 towards Aβ42. These data suggest that
TDIꢀ2760 is effective at blocking the Aβ42ꢀinduced activation of the FXIIꢀmediated contact system.
Figure 5. Effect of TDIꢀ2760 on Aβ42ꢀmediated contact activation. (A) Purified human coagulation
factor XII (FXII) and human plasma prekallikrein (PPK) were used in the assay, and kallikrein activity
was triggered by adding Aβ42 oligomers incubated in the presence or absence of TDIꢀ2760. The final
concentration of Aβ42 oligomers was 1 µM and TDIꢀ2760 was 40 µM in the reaction. Buffer alone
(green) and buffer with TDIꢀ2760 (purple) showed essentially no kallikrein activity. Aβ42 alone (red)
significantly increased kallikrein activity, while TDIꢀ2760 with Aβ42 (blue) led to a significant decrease
in the amount of kallikrein release. (B) Vmax was calculated from the assay in (A). Preꢀincubating TDIꢀ
2760 to Aβ42 oligomers prior to this assay led to a significantly lower substrate (Sꢀ2302TM) conversion
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rate (Vmax) (p<0.001). The experiment was performed in triplicate, and mean ± SEM values are plotted.
(C) Dextran sulfate (DXS, red, 0.1 µg/ml), a positive control for kallikrein activation, was not influenced
by preꢀincubation with TDIꢀ2760 (blue, 40 µM), indicating TDIꢀ2760’s specificity for Aβ42. Statistical
significance was determined by nonꢀparametric unpaired (twoꢀtailed) tꢀ test. p ≤ 0.001 was designated as
(***).
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Discussion
Several studies indicate that Aβ aggregation initiates a cascade of molecular events, eventually leading to
neuronal death and dementia in AD 1, 2, 6ꢀ8. Therefore, targeting Aβ aggregation has increasingly become a
32, 33, 38,
focus of a potential therapeutic approach for AD
49ꢀ54. Despite tremendous efforts, drug
development of Aβ aggregation inhibitors has proven very difficult and the results of ongoing clinical
trials of those inhibitors are mostly inconclusive 55, 56. In this study, we have shown that some Aβ
32, 36ꢀ40
aggregation inhibitors
have the intrinsic ability to disrupt the Aβꢀfibrinogen interaction (Table 1).
These characteristics together would be beneficial not only in altering Aβ aggregationꢀrelated toxicity, but
also in modulating vascular abnormalities that arise from the Aβꢀfibrinogen interaction in AD.
Because our previous work indicated that Aβ oligomers have stronger affinity for fibrinogen than
monomers, we tested whether Aβ aggregation inhibitors could inhibit the Aβꢀfibrinogen interaction in the
present study 27. However, our results in Table 1 suggest that Aβ aggregation inhibition itself is not
36, 41,
sufficient to inhibit the Aβꢀfibrinogen interaction. For example, RSꢀ0406, SEN1576, and tanshinone
42, all of which can strongly inhibit Aβ aggregation and its toxicity, showed negligible efficacy against the
Aβꢀfibrinogen interaction (IC50 > 100 ꢁM) (Table 1). In addition, since we used the oligomeric form of
Aβ in turbidity assays (Figure 2) and kallikrein activity assays (Figure 5), the ability to inhibit Aβ
aggregation, such as these compounds exhibit, may be inconsequential in affecting the result of both
assays. Moreover, the duration of both assays (including the incubation time for compoundꢀAβ mixtures)
was less than an hour. It is very unlikely that Aβ aggregation status affects the result of both the assays
within an hour, because Aβ fibrillization requires several hours at 37°C (Figure 4A). Therefore, we expect
that Aβ binding affinity of compounds could be an important factor in inhibitory efficacy against the Aβꢀ
fibrinogen interaction. In line with this, SEN1269 (5ꢀaryloxypyrimidine) was shown to bind Aβ42 and
subsequently block Aβ42’s βꢀpleated sheet assembly and toxicity 32. Although it is not clear whether
SEN1269 or its analog compounds bind directly to fibrinogen, we found that TDIꢀ2760 does not affect
normal thrombosis in the turbidity assay (Figure 2D) or blood coagulation in an activated partial
thromboplastin time (aPTT) test using human plasma (Figure S2). These results suggest that TDIꢀ2760
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does not have any effect on the activity of coagulation proteases. In addition to fibrin polymer formation
via coagulation cascade, fibrin(ogen) is also involved in other activities, such as binding to platelets via
αIIbβ3 or leukocytes via Macꢀ1. This type of fibrin(ogen) activity is important for hemostasis or innate
immunity 57, 58. Therefore, to become an effective therapeutic molecule, the influence of TDIꢀ2760 on
fibrin(ogen) binding to platelets or leukocytes needs to be examined in future studies.
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The contact activation system, driven by FXII, can also launch a proinflammatory pathway that leads to
the release of bradykinin upon cleavage of HK 17, 48. A significantly higher level of HK cleavage is
reported in AD patient plasma 17. Furthermore, it was recently shown that modulating the innate immunity
response by knocking out the complement component C3 led to neuroprotection and improved cognitive
function in AD mice59. These complement C3ꢀknockout AD mice performed better on learning and
memory tests despite having more cerebral Aβ deposition. The reduced level of proꢀinflammatory
markers in these AD mice highlights the important role of inflammation in AD and suggests that targeting
the inflammatory pathway could be highly beneficial in treating this disease59. Consistent with this
finding, our group has shown that reducing neuroinflammation by depleting FXII improves the cognitive
function of AD mice 25.
Aβ42 can activate FXII 17, 44ꢀ46, which leads to the cleavage of PPK into kallikrein48. AD patient plasma
has increased levels of kallikreinꢀlike activity compared to that of nonꢀdemented control individuals,
further suggesting the crucial role of inflammation in AD pathology17, 19. TDIꢀ2760 lessens the Aβ42ꢀ
driven activation of FXII and subsequent release of kallikrein in vitro (Figure 5). All these results suggest
that TDIꢀ2760 not only alters Aβ42 aggregation but also modulates the other molecular activities of
Aβ42, such as fibrinogen interaction and contact system activation. These molecular events are critical in
driving vascular and inflammatory pathologies in AD. Therefore, the present study suggests that
molecules that can participate in minimizing Aβ42ꢀdriven activation of the contact system and can inhibit
the Aβ42ꢀfibrinogen interaction would be promising drug candidates for the treatment of AD patients
with vascular and inflammatory defects.
Abbreviations:
Aβ, βꢀamyloid; AD, Alzheimer’s disease; aPTT, activated partial thromboplastin time; BSA, bovine
serum albumin; CAA, cerebral amyloid angiopathy; CI, confidence interval; DMSO, dimethyl sulfoxide;
DXS, dextran sulfate; FXI, coagulation factor XI; FXII, coagulation factor XII; HEPES, 4ꢀ(2ꢀ
hydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid; HFIP, hexafluoro isopropanol; HK, high molecular
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weight kininogen; HPLC, highꢀperformance liquid chromatography; IC50, half maximal inhibitory
concentration; LHS, left hand side; PPK, plasma prekallikrein; RHS, rightꢀhand side; SAR, structure
activity relationship; SEM, scanning electron microscopy; TEM, transmission electron microscopy; ThT;
thioflavin T.
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Supporting Information: Methods for thrombin activity assay and aPTT test. Supporting Table S1
contains SAR analysis of SEN1269 analogs by LHS modifications. Three supporting figures (S1ꢀS3)
show the effect of TDIꢀ2760 on thrombin activity and aPTT, and the effect of SEN1269 on Aβ42
aggregation. This material is available free of charge on the ACS Publications website.
Acknowledgements: This work was supported by the Alzheimer’s Drug Discovery Foundation and
Robertson Therapeutic Development Fund for HA; NIH grant NS50537, the TriꢀInstitutional
Therapeutics Discovery Institute, Alzheimer’s Drug Discovery Foundation, Rudin Family Foundation,
and John A. Herrmann for SS. We thank The Rockefeller University’s High Throughput, Spectroscopy,
and Electron Microscopy Resource Centers for access to experimental equipment and technical support.
We thank ZuꢀLin Chen and members of Strickland lab for helpful discussion.
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
Author contributions: PKS performed the experiments, analyzed the results, and wrote the manuscript.
MK designed and conducted the chemical synthesis, analyzed the data, and wrote the manuscript. HBR
performed the experiments. GN performed the experiments. TY perform the experiments. MAF helped
design the experiments, and helped analyze the results. EHN helped analyze the data and wrote the
manuscript. SS helped analyze the results and wrote the manuscript. KA designed and performed the
chemical synthesis and wrote the manuscript. HJA designed and performed the experiments, analyzed the
results, and wrote the manuscript. All the authors edited and approved the final version of the manuscript.
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Table 1. Effect of known Aβ aggregation inhibitors on the Aβ42ꢀfibrinogen interaction. Structures and
other details of known Aβ aggregation inhibitors/βꢀsheet breakers used in this study. These compounds
were tested for their ability to inhibit the Aβ42ꢀfibrinogen interaction by AlphaLISA. The IC50 of these
compounds was determined and is presented with the structure of each compound. AlphaLISA shows that
Aβ aggregation inhibitors also have an inherent ability to block the Aβ42ꢀfibrinogen interaction; however,
the IC50 varies substantially among the inhibitors. All AlphaLISA assays were performed in triplicate, and
the 95% confidence interval (95% CI) used as a statistical parameter.
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Compound
(Structure)
Mol. Wt. AlphaLISA
95% CI
(ꢀM)
Name
(Da)
(IC50)
RSꢀ0406
294.31
>100 ꢁM
ꢀ
ꢀ
Tanshinone
SEN1576
294.34
408.43
>100 ꢁM
>100 ꢁM
ꢀ
Phenyl amino
acetamide
derivative
414.51
293.36
>100 ꢁM
49.8 ꢁM
ꢀ
Aminostyryl
benzofuran
derivative
33.2 – 74.8
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4,5ꢀDianilino
Pthalimide
(DAPH)
329.35
402.45
43.2 ꢁM
36.4 – 51.4
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Nitrophenyl
piperazinyl
derivative
39.5 ꢁM
25.9 – 60.2
Aminophenyl
piperazinyl
derivative
372.47
322.36
21.0 ꢁM
17.8 ꢁM
16.3 – 27.1
16.0 – 19.9
SEN1269
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Table 2. Analogs obtained by rightꢀhand side (RHS) modifications of the SEN1269 scaffold. These
analogs were tested for their efficacy in blocking the Aβ42ꢀfibrinogen interaction using AlphaLISA. IC50
was calculated and compared with that of SEN1269. TDIꢀ2760 (a phenyl analog) showed the lowest IC50
among all the compounds tested. All AlphaLISA assays were performed in triplicate, and the 95%
confidence interval (95% CI) used as a statistical parameter.
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IC50 95% CI
(ꢁM) (ꢁM)
IC50 95% CI
Entry
R
ID
Entry
R
ID
(ꢁM)
(ꢁM)
SEN
1269
16.0 –
17.8
TDIꢀ
3099
24.8ꢀ
34.2
1
7
29.1
19.9
TDIꢀ
2758
5.35 –
6.42
TDIꢀ
3071
60.7 –
78.7
2
3
8
9
69.0
43.5
7.71
TDIꢀ
3066
19ꢀ2 –
25.0
TDIꢀ
3078
39.1 –
48.3
32.4
TDIꢀ
3067
24.9 –
29.0
TDIꢀ
3277
7.74 –
12.7
4
5
6
10
11
9.9
6.2
34.1
TDIꢀ
3082
18.6 –
23.0
TDIꢀ
3275
3.16 –
12.0
28.3
1.36 –
1.67
TDIꢀ
2760
2.05
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