Discovery of a series of selective and cell permeable beta-secretase (BACE1) inhibitors by fragment linking with the assistance of STD-NMR

Article abstract of DOI:10.1016/j.bioorg.2019.103253

Two β-secreatase (BACE1) inhibitors from natural products (cinnamic acid and flavone) were linked to furnish potent, cell permeable BACE1 inhibitors with noncompetitive mode of inhibition, with the assistance of saturated transfer difference (STD)-NMR technique. Some of these conjugates also exhibited selective BACE1 inhibition over other aspartyl proteases such as BACE-2 and renin, as well as poor cytotoxicity. Taken together, conjugates 4 represent a new series of BACE inhibitors warrants further investigation for their potential in Alzheimier's disease therapy.

Full text of DOI:10.1016/j.bioorg.2019.103253

BioorganicChemistry92(2019)103253
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of a series of selective and cell permeable beta-secretase (BACE1)
inhibitors by fragment linking with the assistance of STD-NMR
Wei-Shuo Fanga,1, De-yang Suna, Shuang Yanga, Chen Chengb, Katrin Moschkec,d, Tianqi Lia,
⁎
Shanshan Suna, Stefan F. Lichtenthalerc,d, Jian Huangb, Yinghong Wanga,
a State Key Laboratory of Bioactive Substances and Functions of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union
Medical College (CAMS & PUMC), 2A Nan Wei Road, Beijing 100050, PR China
b College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, PR China
c German Center for Neurodegenerative Diseases (DZNE) and Munich Cluster for Systems Neurology (SyNergy), Technical University of Munich, Feodor-Lynen-Strasse 17,
81377 Munich, Germany
d Neuroproteomics, Klinikum rechts der Isar, Technical University of Munich, 81675 Munich, Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
Two β-secreatase (BACE1) inhibitors from natural products (cinnamic acid and flavone) were linked to furnish
potent, cell permeable BACE1 inhibitors with noncompetitive mode of inhibition, with the assistance of satu-
rated transfer difference (STD)-NMR technique. Some of these conjugates also exhibited selective BACE1 in-
hibition over other aspartyl proteases such as BACE-2 and renin, as well as poor cytotoxicity. Taken together,
conjugates 4 represent a new series of BACE inhibitors warrants further investigation for their potential in
Alzheimier’s disease therapy.
Beta-secretase (BACE1)
STD-NMR
Luteolin
P-hydroxy-cinnamic acid
Noncompetitive inhibition
1. Introduction
the brain. As BACE1 catalyzes the rate-limiting reaction in Aβ biogen-
esis, and BACE1 gene knockout mice lacking Aβ production are viable
Alzheimer's disease (AD) is a progressive neurodegenerative disease
which frequently occurs in the elderly people. It is pathologically
characterized by the presence of extracellular senile plaques consisting
of amyloid-beta (Aβ) deposits, and intracellular neurofibrillary tangles
(NFTs) consisting of hyperphosphorylated tau protein aggregates, and
the ultimate neuronal function impairment or cell death. The incidence
of sporadic AD is continuously growing as the aging population in-
creases, especially in the developed countries, thus making AD one of
the most serious diseases to elderly people [1].
and fertile [4], this protein is regarded as a promising therapeutic target
for AD. The expectation to halt or even prevent AD by targeting BACE1
and subsequent modulation on Aβ, makes it more attractive than some
other symptom-modifying targets, such as acetylcholinesterase (AchE)
and N-methyl D-aspartate (NMDA) receptor [5].
As such, BACE1 inhibitors have been actively pursued for more than
a decade, and the availability of several crystal structures for ligand-
BACE complex released since early 2000 s [6] has speeded up the dis-
covery and optimization of BACE1 inhibitors, especially those active-
site directed peptidomimetic inhibitors. Most of those peptide-based
inhibitors are competitive inhibitors which also suffer from unfavorable
pharmacokineics properties, particularly poor blood brain barrier (BBB)
permeability and low oral bioavailability, preventing them to be pro-
mising drug candidates [7,8].
In the most widely accepted β-amyloid cascade hypothesis for AD
pathogenesis, among many other mechanisms, a peptide consisting of
37–43 amino acids, Aβ, is proposed to aggregate into soluble oligomers
and fibrils which are toxic to neurons and cause neuronal death.
Another protein tau can also be a causative factor independently, pre-
sumably acting downstream of Aβ [2].
Consequently, many non-peptide BACE1 inhibitors were pursued,
some of which have entered clinical trials at different stages, e.g. MK-
8931 and AZD-3293 at phase III trials [9]. However, the diverse toxicity
found in several clinical trials became a serious problem and thus
Aβ is generated by the sequential endoproteolytic cleavage of
amyloid precursor protein (APP), a transmembrane protein expressed in
many tissues and organs, by β-secretase [3] (BACE1) and γ-secretases in
Abbreviations: AD, Alzheimer’s disease; BACE1, beta site amyloid precursor protein cleaving enzyme 1 or beta-secretase; FRET, fluorescence resonance energy
transfer; STD, Saturated Transfer Difference; NP, natural product
⁎ Corresponding author.
E-mail addresses: wfang@imm.ac.cn (W.-S. Fang), wyh@imm.ac.cn (Y. Wang).
1 Institute of Materia Medica, CAMS & PUMC
https://doi.org/10.1016/j.bioorg.2019.103253
Received 20 January 2019; Received in revised form 27 June 2019; Accepted 4 September 2019
Availableonline13September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

W.-S. Fang, et al.
BioorganicChemistry92(2019)103253
to novel non-competitive inhibitors.
The major hurdle for the rational improvement of non-competitive
inhibitors is the structure for ligand-BACE1 complex is not available, so
we sought the help from a NMR technique, saturation transfer differ-
ence (STD). STD-NMR has emerged as a powerful screening tool and a
straightforward way to study the binding epitopes of small molecules
[13,14].
1.1
Luteolin (1)[15], p-hydroxy-cinnamic acid (2)[16] and chromone
glycoside AV-4-1 (3)[17] are several BACE1 inhibitory NPs found by
our and other labs (Fig. 1). During our exploration of their binding
epitopes in complex with BACE1 by STD-NMR, we found 1 and 2 could
bind simultaneously to the BACE1 protein (Fig. 2), whereas 1 and 3 not.
This observation gave us the hint that the binding of 3 to BACE1 is
tolerated (although its activity is not only moderate, e.g., IC50 10−5 M),
whereas the linking of 1 and 2 as fragments at proper position
(northern/northwestern part of the flavone 1, based on the reduced but
retained BACE1 inhibitory activity of the same core but more complex
chromenone glycoside 3) and with an appropriate linker may result in
more potent and possible non-competitive BACE1 inhibitors.
(A) 1H NMR of 1 (0.5 mM) with BACE1 (10 μM); (B) STD-NMR of 1
(0.5 mM) with BACE1 (10 μM); (C) 1H NMR of 2 (2 mM) with BACE1
(10 μM), (D) STD-NMR of 2 (2 mM) with BACE1 (10 μM); (E) STD-NMR
of 1 (0.8 mM) and 2 (2 mM) with BACE1 (10 μM)
Fig. 1. Structures of BACE1 inhibitory natural products and their IC50 in-
hibitory BACE1 in references.
In this work, we designed the conjugates (Fig. 3) through ω-amino
acids and polyethyleneglycols of different length as linkers. Interest-
ingly, some potent and cell permeable inhibitors with poor cytotoxicity
and good enzyme selectivity were found, and their non-competitive
mode of inhibition confirmed.
delayed the progress of drug development [10,11]. The toxicity has
arisen from, at least for some of the drug candidates, the indiscriminate
blocking of almost all BACE1 functions by the active site directed in-
hibitors [3].
Unlike the competitive inhibitors, non-competitive inhibitors
binding to allosteric sites may selectively inhibit specific function of an
enzyme. It is interesting to note that most natural product (NP) derived
BACE1 inhibitors with known mechanisms exhibit non-competitive
mode of inhibition, while a small portion of such inhibitors exhibit the
mixed mode of inhibition [12]. Such NP-based BACE1 inhibitors with
weak to moderate activity are of particular interest during our pursuit
2. Results and discussion
To link two BACE1 binding segments, 1 and 2, we first examined the
STD effect of these two compounds. The effect is almost indis-
tinguishable for different positions on 2, suggesting the surrounding of
Fig. 2. 1H NMR and STD-NMR spectra of 1 and 2.
2

W.-S. Fang, et al.
BioorganicChemistry92(2019)103253
Fig. 3. The design of conjugates 4.
this very small molecule by the protein. In contrast, stronger STD effect
was observed for C-3 and C-6 hydrogens than other part of this mole-
cule in 1, implicating a shorter distance between these positions in the
inhibitor and BACE1 protein. We thus chose to tether 7-OH in the upper
part of 1 to the carboxyl group in 2 through the ω -amino acid linkers of
different length.
Conjugates 4a-d were measured for their BACE1 inhibitory activity.
Whereas the inhibition rate at 10 µM increased as the length of linker
became longer, the inhibition rate of the conjugate 4d with longest
linker (n = 5) decreased to 1.9%. Among these conjugates, 4c with 4-
aminobutyric acid linker is most active.
Although all of the tested conjugates were weakly active, we won-
dered whether inhibitory activity of most active conjugate 4c could be
improved through the incorporation of other linkers. In addition, their
poor water solubility prevented the IC50 measurement as those com-
pounds of 100 μM were not soluble in the assay buffer. Hence, we de-
signed and synthesized another series of conjugates by incorporating
the ethyl glycol to reduce the rigidity of the linker and increase the
water solubility, and also extend the length of linker to maximal four
ethyleneglycols to explore the optimal distance between these two
plausible binding sites occupied by 1 and 2.
The synthetic routes for conjugates 4a-d were outlined in schemes 1
and 2. One building block of the conjugate from 1, 3′,4′-dimethox-
yethyloxymethyl (MEM) luteolin (9), was prepared by a modified
method of literature [18] (Scheme 1). The benzyl protected trihydrox-
ybenzoacetophenone 4 and MEM protected dihydroxybenzaldehyde 5
were condensed to chalcone 6, which was then cyclized, oxidized and
deprotected to furnish 9.
The other building block from 2, 4-MEM-O-cinnamic acid (10), was
synthesized over three steps. By the coupling of 9 with different ω
-amino acid methyl esters, N-amido ω -amino acid methyl esters 12a-d
were furnished. Intermediates 12a-d were then hydrolyzed to remove
methyl ester without breaking the amide bond, coupled with 9 and
deprotected by trimethylsilyl iodide (TMSI) to remove MEM protective
group, affording desired conjugates 4a-d (Scheme 2).
The diphenylmethylidene was chosen as the protective group for
luteolin in 14, which was treated with different glycol tosylate to in-
troduce glycol into 7-position furnishing 15. The terminal hydroxyl
group in 16 was esterified with 4′’-acetyl cinnamic acid (17), and then
deprotection under acidic conditions to afford desired conjugates 4e-h
Scheme 1. Synthesis of intermediate 9. Reagents and conditions: a. 20% KOH aq. ethanol, room temperature, 54%; b. NaOAc, H2O, ethanol, reflux, 46%; c. I2,
pyridine, 100 °C, 69%; d. H2, Pd/C, EtOAc, room temperature, 85%.
Scheme 2. Synthesis of conjugates 4a-d. Reagents and conditions: a. EtOH, conc.H2SO4, 95%. b. MEMCl, DIEA, DCM, rt, 91%. c. LiOH, EtOH, THF, H2O, 95%. d. (i)
SOCl2, MeOH, reflux, (ii) 1-(3-DiMethylaMinopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), dimethylaminopyridine (DMAP), Et3N, CH2Cl2, r.t., 56%-83%. e.
LiOH, EtOH, tetrahydrofuran (THF), H2O, 84%-98%. f. 9, EDCI, DMAP, Et3N, CH2Cl2, 40 °C, 30%-57%. g. Iodotrimethylsilane (TMSI), CH3CN, −20 °C, 67%-88%.
3

W.-S. Fang, et al.
BioorganicChemistry92(2019)103253
Scheme 3. Synthesis of conjugates 4e-h. Reagents and conditions: a. dichlorodiphenylmethane, diphenyl ether, 165 °C, 70%. b. TsCl, Et3N, DCM, 40 °C, 21–23%. c.
K2CO3, CH3CN, reflux, 46%-79%. d. 17, EDCI, DMAP, Et3N, THF, 40 °C, 34%-60%. e. aq. 38% HCl in AcOH (v/v 1:15), r.t., 26–80%.
(Scheme 3).
The poly(ethyleneglycol) linked conjugates 4e-h were first tested
different profile: the increase in Aβ levels was very moderate (< 25%)
for Aβ1-38 and Aβ1-42 and not observed for Aβ1-40 at lower com-
pound concentration and inhibition of Aβ was already seen at 10 µM
and was enhanced at higher concentrations. In parallel to their BACE1
inhibitory activity in enzyme assay, 4e was the most potent and 4c the
least potent in the inhibition of the most toxic species Aβ-42 production
(after normalization to protein). The reference compound 3 showed no
cellular activity at all tested concentrations, 4e had much obvious in-
fluence than luteolin (1) (see Fig. 4).
for their BACE1 inhibitory effects at 10 μM. It is found that the in-
hibitory activity of conjugates 4e-h decreased when the length of linker
increases. Moreover, 4e bearing the single ethylene glycol linker
showed most potent inhibitory activity among all tested conjugates
with an IC50 of 0.25 µM, which was better than both fragments 1 and 2.
To assess the cellular BACE1 inhibitory activity of the conjugates,
4b-d and 4e-h were incubated in APP overexpressing HEK293 cells
comparing with compound 3 and the two fragments 1 and 2 in two
groups and their inhibition on the three main Aβ species Aβ-38, Aβ-40
and Aβ-42, the main amyloidogenic peptides produced by BACE1
cleavage of APP peptide, was measured by an ELISA-like immunoassay
method. Aβ levels were normalized to the protein concentration in the
lysate in order to rule out differences in total cell number per well of the
culture plate. Additionally, the normalized Aβ level was set to 100% for
the DMSO-treated control. At the concentrations of 25 and 50 µM all
conjugates with amino acid linkers, except 4c, dose-dependently re-
duced Aβ levels, although only moderate to low activity was observed
(ca. 55% Aβ remaining at 50 µM). Conjugate 4c showed nearly no
ability to reduce Aβ levels. In another series of conjugates with poly-
glycol linkers, the most potent enzyme inhibitor 4e showed a slightly
Luteolin (1), one segment of the conjugate, is a non-competitive
BACE1 inhibitor. We are interested to know if this fragment of the
conjugate will keep or change the mode of inhibition. The Dixon plot
was used to determine the kinetics of 4e inhibiting BACE1. As shown in
Fig. S3, 4e is still found as a non-competitive inhibitor with a Ki value
of 0.35 µM.
In addition, we hope to understand the interaction of the conjugate
with BACE1, to validate our fragment based strategy and assist further
structural optimization. However, all conjugates 4a-h were not com-
pletely dissolved in the buffer we used to measure STD-NMR, even in
the presence of 30% DMSO-d6 (v/v). Thus we tried to improve the
water solubility of the conjugate through the introduction of mono- and
di-phosphoryl groups into 4e, the most active compound in the series.
Abeta42, C3 control
B
Abeta 42
A
200
150
100
50
120
100
80
60
40
0
20
0.1
2
1
5
10
25
50
-50
0
Inhibitor concentraꢀon (µM
4b 4c 4d 4e 4f 4g 4h
DMSO C3 (2
µM)
1
3
Fig. 4. BACE1 inhibitory activity in cells. (A) The normalized Aβ1-42 level in APP overexpressing HEK293 cells after the treatment of 1, 2, 3, 4b ~ 4 h, compared
with DMSO (100%). (B) Aβ1-42 level for DMSO and the known inhibitor C3 [20].
4

W.-S. Fang, et al.
BioorganicChemistry92(2019)103253
Table 1
Enzymatic inhibition activity of conjugates 4a-4j.
Enzyme Inhibition
Compounds
Inhibition rate at 10 μM
IC50 (μM)
4a
NA
–
–
–
–
4b
10.9% 3.1%
14.7% 5.3%
1.9% 2.8%
92.0% 1.5%
73.0% 5.0%
20.1% 3.1%
NA
4c
4d
4e
0.25
2.22
–
0.16
4f
0.86
Fig. 5. The structures of phosphates 4i and 4j.
4 g
4 h
–
4i
101.7% 3.7%
86.3% 14.5%
62.5% 2.5%
or 66.7% 7.25%
1.31
2.34
1.10
0.28
0.28
1.34
4j
The 4′’-O-phosphate (4i) and 3′,4′-di-phosphate (4j) were prepared
through a common intermediate 18e (4′’-Ac-3′,4′-diphenylmethylene
derivative of 4e) by phosphorylation and deprotection at proper posi-
tion (Fig. 5, Schemes S1, S2).
luteolin (1)
p-hydroxy-cinnamic acid (2)
AV-4-1 (3)
~100
39.0*
The BACE1 inhibitory activities of 4i and 4i were retained, ex-
hibiting IC50 values of 2.13 and 3.93 μM respectively, while enhancing
the solubility in the buffer enabling STD-NMR experiments (Table 1).
The solubility of 4j was better than the compound 4i, and its BACE1
inhibitory activity only a little weaker than 4e. Thus, 4j was chosen to
record its STD-NMR in a pH 4.5 buffer (CD3COOD/CD3COONa), al-
though 20% DMSO-d6 (v/v) as additives are still needed to prepare a
clear solution. We measured the enzymatic inhibition of our compounds
in the presence and absence of DMSO in above buffer, and found their
inhibitions were very similar. Such an observation suggested that the
enzymatic activity was well preserved even in the presence of 20%
DMSO.
* Data from Lv L. et al. Planta Med. 2008, 74: 540-5.
over other structurally related aspartyl proteases has been assessed in
this study. Although the possibility to interact with proteins other than
BACE1 structurally related ones cannot be fully excluded, it is argued
that our compounds, after attachment of a comparably large segment
(cinnamoyl acid + linker, MW 110–310) to another segment/one of the
starting compounds (luteolin, MW ~ 370) may change the way of lu-
teolin interacting with proteins. Nonetheless, the selectivity to a wider
spectrum of proteins could be assessed in further optimization of this
series of conjugates.
In conclusion, we discovered a series of conjugates as cell-perme-
able, low cytotoxic, highly selective non-competitive BACE1 inhibitors
by linking two NP fragments with the assistance of STD-NMR. Such
non-competitive inhibitors may provide an opportunity to decrease APP
processing by BACE1 inhibition, whereas sparing other BACE1 func-
tions, thus providing promising drug candidates.
The experimental STD spectra were shown in Fig. 6 and data in
Table 2. The largest STD effects were observed for the two benzene
rings moiety at the end of the molecule, demonstrated by the highest
intensity enhancement of H-2′, H-3′’, H-5′’ (assigned as 100% intensity).
The STD effects of carbon-carbon double bond (H-11, 12) and chro-
mone core structure (H-3, 6, 8) were a little weaker than the terminal of
two benzene rings, and those of the protons (H-9, 10) on the glycol
linker not observed. Accordingly, it could be postulated that both
terminal in the conjugate are closer to the protein (very possibly in two
sub-pockets), and the 3′ or 4′-phenol and 4′’-phosphate moiety (4″-
phenol and 3′ or 4′-phosphate moiety) on both terminal phenyls may
get involved in the interaction with BACE1 directly.
3. Experimentals
3.1. In vitro BACE1 enzyme assay
The BACE1 FRET assay kit was purchased from the PanVera Co.
(Invitrogen, USA). The assay was carried out according to the supplied
manual with modifications. Briefly, assays were performed in triplicate
in 96-well black plates with a mixture of 10 μL of assay buffer (50 mM
sodium acetate, pH 4.5), 10 μL of BACE1 (1.0 U/ml), 10 μL of the sub-
strate (750 nM, Rh-EVNLDAEFK- Quencherin 50 mM, ammonium bi-
carbonate), and 10 μL of compound dissolved in 10% DMSO. The
fluorescence intensity was measured with a TECAN infinite 200 mi-
croplate reader for 60 min at 25 °C in the dark. The mixture was irra-
diated at 544 nm and the emission intensity recorded at 590 nm. The
percent inhibition (%) was obtained by the following equation:
Inhibition % = (1 − SS/SC) × 100%, where SC is the slope of fluores-
cence change of the control (enzyme, buffer, and substrate) during
60 min, and SS is the slope of fluorescence change of the tested samples
(enzyme, sample solution, and substrate) during 60 min of measure-
ment. IC50 values were calculated from the nonlinear curve fitting of
percentage inhibition against inhibitor concentration using Prism 3.0
software.
As BACE1 is a member of aspartyl protease family, its selective in-
hibition over other closely related proteases is required to avoid pos-
sible severe side effects. Thus, we measured the inhibition of 4e on
BACE-2 and renin, and found 27.6% and 25.9% inhibition rates at a
concentration of 10 μM respectively, corresponding to IC50 over 10 μM
(vs. 0.25 μM on BACE1).
It is also crucial for the BACE1 inhibitors to exert their activities at
sub-toxic concentration, so their cytotoxicity in HEK293 cells were also
assessed by MTT assays. The IC50 for all of the tested compounds are
above 50 μM, among which 4a, b, e, g, i, j and 1, 3 are the least cy-
totoxic (IC50 > 100 μM).
BACE1 has been recognized as a promising therapeutic target for
AD, although the safety of BACE inhibition was in dispute during the
drug development over last decade [19]. Thus, BACE1 inhibitor with
reasonable potency and with the selectivity on APP processing over
other substrates have been actively pursued to avoid unendurable
toxicity. Non-competitive inhibitors, which are able to enhance the
selectivity and reach the reasonable potency by allosteric modulation of
enzyme activity, should be given more attention, although in reality
they are still underexplored.
3.2. Saturation transfer difference (STD) -NMR
As luteolin (1) are known to interact with many other enzymes, such
as carbonic anhydrase [21], tyrosinase [22], α-glucosidase [23], etc.,
our BACE1 inhibitors, either defined as 7-modified analogs or luteolin-
based conjugates, may arise the concern to interact with those proteins.
However, it is worth pointing out that the selective inhibition on BACE1
The sample solutions consisting of 10 μM BACE1 protein and 2 mM
ligand, 50 mM acetate buffer, 150 mM NaCl, and 20% deuterated
DMSO in D2O at pH 4.0 were prepared. All STD-NMR experiments were
performed at 298 K on a Bruker Avance III 500 MHz spectrometer
equipped with a 1.7-mm NMR microprobe. 1H-spectrum was obtained
5

W.-S. Fang, et al.
BioorganicChemistry92(2019)103253
Fig. 6. NMR and STD-NMR spectra of 4j. (A) 1H NMR of 4j (2 mM) with BACE1 (10 μM); (B) STD-NMR of 4j (2 mM) with BACE1 (10 μM).
Funding
This work was supported by the National Natural Science
Table 2
Signal intensity enhancement in STD of 4j.
Proton
Intensity enhancement (%)
Proton
Intensity enhancement (%)
Foundation of China (81273406 to W.-S. F., and 31371331 to J. H.),
the National Major Science and Technology Projects of China (Grant
Nos. 2018ZX09711001-002-004 to Y.W., 2018ZX09711001-001-001,
2018ZX09711001-001-003 to W.-S. F.), the CMAS Innovation Fund for
Medical Sciences (Grant No. 2016-I2M-1-009 to Y.W.) and the Deutsche
Forschungsgemeinschaft (German Research Foundation) within the
framework of the Munich Cluster for Systems Neurology (EXC 2145
SyNergy to S.F.L).
2′
100%
90%
60%
74%
100%
0%
10
0%
5′, 6′
11
53%
90%
58%
100%
3
6
8
9
12
2″, 6″
3″, 5″
first as reference spectrum, and subsequently, an STD spectrum was
obtained. The protein was saturated on-resonance at 0 ppm and off-
resonance at 29 ppm with a cascade of 40 selective Gaussian shaped
pulses, of 50 ms duration with a 4 μs delay between each pulse in all
STD NMR experiments. The total duration of the saturation time was set
to 2 s. A total of 2048 scans per STD NMR experiment were acquired
and a 3-9-19 pusle sequence was used to water suppression. A spin-lock
filter with 9615.38 kHz strength and duration of 30 ms was applied to
suppress protein background.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103253.
References
[1] P. Scheltens, K. Blennow, M.M. Breteler, S.B. De, G.B. Frisoni, S. Salloway,
W.M. Van der Flier, Alzheimer’s disease, Lancet 388 (2016) 505–517, https://doi.
org/10.1016/S0140-6736(15)01124-1.
Other experimentals including the synthesis, enzyme kinetic assay,
cellular activity and cytotoxicity please refer to supplemental in-
formation.
[2] E. Karran, M. Mercken, S.B. De, The amyloid cascade hypothesis for alzheimer’s
disease: an appraisal for the development of therapeutics, Nat. Rev. Drug. Disc. 10
(2011) 698–712.
[3] R. Vassar, P.H. Kuhn, C. Haass, M.E. Kennedy, L. Rajendran, P.C. Wong, Function,
therapeutic potential and cell biology of BACE proteases: current status and future
prospects, J. Neurochem. 130 (2014) 4–28.
Acknowledgements
[4] Y. Luo, B. Bolon, S. Kahn, B.D. Bennett, S. Babu-Khan, P. Denis, W. Fan, H. Kha,
J. Zhang, Y. Gong, L. Martin, J.C. Louis, Q. Yan, W.G. Richards, M. Citron,
R. Vassar, Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal
phenotype and abolished beta-amyloid generation, Nat. Neurosci. 4 (2001)
231–232.
The authors gratefully acknowledge the facilities and assistance
provided by Dr. Lu Shan and Mr. Juan Lv of Bruker Corporation in
Germany, and Mr. Yu-jun He for the technical assistance in enzyme
assays.
[5] T. Silva, J. Reis, J. Teixeira, F. Borges, Alzheimer's disease, enzyme targets and drug
discovery struggles: from natural products to drug prototypes, Ageing. Res. Rev. 15
(2014) 116–145.
[6] L. Hong, G. Koelsch, X. Lin, S. Wu, S. Terzyan, A.K. Ghosh, Structure of the protease
domain of memapsin 2 (β-secretase) complexed with inhibitor, Science 290 (2000)
150–153.
Declaration of Competing Interest
[7] Y. Hamada, Y. Kiso, Advances in the identification of β-secretase inhibitors, Exp.
Opin. Drug. Disc. 8 (2013) 709–731.
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of this article.
[8] J. Yuan, S. Venkatraman, Y. Zheng, B.W. Mckeever, L.W. Dillard, S.B. Singh,
Structure-based design of β-site app cleaving enzyme 1 (BACE1) inhibitors for the
6

W.-S. Fang, et al.
BioorganicChemistry92(2019)103253
treatment of Alzheimer's disease, J. Med. Chem. 56 (2013) 4156–4180.
W.S. Fang, BACE1 (beta -secretase) inhibitory chromone glycosides from Aloe vera
[9] R. Yan, Stepping closer to treating Alzheimer’s disease patients with BACE1 in-
hibitor drugs, Transl. Neurodegeneration 5 (2016) 13–24.
and Aloe nobilis, Planta. Med. 74 (2008) 540–545.
[18] S. Mavel, B.S. Dikic, P. Emond, I. Greguric, A.G. de Gracia, F. Mattner, Synthesis
and biological evaluation of a series of flavone derivatives as potential radioligands
for imaging the multidrug resistance-associated protein 1 (ABCC1/MRP1), Bioorg.
Med. Chem. 14 (2006) 1599–1607.
[10] H. Wang, R. Li, Y. Shen, β-Secretase: its biology as a therapeutic target in diseases,
Trends. Pharmacol. Sci. 34 (2013) 215–225.
[11] G. Evin, A. Barakat, Critical analysis of the use of β-site amyloid precursor protein-
cleaving enzyme 1 inhibitors in the treatment of Alzheimer’s Disease, Degener.
Neurol. Neuromuscul. Dis. 4 (2014) 1–10.
[19] K.W. Menting, J.A. Claassen, β-Secretase inhibitor: a promising novel therapeutic
drug in Alzheimer's disease, Front. Aging. Neurosci. 6 (2014) 165.
[20] S.J. Stachel, C.A. Coburn, T.G. Steele, K.G. Jones, E.F. Loutzenhiser, A.R. Gregro,
Structure-based design of potent and selective cell-permeable inhibitors of human
beta-secretase (BACE-1), J. Med. Chem. 47 (2004) 6447–6450.
[12] P. Williams, A. Sorribas, M.J. Howes, Natural products as a source of Alzheimer's
drug leads, Nat. Prod. Rep. 28 (2010) 48–77.
[13] N.R. Krishna, V. Jayalakshmi, Quantitative analysis of STD-NMR spectra of re-
versibly forming ligand-receptor complexes, Top. Curr. Chem. 273 (2008) 15–54.
[14] A. Viegas, J. Manso, M.C. Corvo, M.M. Marques, E.J. Cabrita, Binding of ibuprofen,
ketorolac, and diclofenac to cox-1 and cox-2 studied by saturation transfer differ-
ence NMR, J. Med. Chem. 54 (2011) 8555–8562.
[21] L. Derya Ekinci, Deniz Ekinci Karagoz, M. Senturk, C.T. Supuran, Carbonic anhy-
drase inhibitors: in vitro inhibition of α isoforms (hCA I, hCA II, bCA III, hCA IV) by
flavonoids. J. Enzyme. Inhib, Med. Chem. 28 (2013) 283–288.
[22] L. Zhang, X. Zhao, G.J. Tao, J. Chen, Z.P. Zheng, Investigating the inhibitory ac-
tivity and mechanism differences between norartocarpetin and luteolin for tyr-
osinase: a combinatory kinetic study and computational simulation analysis, Food.
Chem. 223 (2017) 40–48.
[15] S.H. Choi, J.M. Hur, E.J. Yang, M. Jun, H.J. Park, K.B. Lee, Beta-secretase (BACE1)
inhibitors from Perilla frutescens var. acuta, Arch. Pharma. Res. 31 (2008)
183–187.
[16] K. Youn, M. Jun, Inhibitory effects of key compounds isolated from Corni fructus on
BACE1 activity, Phytother. Res. 26 (2012) 1714–1718.
[23] J.K. Yan, G.W. Zhang, J.H. Pan, Y.J. Wang, α-Glucosidase inhibition by luteolin:
kinetics, interaction and molecular docking, Int. J. Biol. Macromol. 64 (2014)
213–223.
[17] L. Lv, Q.Y. Yang, Y. Zhao, C.S. Yao, Y. Sun, E.J. Yang, K.S. Song, J.I. Mook,
7