Screening Platform Based on Inductively Coupled Plasma Mass Spectrometry for β-Site Amyloid Protein Cleaving Enzyme 1 (BACE1) Inhibitors

Article abstract of DOI:10.1021/acschemneuro.0c00816

β-Site amyloid protein cleaving enzyme 1 (BACE1) is a promising therapeutic target for developing inhibitors to alleviate Alzheimer's disease (AD). Herein, we established an inductively coupled plasma mass spectrometry (ICPMS)-based inhibitor screening platform. A biotin-labeled lanthanide-coded peptide probe (LCPP; biotin-PEG2-EVNLDAEC-DOTA-Ln) was designed to determine the activity of BACE1 and evaluate the degree of inhibition of inhibitors. The platform was first validated with two commercially available inhibitors (BSI I and BSI IV) in terms of IC50 values and then applied to two newly designed inhibitors (inhibitors II and III) based on the crystal structure of BACE1 interacting with inhibitor I, and each of them contained an acylguanidine core structure. We found that their inhibition effects were improved as evaluated by the sensitive and accurate LCPP-ICPMS platform, demonstrating its ability for new drug screening.

Full text of DOI:10.1021/acschemneuro.0c00816

pubs.acs.org/chemneuro
Letter
Screening Platform Based on Inductively Coupled Plasma Mass
Spectrometry for β‑Site Amyloid Protein Cleaving Enzyme 1 (BACE1)
Inhibitors
Xin Jin, Limin Yang,* Xiaowen Yan,* and Qiuquan Wang*
Cite This: ACS Chem. Neurosci. 2021, 12, 1093−1099
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: β-Site amyloid protein cleaving enzyme 1 (BACE1)
is a promising therapeutic target for developing inhibitors to
alleviate Alzheimer’s disease (AD). Herein, we established an
inductively coupled plasma mass spectrometry (ICPMS)-based
inhibitor screening platform. A biotin-labeled lanthanide-coded
peptide probe (LCPP; biotin-PEG2-EVNLDAEC-DOTA-Ln) was
designed to determine the activity of BACE1 and evaluate the
degree of inhibition of inhibitors. The platform was first validated
with two commercially available inhibitors (BSI I and BSI IV) in
terms of IC50 values and then applied to two newly designed
inhibitors (inhibitors II and III) based on the crystal structure of
BACE1 interacting with inhibitor I, and each of them contained an
acylguanidine core structure. We found that their inhibition effects were improved as evaluated by the sensitive and accurate LCPP−
ICPMS platform, demonstrating its ability for new drug screening.
KEYWORDS: Inductively coupled plasma mass spectrometry (ICPMS), BACE1, BACE1 inhibitor, lanthanide, element-tag
ACE1, also known as β-site amyloid precursor protein
B(APP)-cleaving enzyme 1 or β-secretase, is the rate-
limiting enzyme that produces toxic peptides Aβ40 and Aβ42,
according to the amyloid cascade hypothesis.1,2 Several studies
observed a lower production of Aβ with minor effects on
normal physiological processes of mice whose BACE1 gene
was knocked out.3,4 Recent studies revealed that BACE1 levels
increased in the cortices and plasma of Alzheimer’s disease
(AD) patients.5,6 Therefore, BACE1 is considered as a
promising therapeutic target for AD treatment, and a growing
number of BACE1 inhibitors with different structures have
been constantly reported.7−12
The development of a simple, sensitive, and reliable method
to detect BACE1 and screen its inhibitors is crucial for clinical
diagnosis and treatment of AD. Until now, several methods
such as enzyme-linked immunosorbent assay, surface plasmon
resonance (SPR), and fluorescence resonance energy transfer
(FRET) have been developed for this purpose.13−16 Among
them, FRET is most frequently used one mainly due to its
sensitivity and high-throughput capability. However, conven-
tional FRET assay presents shortcomings such as narrow
Stokes shifts and spectral overlap of near-UV excitation
wavelengths of some fluorophores with the absorption peaks
of many small background molecules containing aromatic
rings, so the choice of suitable donor/acceptor pairs has to be
varied from study to study for their applications.16−19
Furthermore, the peptide substrates should be carefully
optimized to achieve high FRET efficiency, avoiding the
frequently encountered steric hindrance and low solubility
issues owing to the presence of hydrophobic fluorophores at
each end of the peptide substrates.16,18 Although use of
quantum dots and layered tungsten disulfides has been
attempted to solve these problems, they could not be used
in acid pH medium in which BACE1 exhibits higher
activity.14,16 More seriously, the synthetic and natural
inhibitors containing aromatic rings could absorb or quench
the light signals, leading to false-positive or false-negative
results in some cases.20,21
Inductively coupled plasma mass spectrometry (ICPMS) is
an ideal technology capable of determining multiple metallic
elements simultaneously at ppt level with high accuracy and
broad dynamic range.22 In ICP, a hard ion source, molecules
are completely broken down into atomic ions; the signal
response is therefore independent of species and matrix.23,24
Because of these unique features, ICPMS has been applied to
the quantification of proteins, DNA, and bacteria as well as
Received: December 24, 2020
Accepted: March 23, 2021
Published: March 25, 2021
https://doi.org/10.1021/acschemneuro.0c00816
© 2021 American Chemical Society
ACS Chem. Neurosci. 2021, 12, 1093−1099
1093

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Letter
viruses with the aid of diverse element-tagging strategies.25−33
On the other hand, metal-tagged substrates have also been
applied to measure protease activity, demonstrating the
advantage of ICPMS compared with the reported optical
methods for enzyme assay.34,35 We previously reported an
ICPMS-based multiplex protease assay through Ln-coded
protease-specific peptide-nanoparticle probes, during which
only a centrifugation step was needed to separate the cleaved
probes from the uncleaved ones for ICPMS analysis.35
Herein, we established an ICPMS-based inhibitor screening
platform enabling not only BACE1 activity assay but also
inhibitor screening through a Ln-coded peptide probe (LCPP,
Scheme 1A). To start with, we chose EVNLDAE, the Swedish
1B). When a BACE1 inhibitor is present, BACE1 activity is
blocked, resulting in reduced LCPP cleavage and therefore the
corresponding Ln ICPMS signal decrease (Scheme 1B). The
decrease in the ICPMS Ln-signal intensity is correlated with
the degree of inhibition of the inhibitor, meaning that stronger
inhibitors cause lower Ln-signal on ICPMS. In this way, we are
able not only to determine the activity of BACE1 on the
LCPP−ICPMS platform but also to assess the inhibition effect
of its potential inhibitors, thus effectively screening BACE1
inhibitors.
In order to confirm that BACE1 could hydrolyze LCPP, in a
preliminary experiment we incubated 40 nM BACE1 and 10
μM LCPP in sodium acetate buffer and monitored the cleaved
products by HPLC-ESI-MS. As shown in Figure 1, LCPP was
Scheme 1. (A) Structure of Ln-Coded Peptide Probe
(LCPP) and (B) Schematic Illustration of ICPMS-Based
BACE1 Assay and Inhibitor Screening Using LCPP
Figure 1. Selected ion chromatograms from HPLC-ESI-MS
monitoring the cleaved products of LCPP. LCPP (10 μM, red
peak) and BACE1 (40 nM) were first incubated in sodium acetate
buffer (20 mM, pH 4.5) at 37 °C for 120 min; the cleaved products
were then monitored by HPLC-ESI-MS. Peaks 1 and 2 represent
DAEC-MMA-DOTA-Eu (blue peak) and biotin-EVNL (black peak),
respectively.
precisely hydrolyzed by BACE1 at the cutting site between L
and D. The two cleaved products of DAEC-MMA-DOTA-Eu
(peak 1 at 22.0 min) and biotin-EVNL (peak 2 at 27.5 min)
were confirmed by ESI-MS (Figure S2), demonstrating the
feasibility of our designed probe for BACE1 cleavage.
mutated sequence of APP that can be specifically recognized
by BACE1 as the substrate.36 It was further chemically
modified to add cysteine (C) to glutamic acid (E) on the C-
terminal end, resulting in EVNLDAEC. Afterward, a biotin
group was conjugated to the amino group on the N-terminal
end using biotin-PEG2-NHS, the hydrophilic PEG2 linker of
which may improve the solubility of the probe in aqueous
solutions. In order to read out element signal in ICPMS, we
conjugated lanthanide-loaded 1,4,7,10-tetraazacyclododecane-
1,4,7-tris(acetic acid)-10-maleimidoethylacetamide (MMA-
DOTA-Ln) to biotin-PEG2-EVNLDAEC on the cysteine
residue based on a Michael addition reaction to obtain
LCPP (Scheme S1). Detailed procedures can be found in
Methods section and ESI-MS spectra for the synthesized
biotin-peptide-MMA-DOTA-Ln (Ln = Eu, La, Pr, Nd) LCPP
probes are shown in Figure S1 in the Supporting Information.
When BACE1 meets LCPP, it cleaves LCPP specifically at
the peptide bond between leucine (L) and aspartic acid (D),
resulting in biotin-EVNL and DAEC-MMA-DOTA-Ln
(Scheme 1A). The resulting biotin-EVNL and the remaining
uncleaved LCPP can be pulled down using streptavidin-
functionalized magnetic beads because of the strong
interaction between biotin and streptavidin, while the
DAEC-MMA-DOTA-Ln in the supernatant can be subjected
to ICPMS quantification via the determination of Ln (Scheme
Next, we optimized the reaction conditions to improve the
probe performance. The results obtained from the experiments
on time-dependent cleavage of LCPP by BACE1 indicated that
the ICPMS signal of BACE1-cleaved DAEC-MMA-DOTA-Eu
increased along with the increase in the incubation time from 0
to 60 min, and then gradually reached a plateau after 60 min
(Figure S3A). We therefore chose 60 min as the incubation
time for the following experiments. The pH value of the
reaction buffer has a significant influence on BACE1 activity.
BACE1 exerts its enzymatic activity through a general acid−
base mechanism, in which Asp228 acts as a base to activate a
bridging catalytic water, while Asp32 acts as an acid to
protonate the substrate carbonyl group.37−39 It was reported
that BACE1 cleavage occurs in cellular acid compartments, for
example, endosomal or trans-Golgi network,40 so we chose pH
values varying from 3.5 to 6.0 to study the pH-dependent
BACE1 activity. As shown in Figure S3B, the maximum 153Eu
signal was obtained at pH 4.5, which is consistent with the
results reported in the literature.41,42 Previous simulation
studies revealed a detailed mechanism of the pH-regulated
enzymatic activity. When the catalytic dyad is monoprotonated
at pH 4.5, a binding-competent state is highly occupied, which
is conducive to substrate binding.43 We therefore chose 4.5 as
the optimal pH in the subsequent experiments.
1094
https://doi.org/10.1021/acschemneuro.0c00816
ACS Chem. Neurosci. 2021, 12, 1093−1099

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Letter
To further confirm the specificity of LCPP toward BACE1, 1
μM probe was incubated with 20 nM pepsin, trypsin, papain,
pectinase, bovine serum albumin (BSA), and BACE1. As
depicted in Figure 2A, significant 153Eu signal was determined
Figure 2. (A) Specificity of LCPP toward BACE1. LCPP was
incubated with 20 nM pepsin, trypsin, papain, pectinase, BSA, or
BACE1. (B) Linear relationship between 153Eu signals and the
concentration of BACE1. Experiments were performed in sodium
acetate buffer (20 mM, pH 4.5) containing 1 μM LCPP (n = 3).
only in the presence of BACE1, while other enzymes and
proteins produced negligible signals under the same reaction
conditions, demonstrating the high specificity of LCPP toward
BACE1.
Figure 3. Sigmoidal dose response curves of BACE1 activity versus
logarithmic concentration of the compositionally and structurally
different BSI I (A), BSI IV (B), and donepezil (C). Experiments were
performed in sodium acetate buffer (20 mM, pH 4.5) containing 1
μM LCPP and 20 nM BACE1.
After the LCPP working conditions were optimized, we
applied the probe for BACE1 assay. As shown in Figure 2B,
with increase of the BACE1 concentration from 4 to 40 nM, a
linear increase of 153Eu signal intensity from the cleaved probe
was detected by ICPMS. The limit of detection (LOD, 3σ) was
calculated to be 0.5 nM BACE1, which is much lower than the
results obtained using quantum dot−gold nanoparticle
assembly (0.15 μM) and SPR (10 nM) reported in the
literature.14,16 In addition, the LOD obtained from the FITC-
labeled probe that we synthesized (Figure S4A) was found to
be 22 nM BACE1. The possible reason for the lower sensitivity
obtained from the FITC-labeled probe is the lower
fluorescence emission of FITC (Figure 4B) at pH 4.5, the
optimal pH for BACE1 (Figure S3B).
In order to validate that this probe can be applied to screen
BACE1 inhibitors, two commercially available BACE1
inhibitors were selected to evaluate their IC50 values. BSI I is
a statine-based peptide mimetic inhibitor with an IC50 value of
30 nM, while BSI IV is a cell-permeable isophthalamide
compound containing a hydroxyethylamine moiety with an
IC50 value of 29 nM.7,13 After BACE1 was first treated with
different concentrations of BSI I and BSI IV for 30 min, the
IC50 values were measured on the established LCPP−ICPMS
platform and determine to be 32 3 nM for BSI I (Figure 3A,
n = 3) and 37 2 nM for BSI IV (Figure 3B, n = 3). These
results demonstrated the ability of LCPP−ICPMS to screen
BACE1 inhibitors. In addition to the well-known BACE1
inhibitors BSI I and BSI IV, we also chose donepezil, which is
an acetylcholinesterase inhibitor, to further validate the
LCPP−ICPMS screening platform. No inhibitory effect was
observed even when the concentration of donepezil was up to
1 mM (Figures 3C). This is a correct result because donepezil
is an inhibitor of acetylcholinesterase but not of BACE1, being
consistent with results accessed using unlabeled substrates by
CE-MS and MALDI-MS.44,45 However, an inhibition effect of
donepezil in sub-micromolar range toward BACE1 using the
FRET substrate was reported.20,46 These conflicting results
might be due to the quenching of fluorophore by donepezil in
a concentration-dependent manner.44−47 Conclusively, our
study confirmed that accurate result could be obtained on the
established LCPP−ICPMS platform.
Furthermore, on the basis of the crystal structure of BACE1
complexed with acylguanidine-based inhibitor I (IC50 = 3.7
μM,48 Figure 4A, http://www1.rcsb.org/structure/2QU2), we
designed two new inhibitors that are expected to have higher
inhibitory ability toward BACE1. The acylguanidine moiety of
inhibitor I forms four key hydrogen-bonding interactions with
the catalytic aspartic acids, Asp32 and Asp228. The two aryl
groups (P1, P2′) extend into the S1 and S2′ pockets,
respectively, and the para position of the P1 phenyl group
projects directly toward the unoccupied S3 pocket, providing
an opportunity to add substituents to the P1 phenyl that may
extend into the S3 pocket; therefore additional interactions will
increase the binding affinity. On the other hand, the S2′ pocket
provides access to more polar and charged groups, thus
offering the potential to form additional hydrogen bonds
directly with BACE1. Moreover, one of the nitrogen groups of
the acylguanidine moiety faces away from the catalytic residues
toward the S1′ pocket; substitution on this nitrogen might
allow access to the unoccupied S1′ pocket, thus presenting
potential opportunities to form polar or hydrogen-bonding
interactions. Based on these possibilities, inhibitor II was
designed by introducing two F atoms on the P2′ phenyl group
to facilitate hydrogen bonds with the S2′ pocket (Figure 4C),
while inhibitor III was designed by introducing a para-n-
butoxyphenyl substitution on the P1 phenyl group and a
propanol substitution of the acylguanidine moiety to extend
into the S3 pocket and S1′ pocket (Figure 4D), respectively.
We further replaced the P2′ phenyl with pyrimidine to
facilitate a hydrogen bond with the S2′ pocket (Figure 4D).
The detailed chemical synthesis and characterization are
shown in Scheme S2, Scheme S3 and Figures S5−S18.
1095
https://doi.org/10.1021/acschemneuro.0c00816
ACS Chem. Neurosci. 2021, 12, 1093−1099

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Letter
peptide substrate of which one end is tagged with a DOTA-Ln
that can be encoded with different Ln ions on demand and the
other end is labeled with a biotin group enabling the highly
efficient separation of the cleaved Ln from uncleaved LCPP for
accurate ICPMS quantification. Such a design allows us to
screen BACE1 inhibitors via switching the determination of
the hydrolyzed polypeptide fragments to quantification of Ln
on ICPMS. To the best of our knowledge, this is the first
example applying ICPMS for inhibitor screening. This
methodology should be very much expected to be applied to
the screening of other enzymes inhibitors, not limited to
BACE1 demonstrated here.
METHODS
■
Synthesis of LCPP and FITC-Labeled Probe. Triethylamine
(TEA, 4.3 μL, 32.4 μmol) and biotin-NHS ester (8.1 mg, 16.2 μmol)
were added into peptide EVNLDAEC (9.6 mg, 10.8 μmol) with
anhydrous N,N-dimethylformamide (DMF, 100 μL) as solvent. After
overnight reaction, the mixture was purified using the semipreparative
HPLC with a TechMate C18-ST column (10.0 i.d. × 250 mm in
length, 5 μm particle size), during which H2O/ACN mixture was used
as the mobile phase with a gradient elution program from 70/30
H2O/ACN to 20/80 H2O/ACN in 30 min at a flow rate of 4.0 mL/
min, followed by reduced pressure distillation and freeze-drying to
give biotin-EVNLDAEC (10.0 mg, yield 72%). Then, the obtained
biotin-EVNLDAEC (10.0 mg, 7.82 μmol) and MMA-DOTA-Eu
(13.5 mg, 20 μmol) were dissolved in ammonium acetate buffer (0.2
mL, 100 mM, pH 6.4) and stirred for 3 h at 37 °C and then purified
by HPLC to obtain biotin-peptide-DOTA-Eu (13.0 mg, yield 85%).
Biotin-peptide-MMA-DOTA-Ln (Ln = La, Pr, Nd) were prepared in
the same way as biotin-peptide-MMA-DOTA-Eu. (Scheme S1) They
were characterized using ESI-MS (Figure S1). The synthetic route for
FITC-labeled probe was similar to that for LCPP just using FITC
instead of MMA-DOTA-Ln. The obtained biotin-EVNLDAEC (5.0
mg, 3.9 μmol) and MMA-FITC (4.3 mg, 10 μmol) were dissolved in
ammonium acetate buffer (0.2 mL, 100 mM, pH 6.4) and stirred for 3
h at 37 °C, then purified by HPLC to obtain FITC-labeled probe (5.0
mg, yield 75%).
Synthesis of 1-(2,4-Difluorophenyl)-4-phenylbutane-1,4-
dione (Compound 1). To a solution of ZnCl2 (5.0 g, 36.7 mmol)
in toluene (40 mL) was added diethylamine (DEA, 2.9 mL, 27.5
mmol) followed by t-BuOH (2.6 mL, 27.5 mmol). The mixture was
stirred at room temperature (RT) for 2 h until it was mostly
dissolved; then acetophenone (3.3 mL, 27.5 mmol) and 2-bromo-
2′,4′-difluoroacetophenone (4.3 g, 18.4 mmol) were added to the
reaction mixture for 6 h reaction under stirring, and the reaction
mixture was diluted with water (40 mL), extracted with CH2Cl2 (3 ×
40 mL), dried with MgSO4, and filtered through a funnel in sequence.
The filtrate was concentrated in vacuo and purified by silica gel flash
column chromatography (EtOAc/petroleum ether (PE), 1/40) to
give a yellow powder, compound 1 (2.1 g, yield 42%) (Scheme S2).
1H NMR (500 MHz, CDCl3) δ 8.07−7.99 (m, 2H), 7.96 (td, J = 8.6,
6.7 Hz, 1H), 7.61−7.54 (m, 1H), 7.47 (t, J = 7.7 Hz, 2H), 7.01−6.92
(m, 1H), 6.89 (ddd, J = 11.1, 8.7, 2.5 Hz, 1H), 3.42 (dhept, J = 9.5,
2.8 Hz, 4H). 13C NMR (125 MHz, CDCl3) δ 198.4, 195.3 (d, J = 4.7
Hz), 166.8 (d, J = 12.2 Hz), 164.8 (d, J = 12.4 Hz), 163.9 (d, J = 12.6
Hz), 161.9 (d, J = 12.5 Hz), 136.7, 133.1, 132.7 (dd, J = 10.6, 4.3 Hz),
128.6, 128.1, 122.0 (dd, J = 13.2, 3.6 Hz), 112.2 (dd, J = 21.4, 3.5
Hz), 104.8 (dd, J = 27.7, 25.4 Hz), 37.2 (d, J = 8.4 Hz), 32.5 (d, J =
2.3 Hz). MS calcd for C16H12F2O2 [M + H]+ m/z 275.0878, found
275.0870.
Synthesis of 2-(2-(2,4-Difluorophenyl)-5-phenyl-1H-pyrrol-
1-yl) Acetic Acid (Compound 2). A solution of compound 1 (2.1 g,
7.5 mmol) and glycine (1.7 g, 22.5 mmol) in acetic acid (20 mL) was
refluxed at 130 °C overnight. The mixture was naturally cooled to RT,
diluted with H2O (20 mL), extracted with EtOAc (3 × 20 mL), dried
with MgSO4, and filtered through a funnel in sequence. The filtrate
was concentrated in vacuo and purified by silica gel flash column
Figure 4. (A) The bonding mode of inhibitor I with BACE1. X-ray
crystal structure of inhibitor I binding with BACE1 (PDB code 2QU2,
left); the relative locations of the different binding pockets of the
active site are labeled, and key amino acid residues and key binding
interactions (right) are given. Sigmoidal dose response curves of
BACE1 activity versus logarithmic concentration of inhibitor I (B),
inhibitor II (C), and inhibitor III (D). Experiments were performed in
sodium acetate buffer (20 mM, pH 4.5) containing 1 μM LCPP and
20 nM BACE1.
Considering that ICPMS can easily distinguish and quantify
multiple Ln, we encoded LCPP with different Ln ions to
measure the IC50 values of the newly synthesized BACE1
inhibitors. As shown in Scheme S4, to determine the IC50
values of the acylguanidine-based inhibitors using three
different Ln-encoded LCPPs (Ln = La, Pr, Nd), the BACE1-
cleaved DAEC-MMA-DOTA-Ln in the supernatants were
combined and analyzed by ICPMS simultaneously. The
determined IC50 values of inhibitors I, II, and III were 1.01
0.25 μM, 186 2 nM, and 382 2 nM (n = 3) (Figure 4B−
D), suggesting that inhibitor II resulting from the simple
introduction of two F atoms on P2′ phenyl group of inhibitor I
is more efficacious than inhibitor III with the more
complicated structure. Clearly, sophisticated design of
BACE1 inhibitory molecules is much more desired in the
future, and they can be precisely evaluated on the established
LCPP−ICPMS platform.
In summary, we have successfully developed a sensitive and
accurate LCPP−ICPMS platform for not only BACE1 assay
but also BACE1 inhibitor screening. The LCPP features a
1096
https://doi.org/10.1021/acschemneuro.0c00816
ACS Chem. Neurosci. 2021, 12, 1093−1099

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Letter
chromatography (EtOAc/PE/HCOOH, 1/10/0.01) to obtain com-
pound 2 as a white powder (2.3 g, yield 98%) (Scheme S2). 1H NMR
(500 MHz, CDCl3) δ 7.42−7.28 (m, 3H), 6.89 (t, J = 8.7 Hz, 1H),
6.33 (d, J = 3.5 Hz, 1H), 6.31 (d, J = 3.6 Hz, 1H). 13C NMR (125
MHz, CDCl3) δ 174.6, 163.9 (d, J = 12.1 Hz), 161.9 (d, J = 12.0 Hz),
161.1 (d, J = 12.2 Hz), 159.1 (d, J = 12.5 Hz), 137.0, 133.5 (dd, J =
9.2, 4.3 Hz), 132.7, 129.2, 128.7, 128.6, 127.7, 117.2−116.5 (m),
111.7 (dd, J = 20.8, 4.1 Hz), 111.2, 109.8, 104.3 (t, J = 25.9 Hz), 46.5
(d, J = 3.1 Hz). MS calcd for C18H13F2NO2 [M + H]+ m/z 314.0987,
found 314.1020.
6.24 (d, J = 3.8 Hz, 1H), 5.15−4.96 (m, 2H), 4.01 (t, J = 6.4 Hz, 2H),
1.79−1.63 (m, 2H), 1.52−1.35 (m, 2H), 0.94 (t, J = 7.4 Hz, 3H). 13C
NMR (125 MHz, CDCl3) δ 198.6, 196.8, 163.2, 160.0, 157.7, 130.4,
129.5, 123.0, 114.2, 67.9, 33.1, 32.7, 31.1, 19.2, 13.8. MS calcd for
C18H20N2O3 [M + H]+ m/z 313.1547, found 313.1467.
Synthesis of 2-(2-(4-Butoxyphenyl)-5-(pyrimidin-2-yl)-1H-
pyrrol-1-yl) Acetic Acid (Compound 6). The synthetic procedure
for compound 6 (Scheme S3) was the same as that for compound 2.
1H NMR (500 MHz, DMSO-d6) δ 8.68 (d, J = 4.9 Hz, 2H), 7.41−
7.24 (m, 2H), 7.22−7.08 (m, 2H), 7.08−6.93 (m, 2H), 6.24 (d, J =
3.8 Hz, 1H), 4.01 (t, J = 6.4 Hz, 2H), 1.76−1.63 (m, 2H), 1.52−1.33
(m, 2H), 0.94 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz, DMSO-d6) δ
171.5, 159.9, 159.0, 157.3, 140.4, 131.7, 130.7, 124.6, 117.8, 115.3,
115.1, 109.5, 79.6, 67.7, 49.4, 31.2, 19.2, 14.1. MS calcd for
C21H22N2O3 [M + H]+ m/z 352.1656, found 352.1413.
Synthesis of 1-(3-Hydroxypropyl) Guanidine (Compound
7). Compound 7 (Scheme S3) was prepared according to a published
procedure.45
Synthesis of N-(Diaminomethylene)-2-(2-(2,4-difluorophen-
yl)-5-phenyl-1H-pyrrol-1-yl) Acetamide (Inhibitor II). To a
solution of compound 2 (2.3 g, 7.4 mmol) in anhydrous DMF (4.0
mL) was added 1,1′-carbonyldiimidazole (CDI, 1.7 g, 10.5 mmol),
and the mixture was stirred at RT for 1 h. To the reaction mixture was
added a solution of guanidine hydrochloride (2.5 g, 26.1 mmol) and
TEA (3.6 mL, 26.1 mmol) in DMF (6.5 mL). After stirring for 6 h,
the above mixture was poured into H2O (10 mL). The aqueous phase
was extracted with EtOAc (3 × 10 mL), and the combined organic
layers were dried with MgSO4 and then filtered through a funnel. The
filtrate was concentrated in vacuo and purified by silica gel flash
column chromatography (EtOAc/PE/HCOOH, 2/8/0.01) to obtain
Synthesis of (E)-N-(Amino((3-hydroxypropyl)amino)-
methylene)-2-(2-(4-butoxyphenyl)-5-(pyrimidin-2-yl)-1H-pyr-
rol-1-yl) Acetamide (Inhibitor III). Compound 6 (386.0 mg, 1.1
mmol) and CDI (0.9 g, 5.5 mmol) were dissolved in anhydrous DMF
(5.0 mL), and the mixture was stirred at RT for 0.5 h. Compound 7
(1.3 g, 11.0 mmol), TEA (1.5 mL, 11.0 mmol), and 4-
dimethylaminopyridine (DMAP, 13.4 mg, 0.1 mmol) were added to
the reaction mixture, and the mixture was stirred for 12 h at RT. Then
the mixture was concentrated in vacuo and purified by silica gel flash
column chromatography to obtain inhibitor III (50.0 mg, yield
1
inhibitor II (2.6 g, yield 98%) (Scheme S2). H NMR (500 MHz,
CDCl3) δ 7.88 (d, J = 3.5 Hz, 4H), 7.33−7.27 (m, 4H), 7.25 (ddd, J =
8.6, 5.2, 2.3 Hz, 2H), 6.82 (ddt, J = 16.3, 9.1, 4.6 Hz, 2H), 6.29−6.24
(m, 2H), 4.58 (s, 2H). 13C NMR (125 MHz, CDCl3) δ 172.5, 163.9
(d, J = 12.2 Hz), 161.9 (d, J = 12.2 Hz), 161.1 (d, J = 12.5 Hz), 159.1
(d, J = 12.2 Hz), 156.2, 137.3, 134.0−133.1 (m), 132.6, 129.1, 128.8,
128.7, 127.8, 116.7 (dd, J = 15.0, 4.4 Hz), 111.8 (d, J = 24.5 Hz),
111.5, 110.0, 104.4 (t, J = 25.8 Hz), 49.1. MS calcd for C19H16F2N4O
[M + H]+ m/z 355.1370, found 355.1365.
Synthesis of 1-(4-Butoxyphenyl)-3-(dimethylamino) Prop-
an-1-one (Compound 3). A solution of 4′-butoxyacetophenone
(2.5 g, 13.0 mmol), dimethylamine hydrochloride (DMA, 1.4 g, 14.6
mmol), paraformaldehyde (PA, 0.6 g, 19.7 mmol), and 12 N HCl (25
μL) in ethanol (10 mL) was refluxed at 100 °C overnight. The
solution was naturally cooled to RT, and the ethanol was evaporated
under vacuum; a 20% aqueous solution of NaOH was added to adjust
pH = 10, and then the residue was extracted with EtOAc (3 × 20
mL). The combined organic layers were dried with MgSO4 and
filtered through a funnel. The filtrate was concentrated in vacuo and
purified by silica gel flash column chromatography (EtOAc/MeOH,
10/1) to give compound 3 (1.6 g, yield 49%) (Scheme S3). 1H NMR
(500 MHz, CDCl3) δ 7.93 (d, J = 9.0 Hz, 2H), 6.92 (d, J = 8.9 Hz,
2H), 4.02 (t, J = 6.5 Hz, 2H), 3.10 (dd, J = 8.0, 6.8 Hz, 2H), 2.75 (dd,
J = 8.0, 6.8 Hz, 2H), 2.29 (s, 4H), 1.79 (dq, J = 8.2, 6.7 Hz, 2H),
1.55−1.44 (m, 2H), 0.98 (t, J = 7.4 Hz, 3H). 13C NMR (125 MHz,
CDCl3) δ 197.6, 163.1, 130.3, 129.8, 114.2, 67.9, 54.6, 45.5, 36.5,
31.1, 19.2, 13.8. MS calcd for C15H23NO2 [M + H]+ m/z 250.1802,
found 250.1750.
Synthesis of Pyrimidine-2-carbaldehyde (Compound 4). To
a solution of 2-iodoxybenzoic acid (IBX, 7.6 g, 27.3 mmol) in ACN
(20 mL) was added 2-pyrimidinemethanol (1.0 g, 9.1 mmol), and the
reaction mixture was stirred at 80 °C for 3 h in N2 atmosphere, then
filtered through a funnel and concentrated in vacuo to give crude
compound 4 (Scheme S3), which was directly used for next step of
synthesis.
Synthesis of 1-(4-Butoxyphenyl)-4-(pyrimidin-2-yl) Butane-
1,4-dione (Compound 5). To a well stirred solution of compound 3
(1.6 g, 6.4 mmol) in 1,4-dioxane (10 mL) was added compound 4
(0.8 g, 7.4 mmol), 3-ethyl-5-(2-hydroxyethyl)-4-methylthiazolium
bromide (EHMB, 0.5 g, 2.0 mmol), and TEA (5 mL). The reaction
mixture was refluxed at 120 °C for 16 h, the solution was naturally
cooled to RT and diluted with H2O (20 mL), then the mixture was
extracted with EtOAc (3 × 30 mL). The combined organic layers
were dried over anhydrous MgSO4, filtered through a funnel, and
concentrated in vacuo. The residue was purified by silica gel flash
column chromatography to give compound 5 (1.0 g, yield 50%)
(Scheme S3). 1H NMR (500 MHz, DMSO-d6) δ 8.68 (d, J = 4.9 Hz,
2H), 7.37−7.26 (m, 2H), 7.22−7.12 (m, 2H), 7.06−6.97 (m, 2H),
1
10.1%) (Scheme S3). H NMR (500 MHz, CD3OD) δ 8.59 (d, J =
4.9 Hz, 1H), 7.35 (d, J = 8.7 Hz, 1H), 7.24 (dd, J = 8.8, 3.7 Hz, 1H),
7.04 (dd, J = 11.5, 6.7 Hz, 1H), 6.96 (t, J = 6.3 Hz, 1H), 6.23 (dd, J =
7.5, 3.9 Hz, 1H), 5.08 (d, J = 12.6 Hz, 1H), 4.01 (t, J = 6.4 Hz, 1H),
3.61 (dt, J = 29.5, 4.2 Hz, 1H), 3.30 (ddd, J = 15.8, 8.4, 4.2 Hz, 2H),
1.82−1.71 (m, 2H), 1.52 (dq, J = 14.8, 7.4 Hz, 1H), 1.29 (d, J = 4.6
Hz, 1H), 1.00 (td, J = 7.4, 2.6 Hz, 2H). 13C NMR (214 MHz,
CD3OD) δ 171.2, 159.6, 159.4, 156.5, 156.5, 141.1, 130.3, 130.3,
124.4, 116.8, 115.1, 114.3, 109.1, 67.4, 67.4, 58.5, 58.3, 48.9, 48.4,
38.2, 31.1, 31.0, 18.9, 12.8. MS calcd for C24H30N6O3 [M + H]+ m/z
451.2452, found 451.2351.
Measurement of the IC50 Values of Inhibitors I, II, and III.
The inhibitors at different concentrations were incubated with 20 nM
BACE1 in CH3COONa (20 mM, pH 4.5) containing 0.01% Triton
X-100 at 37 °C for 30 min, and then 1 μM LCPP (La for inhibitor I,
Pr for inhibitor II, Nd for inhibitor III) was added, and the reaction
was incubated at 37 °C for 60 min with gentle shaking (600 rpm).
Subsequently, streptavidin magnetic beads and MOPS buffer (100
mm, pH 7.4) were added to the mixture, and it was incubated on a
rotary mixer for 15 min and set on the magnet for 3 min for
separation of the uncleaved LCPP from the cleaved DAEC-MMA-
DOTA-Ln (Ln = La, Pr, Nd). Finally, the supernatants were mixed
and diluted with 2% HNO3 for ICPMS measurement to obtained the
inhibitor-mediated BACE1 activity. Sigmoidal dose response curves of
the inhibition percentage (%) versus logarithmic concentration of
inhibitor I, inhibitor II, and inhibitor III were thus constructed
(Figure 4). From the sigmoidal dose response curves, the IC50 value
was obtained.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acschemneuro.0c00816.
Materials and instrumentation, synthetic routes, meas-
urement of the IC50 values of inhibitors I, II, and III,
ESI-MS of biotin-peptide-MMA-DOTA-Ln (Eu, La, Pr,
Nd), ESI-MS of LCPP, DAEC-MMA-DOTA-Eu, and
biotin-EVNL, time and pH-dependent BACE1 proteo-
lytic activity, structure of FITC-labeled probe and
fluorescence responses from a series of BACE1
1097
https://doi.org/10.1021/acschemneuro.0c00816
ACS Chem. Neurosci. 2021, 12, 1093−1099

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Letter
concentrations, and 1H and 13C NMR of the synthesized
compounds (PDF)
Abeta and alters APP processing in wild-type mice. J. Neurochem. 99,
1555−1563.
(5) Yang, L. B., Lindholm, K., Yan, R. Q., Citron, M., Xia, W. M.,
Yang, X. L., Beach, T., Sue, L., Wong, P., Price, D., et al. (2003)
Elevated beta-secretase expression and enzymatic activity detected in
sporadic Alzheimer disease. Nat. Med. 9, 3−4.
(6) Shen, Y., Wang, H. B., Sun, Q. Y., Yao, H. H., Keegan, A. P.,
Mullan, M., Wilson, J., Lista, S., Leyhe, T., Laske, C., et al. (2018)
Increased plasma beta-secretase 1 may predict conversion to
Alzheimer’s disease dementia in individuals with mild cognitive
impairment. Biol. Psychiatry 83, 447−455.
(7) Sinha, S., Anderson, J. P., Barbour, R., Basi, G. S., Caccavello, R.,
Davis, D., Doan, M., Dovey, H. F., Frigon, N., Hong, J., et al. (1999)
Purification and cloning of amyloid precursor protein beta-secretase
from human brain. Nature 402, 537−540.
(8) Hong, L., Koelsch, G., Lin, X. L., Wu, S. L., Terzyan, S., Ghosh,
A. K., Zhang, X. C., and Tang, J. (2000) Structure of the protease
domain of memapsin 2 (beta-secretase) complexed with inhibitor.
Science 290, 150−153.
(9) Ghosh, A. K., and Osswald, H. L. (2014) BACE1 (β-secretase)
inhibitors for the treatment of Alzheimer’s disease. Chem. Soc. Rev. 43,
6765−6813.
(10) Das, B., and Yan, R. Q. (2019) A close look at BACE1
inhibitors for Alzheimer’s disease treatment. CNS Drugs 33, 251−263.
(11) Chen, J., Wang, J., Yin, B., Pang, L., Wang, W., and Zhu, W.
(2019) Molecular mechanism of binding selectivity of inhibitors
toward BACE1 and BACE2 revealed by multiple short molecular
dynamics simulations and free-energy predictions. ACS Chem.
Neurosci. 10, 4303−4318.
(12) Chen, J., Yin, B., Wang, W., and Sun, H. (2020) Effects of
disulfide bonds on binding of inhibitors to β-amyloid cleaving enzyme
1 decoded by multiple replica accelerated molecular dynamics
simulations. ACS Chem. Neurosci. 11, 1811−1826.
AUTHOR INFORMATION
Corresponding Authors
■
Limin Yang − Department of Chemistry and the MOE Key
Laboratory of Spectrochemical Analysis & Instrumentation,
College of Chemistry and Chemical Engineering, Xiamen
University, Xiamen 361005, China; Email: lmyang@
xmu.edu.cn
Xiaowen Yan − Department of Chemistry and the MOE Key
Laboratory of Spectrochemical Analysis & Instrumentation,
College of Chemistry and Chemical Engineering, Xiamen
University, Xiamen 361005, China; orcid.org/0000-
0001-6608-6044; Email: xwyan@xmu.edu.cn
Qiuquan Wang − Department of Chemistry and the MOE Key
Laboratory of Spectrochemical Analysis & Instrumentation,
College of Chemistry and Chemical Engineering, Xiamen
University, Xiamen 361005, China; orcid.org/0000-
0002-5166-4048; Email: qqwang@xmu.edu.cn; Fax: +86
592 2187400
Author
Xin Jin − Department of Chemistry and the MOE Key
Laboratory of Spectrochemical Analysis & Instrumentation,
College of Chemistry and Chemical Engineering, Xiamen
University, Xiamen 361005, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acschemneuro.0c00816
(13) Pietrak, B. L., Crouthamel, M. C., Tugusheva, K., Lineberger, J.
E., Xu, M., DiMuzio, J. M., Steele, T., Espeseth, A. S., Stachel, S. J.,
Coburn, C. A., et al. (2005) Biochemical and cell-based assays for
characterization of BACE-1 inhibitors. Anal. Biochem. 342, 144−151.
(14) Yi, X. Y., Hao, Y. Q., Xia, N., Wang, J. X., Quintero, M., Li, D.,
and Zhou, F. M. (2013) Sensitive and continuous screening of
inhibitors of β-site amyloid precursor protein cleaving enzyme 1
(BACE1) at single SPR chips. Anal. Chem. 85, 3660−3666.
(15) Folk, D. S., Torosian, J. C., Hwang, S., McCafferty, D. G., and
Franz, K. J. (2012) Monitoring β-secretase activity in living cells with
a membrane-anchored FRET probe. Angew. Chem., Int. Ed. 51,
10795−10799.
(16) Choi, Y., Cho, Y., Kim, M., Grailhe, R., and Song, R. (2012)
Fluorogenic quantum dot-gold nanoparticle assembly for beta
secretase inhibitor screening in live cell. Anal. Chem. 84, 8595−8601.
(17) Kennedy, M. E., Wang, W. Y., Song, L. X., Lee, J., Zhang, L. L.,
Wong, G., Wang, L. Y., and Parker, E. (2003) Measuring human beta-
secretase (BACE1) activity using homogeneous time-resolved
fluorescence. Anal. Biochem. 319, 49−55.
(18) Zuo, X. W., Dai, H. X., Zhang, H. G., Liu, J. J., Ma, S. D., and
Chen, X. G. (2018) A peptide-WS2 nanosheet based biosensing
platform for determination of β-secretase and screening of its
inhibitors. Analyst 143, 4585−4591.
(19) Mancini, F., Naldi, M., Cavrini, V., and Andrisano, V. (2007)
Multiwell fluorometric and colorimetric microassays for the
evaluation of beta-secretase (BACE-1) inhibitors. Anal. Bioanal.
Chem. 388, 1175−1183.
Author Contributions
X.Y., L.Y., and Q.W. conceived and supervised the research
design and details. X.J. carried out the experiments and
analyzed the results. The manuscript was first prepared by X.J.,
L.Y. and X.Y. and then revised by X.Y. and Q.W.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This study was financially supported by the National Natural
Science Foundation of China (21535007, 21874112, and
22074127) and the Fundamental Research Funds for the
Central Universities (20720200073). We thank the Founda-
tion for Innovative Research Groups of the National Natural
Science Foundation of China (21521004) and Program for
Changjiang Scholars and Innovative Research Team in
University (PCSIRT, IRT13036) for partial financial support.
REFERENCES
■
(1) Mattson, M. P. (2004) Pathways towards and away from
Alzheimer’s disease. Nature 430, 631−639.
(2) Cai, H., Wang, Y. S., McCarthy, D., Wen, H. J., Borchelt, D. R.,
Price, D. L., and Wong, P. C. (2001) BACE1 is the major beta-
secretase for generation of Abeta peptides by neurons. Nat. Neurosci.
4, 233−234.
(3) Luo, Y., Bolon, B., Kahn, S., Bennett, B. D., Babu-Khan, S.,
Denis, P., Fan, W., Kha, H., Zhang, J. H., Gong, Y. H., et al. (2001)
Mice deficient in BACE1, the Alzheimer’s β-secretase, have normal
phenotype and abolished β-amyloid generation. Nat. Neurosci. 4,
231−232.
(4) Nishitomi, K., Sakaguchi, G., Horikoshi, Y., Gray, A. J., Maeda,
M., Hirata-Fukae, C., Becker, A. G., Hosono, M., Sakaguchi, I.,
Minami, S. S., et al. (2006) BACE1 inhibition reduces endogenous
(20) Mancini, F., De Simone, A., and Andrisano, V. (2011) Beta-
secretase as a target for Alzheimer’s disease drug discovery: an
overview of in vitro methods for characterization of inhibitors. Anal.
Bioanal. Chem. 400, 1979−1996.
́
(21) Łobinski, R., Schaumlöffel, D., and Szpunar, J. (2006) Mass
spectrometry in bioinorganic analytical chemistry. Mass Spectrom. Rev.
25, 255−289.
(22) Svantesson, E., Pettersson, J., and Markides, K. E. (2002) The
use of inorganic elemental standards in the quantification of proteins
1098
https://doi.org/10.1021/acschemneuro.0c00816
ACS Chem. Neurosci. 2021, 12, 1093−1099

ACS Chemical Neuroscience
pubs.acs.org/chemneuro
Letter
and biomolecular compounds by inductively coupled plasma
spectrometry. J. Anal. At. Spectrom. 17, 491−496.
(41) Vassar, R., Bennett, B. D., Babu-Khan, S., Kahn, S., Mendiaz, E.
A., Denis, P., Teplow, D. B., Ross, S., Amarante, P., Loeloff, R., et al.
(1999) Beta-secretase cleavage of Alzheimer’s amyloid precursor
protein by the transmembrane aspartic protease BACE. Science 286,
735−741.
(23) Xu, M., Yang, L. M., and Wang, Q. Q. (2008) Quantification of
selenium-tagged proteins in human plasma using species-unspecific
isotope dilution ICP-DRC-qMS coupled on-line with anion exchange
chromatography. J. Anal. At. Spectrom. 23, 1545−1549.
(24) Guo, Y. F., Xu, M., Yang, L. M., and Wang, Q. Q. (2009)
Strategy for absolute quantification of proteins: CH3Hg+ labeling
integrated molecular and elemental mass spectrometry. J. Anal. At.
Spectrom. 24, 1184−1187.
(42) Gruninger-Leitch, P. F., Schlatter, D., Kung, E., Nelböck, P.,
̈ ̈
and Döbeli, H. (2002) Substrate and inhibitor profile of BACE (beta-
secretase) and comparison with other mammalian aspartic proteases.
J. Biol. Chem. 277, 4687−4693.
(43) Ellis, C. R., and Shen, J. (2015) pH-dependent population shift
regulates BACE1 activity and inhibition. J. Am. Chem. Soc. 137, 9543−
9546.
(25) Liu, R., Zhang, S. X., Wei, C., Xing, Z., Zhang, S. C., and Zhang,
X. R. (2016) Metal stable isotope tagging: renaissance of radio-
immunoassay for multiplex and absolute quantification of biomole-
cules. Acc. Chem. Res. 49, 775−783.
(26) Baranov, V. I., Quinn, Z. A., Bandura, D. R., and Tanner, S. D.
(2002) The potential for elemental analysis in biotechnology. J. Anal.
At. Spectrom. 17, 1148−1152.
(27) Prange, A., and Pröfrock, D. (2008) Chemical labels and
natural element tags for the quantitative analysis of biomolecules. J.
Anal. At. Spectrom. 23, 432−459.
̌
̌
(44) Schejbal, J., Slezácková, L., Remínek, R., and Glatz, Z. (2017) A
capillary electrophoresis-mass spectrometry based method for the
screening of β-secretase inhibitors as potential Alzheimer’s disease
therapeutics. J. Chromatogr. A 1487, 235−241.
(45) Liu, R. F., Liu, Y. C., Meng, J. W., Zhu, H. Y., and Zhang, X. H.
(2017) A microfluidics-based mobility shift assay to identify new
inhibitors of β-secretase for Alzheimer’s disease. Anal. Bioanal. Chem.
409, 6635−6642.
(46) Viayna, E., Gomez, T., Galdeano, C., Ramirez, L., Ratia, M.,
Badia, A., Clos, M. V., Verdaguer, E., Junyent, F., Camins, A., et al.
(2010) Novel huprine derivatives with inhibitory activity toward β-
amyloid aggregation and formation as disease-modifying anti-
Alzheimer drug candidates. ChemMedChem 5, 1855−1870.
(47) Shoichet, B. K. (2006) Interpreting steep dose-response curves
in early inhibitor discovery. J. Med. Chem. 49, 7274−7277.
(48) Cole, D. C., Manas, E. S., Stock, J. R., Condon, J. S., Jennings,
L. D., Aulabaugh, A., Chopra, R., Cowling, R., Ellingboe, J. W., Fan, K.
Y., et al. (2006) Acylguanidines as small-molecule beta-secretase
inhibitors. J. Med. Chem. 49, 6158−6161.
(28) Bettmer, J., Montes-Bayón, M., Ruiz-Encinar, J., Fernández-
Sánchez, M. L., Fernández de la Campa, M. d. R., and Sanz-Medel, A.
(2009) The emerging role of ICP-MS in proteomic analysis. J.
Proteomics 72, 989−1005.
(29) Luo, Y. C., Yan, X. W., Huang, Y. S., Wen, R. B., Li, Z. X., Yang,
L. M., Yang, C. J., and Wang, Q. Q. (2013) ICP-MS-based multiplex
and ultrasensitive assay of viruses with lanthanide-coded biospecific
tagging and amplification strategies. Anal. Chem. 85, 9428−9432.
(30) Yan, X. W., Yang, L. M., and Wang, Q. Q. (2013) Detection
and quantification of proteins and cells by use of elemental mass
spectrometry: progress and challenges. Anal. Bioanal. Chem. 405,
5663−5670.
(31) de Bang, T. C., and Husted, S. (2015) Lanthanide elements as
labels for multiplexed and targeted analysis of proteins, DNA and
RNA using inductively-coupled plasma mass spectrometry. TrAC,
Trends Anal. Chem. 72, 45−52.
(32) Calderón-Celis, F., Encinar, J. R., and Sanz-Medel, A. (2018)
Standardization approaches in absolute quantitative proteomics with
mass spectrometry. Mass Spectrom. Rev. 37, 715−737.
(33) Liang, Y., Liu, Q., Zhou, Y., Chen, S., Yang, L. M., Zhu, M., and
Wang, Q. Q. (2019) Counting and recognizing single bacterial cells
by a lanthanide-encoding inductively coupled plasma mass spectro-
metric approach. Anal. Chem. 91, 8341−8349.
(34) Lathia, U. S., Ornatsky, O., Baranov, V., and Nitz, M. (2011)
Multiplexed protease assays using element-tagged substrates. Anal.
Biochem. 408, 157−159.
(35) Yan, X. W., Yang, L. M., and Wang, Q. Q. (2011) Lanthanide-
coded protease-specific peptide-nanoparticle probes for a label-free
multiplex protease assay using element mass spectrometry: a proof-of-
concept study. Angew. Chem., Int. Ed. 50, 5130−5133.
(36) Mullan, M., Crawford, F., Axelman, K., Houlden, H., Lilius, L.,
Winblad, B., and Lannfelt, L. (1992) A pathogenic mutation for
probable Alzheimer’s disease in the APP gene at the N-terminus of
beta-amyloid. Nat. Genet. 1, 345−347.
(37) Barman, A., Schurer, S., and Prabhakar, R. (2011) Computa-
̈
tional modeling of substrate specificity and catalysis of the beta-
secretase (BACE1) enzyme. Biochemistry 50, 4337−4349.
(38) Toulokhonova, L., Metzler, W. J., Witmer, M. R., Copeland, R.
A., and Marcinkeviciene, J. (2003) Kinetic studies on β-site amyloid
precursor protein-cleaving enzyme (BACE): confirmation of an iso-
mechanism. J. Biol. Chem. 278, 4582−4589.
(39) Kocak, A., Erol, I., Yildiz, M., and Can, H. (2016)
Computational insights into the protonation states of catalytic dyad
in BACE1-acyl guanidine based inhibitor complex. J. Mol. Graphics
Modell. 70, 226−235.
(40) Westmeyer, G. G., Willem, M., Lichtenthaler, S. F., Lurman, G.,
Multhaup, G., Assfalg-Machleidt, I., Reiss, K., Saftig, P., and Haass, C.
(2004) Dimerization of beta-site beta-amyloid precursor protein-
cleaving enzyme. J. Biol. Chem. 279, 53205−53212.
1099
https://doi.org/10.1021/acschemneuro.0c00816
ACS Chem. Neurosci. 2021, 12, 1093−1099