Modulation of microglial immune responses by a novel thiourea derivative

Article abstract of DOI:10.1016/j.cbi.2010.07.006

Increasing evidence indicates that microglial activation plays an important role in the pathogenesis of Alzheimer's disease (AD). In AD, activated microglia may facilitate the clearance of β-amyloid (Aβ), a neurotoxic component in AD pathogenesis. However, microglial activation comes at the cost of triggering neuro-inflammation, which contributes to cerebral dysfunction. Thus, pharmacological approaches that can achieve a favorable combination of a reduced microglia-mediated neuro-inflammation, and an enhanced Aβ clearance may be beneficial for preventing the progression of the disease. Here, we show that some newly synthesized compounds may exert such a combination of functions. Using mouse primary microglia and RAW264.7 cells, we found that some thiourea derivatives significantly enhanced microglial Aβ phagocytosis and suppressed microglial immune responses, as evidenced by the reduced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Of note, some commercially available inhibitors for iNOS and/or COX-2, such as ibuprofen, dextromethorphan, and N^G-methyl-l-arginine (l-NMA), show negligible effects on microglial Aβ phagocytosis. Among the thiourea derivatives, our data show that a lead compound, designated as compound #326, (1-Naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-thiourea) appears to be the most potent in promoting Aβ phagocytosis and in inhibiting the LPS-induced expression of iNOS and COX-2 (when used at concentrations in the low μM range). The potency of compound #326 may have beneficial effects on modulating microglial activation in AD. The structure-activity relationship indicates that the thiourea group, alkyl linker, and the hydrophobic aryl group largely influence the dual functions of the compounds. These findings may indicate a structural basis for the improved design of future drug therapies for AD.

Full text of DOI:10.1016/j.cbi.2010.07.006

Chemico-Biological Interactions 188 (2010) 228–236
Contents lists available at ScienceDirect
Chemico-Biological Interactions
journal homepage: www.elsevier.com/locate/chembioint
Modulation of microglial immune responses by a novel thiourea derivative
Jyh-Haur Cherna, Pei-Chien Hsub, Li-Wen Wanga, Huey-Jen Tsayb, Iou-Jiun Kanga, Feng-Shiun Shiec,∗
a Division of Biotechnology and Pharmaceutical Research, National Health Research Institutes, Zhunan, Taiwan, ROC
b Institute of Neuroscience, National Yang-Ming University, Taipei, Taiwan, ROC
c Division of Mental Health and Addiction Medicine, the Institute of Population Health Sciences, Zhunan, Taiwan, ROC
a r t i c l e i n f o
a b s t r a c t
Article history:
Increasing evidence indicates that microglial activation plays an important role in the pathogenesis of
Alzheimer’s disease (AD). In AD, activated microglia may facilitate the clearance of ␤-amyloid (A␤), a
neurotoxic component in AD pathogenesis. However, microglial activation comes at the cost of trigger-
ing neuro-inflammation, which contributes to cerebral dysfunction. Thus, pharmacological approaches
that can achieve a favorable combination of a reduced microglia-mediated neuro-inflammation, and an
enhanced A␤ clearance may be beneficial for preventing the progression of the disease. Here, we show that
some newly synthesized compounds may exert such a combination of functions. Using mouse primary
microglia and RAW264.7 cells, we found that some thiourea derivatives significantly enhanced microglial
A␤ phagocytosis and suppressed microglial immune responses, as evidenced by the reduced expression of
inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2). Of note, some commercially avail-
able inhibitors for iNOS and/or COX-2, such as ibuprofen, dextromethorphan, and NG-methyl-l-arginine
(l-NMA), show negligible effects on microglial A␤ phagocytosis. Among the thiourea derivatives, our
data show that a lead compound, designated as compound #326, (1-Naphthalen-1-yl-3-[5-(3-thioureido-
phenoxy)-pentyl]-thiourea) appears to be the most potent in promoting A␤ phagocytosis and in inhibiting
the LPS-induced expression of iNOS and COX-2 (when used at concentrations in the low ␮M range). The
potency of compound #326 may have beneficial effects on modulating microglial activation in AD. The
structure–activity relationship indicates that the thiourea group, alkyl linker, and the hydrophobic aryl
group largely influence the dual functions of the compounds. These findings may indicate a structural
basis for the improved design of future drug therapies for AD.
Received 16 April 2010
Received in revised form 2 July 2010
Accepted 2 July 2010
Available online 14 July 2010
Keywords:
Microglial activation
Alzheimer’s disease
Treatment
Thiourea
© 2010 Elsevier Ireland Ltd. All rights reserved.
1. Introduction
secretion of cytokines, transforming growth factor (TGF) ␣, gluta-
mate, and neurotrophic factors, such as nerve growth factor (NGF),
Microglia are immune cells in the brain. Under physiological
conditions, microglia actively monitor and respond to changes
in the microenvironment [1]. In various neuro-inflammatory dis-
eases, especially Alzheimer’s disease (AD), microglia have been
suggested as a primary source of effectors and regulatory immune
mediators in the brain due to their functions in producing sev-
eral molecules, including pro- and anti-inflammatory cytokines and
chemokines, prostanoids, neurotrophic factors, glutamate, and var-
ious free radicals [2–7]. Indeed, the plethora of functions attributed
to microglia makes their activation a double-edged sword. On
the one hand, microglia can benefit neuronal survival by reduc-
ing the exposure to neurotoxic agents via phagocytosis and by the
brain-derived neurotrophic factor (BDNF), and neurotrophic fac-
tor (NT)-3/4 to promote synaptic function. On the other hand, the
increased activity of cyclooxygenase (COX) and inducible nitric
oxide synthase (iNOS) and the production of pro-inflammatory
cytokine/chemokines and prostanoids, especially interleukin (IL)-
1␤, IL-6, tumor necrosis factor (TNF)␣, and prostaglandin (PG) E2,
contribute to neuro-inflammation and elevated oxidative stress
during microglial activation. These effects do not appear to be
limited to establish, functional neurons. It has been reported that
inflammation, through effects on neural stem cells, may inhibit
hippocampal neurogenesis that may, in turn, lead to a decline in
cognitive function in AD patients [8]. Similarly, the generation of
pro-NGF and the mature form of NGF by microglia is responsible
for inducing developmental Purkinje cell death and retinal degen-
eration [9].
∗
Corresponding author at: Division of Mental Health and Addiction Medicine, the
In regards to the microglial activation of induced oxidative
stress, reactive oxygen species (ROS), such as superoxide anion
(O2−), hydroxyl radical (OH−), and hydrogen peroxide (H2O2), are
increased. Microglial superoxide is generated from a membranous
Institute of Population Health Sciences, National Health Research Institutes, No.35
Keyan Road, Zhunan Town, Miaoli County 350, Taiwan, ROC.
Tel.: +886 1 37 246 166x36709.
E-mail address: fshie@nhri.org.tw (F.-S. Shie).
0009-2797/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.cbi.2010.07.006

J.-H. Chern et al. / Chemico-Biological Interactions 188 (2010) 228–236
229
nicotinamide adenine dinucleotide phosphate (NADPH) oxidase
complex and the released superoxide can directly damage neu-
rons [10,11]. Oxidative stress may lead to an increased expression
of pro-inflammatory genes, such as nuclear factor kappa B (NF␬B)
and activator protein 1 (AP-1), which leads to the increased pro-
duction of TNF␣ and PGE2 and elevated iNOS activity in microglia
[12,13]. Massive NO release from activated microglia having ele-
vated iNOS activity inhibits neuronal mitochondrial respiration
through a reversible binding to cytochrome oxidase in competition
with oxygen, resulting in reduced energy production, sensitization
to hypoxia, and excitotoxicity. In addition, NO derivatives, such as
peroxynitrite (ONOO−), nitrogen dioxide (NO2), dinitrogentrioxide
(N2O3), and S-nitrosothiols (SNOs), can also lead to neurotoxicity
through the inhibition of mitochondrial respiration or the activa-
tion of the mitochondrial permeability transition [14,15].
activation may not be the optimal therapeutic strategy for AD.
Therefore, any treatment that can achieve a favorable combina-
tion of the reduction of microglia-mediated neuro-inflammation
and the enhancement of the phagocytic activity of microglia may
be beneficial in preventing the progression of the disease. To
achieve the desired effects by a pharmacological mean, we syn-
thesized a series of thiourea derivatives and examined their effects
on microglial immune responses, including anti-inflammation and
phagocytic activity, using a cell-based enzyme-linked immunosor-
bent assay (ELISA) and flow cytometry. In this study, we show that
a lead compound appears to possess such dual functions.
2. Materials and methods
2.1. Reagents
The number of inflammatory and oxidative processes that
develop with inappropriate microglial activation provide a strong
rationale for the control of microglial activation as a prophylac-
tic or interventional strategy, especially in the course of AD [16].
AD is pathologically characterized by the appearance of neurofib-
rillary tangles and senile plaques, which predominantly consist of
␤-amyloid (A␤) [17]. In the brain of patients with AD, microglial
activation is characterized by morphological changes and by the
production of various effectors that contribute to massive oxida-
tive stress during pathological events. Microglial activation appears
to be a key step in the self-reinforcing cycles of the biochemical
pathologies in cerebrum that ultimately manifest clinically as AD.
Despite the abundant presence of activated microglia around the
plaques and the potential neuro-protective effect of microglial acti-
vation, through their ability of facilitating the clearance of A␤ via
phagocytosis, microglia that fail to phagocytose A␤ were found
in the AD brain and were identified in a mouse model [18]. One
interpretation is that microglial phagocytic activity may be inhib-
ited by the resulting consequences of neuro-inflammation. Others
and we have shown that microglial phagocytosis may be regu-
lated by signaling through a PGE2 receptor that is enhanced during
microglial activation [16,19]. Thus, in AD pathogenesis, the bene-
ficial functions of microglial activation appear to be overwhelmed
by the deteriorating effects, including increased oxidative stress
and the production of an assortment of effector molecules, lead-
ing to neuro-inflammation and, ultimately, to brain dysfunction.
Therefore, understanding the regulatory mechanism underlying
microglial activation is of importance to the development of the
most effective strategies to counteract AD.
Poly-d-lysine was obtained from BD Biosciences. Papain and
DNase I were obtained from Worthington Biochemical. Synthetic
A␤1–42 peptide came from Bachem and fluorescein isothiocyanate
(FITC)-labeled A␤1–42 was obtained from rPeptide. Antibodies
for iNOS, COX-2, A␤ (clone 6E10), and ␤-actin were obtained
from BD Transduction Lab, Abcam, Signet, and Novus Biologi-
cals, respectively. Fetal bovine serum was obtained from Hyclone
and heat-inactivated as described elsewhere [19]. Lipopolysaccha-
ride (LPS, Escherichia Coli O55:B5) was obtained from Calbiochem.
Culture media and penicillin/streptomycin were ordered from
Invitrogen. All other chemicals were from Sigma–Aldrich unless
stated otherwise.
2.2. General procedure for the preparation of thiourea derivatives
and the chemical structure of the lead compound #326
Synthesis of a series of the thiourea derivatives was achieved
via the key reaction of the thiourea group formation of the aniline
with 1,1ꢀ-thiocarbonyl diimidazole (TCDI) and aqueous ammonia
(25%), as indicated in Synthetic Scheme 1. Briefly, reduction of
the nitro compound (1) with tin (II) chloride in absolute ethanol
at 70 ◦C overnight gave the aniline (2). Subsequently, the ani-
line (2) was treated with 1,1ꢀ-thiocarbonyl diimidazole (TCDI) in
dichloromethane at room temperature followed by reaction with
25% ammonia solution to give the desired thiourea compounds (3).
The resulting compounds were then subjected to tests for biologi-
cal function in modulation of microglial activation [see supplement
Fig. 1 for the structures of the compounds].
To date, there is no cure for AD. Current Food and Drug Admin-
istration (FDA) approved drugs that target the symptoms of the
disease generally have only modest efficacy, and the effects of these
drugs do not persist [20]. Thus, a variety of “disease-modifying”
therapies have been proposed based on what is understood about
the pathogenesis of AD. Many of these treatments focus on the
metabolism of A␤ and, to a lesser extent, on the metabolism of
tau [21,22]. The theory that microglial activation is a key com-
ponent of the disease process has led to a renewed focus on
anti-inflammatory therapies, with an emphasis on treatments
that modulate microglial activation. Several compounds, including
botanical polyphenols, tetracycline derivatives, non-steroidal anti-
inflammatory drugs (NSAIDs), 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA) reductase inhibitors (statins), and tocopherol (vitamin
E), have been reported to accomplish their effects through various
receptors and associated signaling pathways [23–28]. NSAIDs are
thought to act primarily through the inhibition of COX enzymes
that catalyze the committed step in prostaglandin (PG) and throm-
boxane (Tx) synthesis [29,30]. However, several studies show no
or only partial efficacy of these compounds in AD therapy, sug-
gesting that anti-inflammation may not be the sole therapeutic
target for the disease and that the complete inhibition of microglial
Chemical structure of the lead compound #326:
(1-Naphthalen-1-yl-3-[5-(3-thioureido-phenoxy)-pentyl]-
thiourea).
2.3. Cell cultures
Primary microglia cultures were derived from the cortices of
P1–P3 neonates of BALB/c mice. Animals were handled according
to the regulations of the National Health Research Institutes Ani-
mal Care and Use Committee. The method for cell isolation and
culture was reported previously [19]. Briefly, cells were dissociated
using an enzyme solution containing Dulbecco’s modified Eagle’s
medium (DMEM), ethylenediaminetetraacetic acid (0.5 mmol/l), l-
cysteine (0.2 mg/ml), papain (15 U/ml), and DNase I (200 ␮g/ml);
cells were titrated after dissociation. The culture medium (DMEM
with 10% fetal bovine serum, 100 U/ml penicillin, and 100 ␮g/ml
streptomycin) was changed after 24 h of initial seeding. On the

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J.-H. Chern et al. / Chemico-Biological Interactions 188 (2010) 228–236
Scheme 1. Synthetic scheme 1.
14th day in vitro (DIV), microglia were separated from the under-
lying astrocytic monolayer by gentle agitation; this method takes
advantage of the differential adhesive properties of these two types
of cells. Cells were seeded overnight followed by an additional
24-h incubation in serum-free DMEM containing penicillin and
streptomycin. Purity of the microglia was measured as described
elsewhere [19], and cultures were >99% positive for CD11b. RAW
264.7 cells were maintained in primary microglial culture medium
with 2 mM of glutamine.
doses of LPS, as indicated. To test the inhibitory potency of the
newly synthesized compounds on microglial activation, cells were
seeded overnight at 1 × 105 cell per well in primary microglial
culture medium followed by a 1-h pretreatment with the com-
pounds. An additional 24-h treatment of 10 ng/ml LPS in serum-free
DMEM was performed. Cells were fixed with 4% paraformalde-
hyde in PBS for 30 min followed by incubation with 3% H2O2 for
20 min. Without an additional blocking step, cells were then probed
with an antibody against iNOS (diluted 1:500 in PBS containing
0.1% Tween 20) overnight. After a 30-min wash with buffer con-
taining PBS and 0.05% Tween 20 (wash buffer), the cells were
probed with an HRP-conjugated secondary antibody (1:5000 in
PBS containing 0.1% Tween 20) for 2 h and then washed for 30 min
with wash buffer. Cells were then incubated with a reagent con-
taining 10-acetyl-3,7-dihydroxyphenoxazine (ADPH, AnaSpec) and
4ꢀ-6-diamidino-2-phenylindole (DAPI, 0.05 ␮g/ml) for 20 min; sub-
sequent fluorescent analysis utilized a plate reader (SpectraMax
M2, Molecular Devices). Fluorescent units for non-specific back-
ground controls (in which the primary antibody was omitted) were
subtracted from the experimental data, and the experimental data
was then normalized by the intensity of DAPI fluorescence. Data
are presented as a percentage of the positive controls treated with
LPS without compound pretreatments.
2.4. Viability assay
The effects of the drugs on cell viability were determined
by vibrant MTT assay (Molecular Probes). Primary microglia and
RAW264.7 cells were seeded in 96-well culture plates overnight fol-
lowed by incubation with drugs for an additional 24 h. Afterwards,
the cells were subjected to the MTT assay according to the manufac-
turer’s instructions. The samples were subjected to analysis using
a plate reader (SpectraMax M2, Molecular Devices). Cell viability is
presented as a percentage of non-treated control viability.
2.5. Cell-based ELISA for microglial activation
To establish the cell-based ELISA assay, primary microglia and
RAW264.7 cells were used and compared. Expression of iNOS
and COX-2 in response to LPS treatments was used as indica-
tion of microglial activation. Cells were equally seeded in 96-well
plates at 1 × 105 cells per well and were incubated overnight. This
was followed by treatments for an additional 24 h with various
2.6. Western blot analysis
RAW264.7 cells were seeded overnight at 3 × 105 cells per well
in 24-well plates followed by pretreatment with the compounds as
indicated for 1 h followed by LPS treatment (10 ng/ml) for an addi-

J.-H. Chern et al. / Chemico-Biological Interactions 188 (2010) 228–236
231
tional 24 h. Cells were harvested with a lysis buffer (50 mM Tris,
150 mM NaCl, 0.02% NaN3, 0.5% Nonidet P-40 [NP-40], 0.5% Triton
X-100, and protease inhibitor mixture) and protein concentrations
were determined using a Bio-Rad protein assay (Bio-Rad). To
measure the levels of iNOS and COX-2, cell lysates were subjected
to sodium dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE; 7.5% Tris–HCl) followed by Western blot analysis
using antibodies against mouse iNOS (BD Transduction Lab) and
COX-2 (Abcam) at dilutions of 1:2000 and 1:1000, respectively.
To quantify A␤ levels, cell lysates were subjected to 16.5% tricine
SDS-PAGE [19]. An antibody against ␤-actin (1:50,000) was used
to normalize the quantity of protein in each sample. Enhanced
chemiluminescence (ECL, Amersham) was used for Western blot
detection. Corresponding bands were quantified with Image J
software from the NIH. Data are presented as a percentage of
LPS-treated cells without any drug treatment.
percentage of live cells (negative for trypan blue). Three indepen-
dent experiments were performed for each condition.
2.10. Effects of compound #326 on zymosan phagocytosis
Assay for zymosan phagocytosis was performed according to the
literature with some modifications [31,32]. RAW264.7 cells were
seeded overnight at 3 × 105 cells per well in 4-well chamber slides
(Lab-Tek) followed by incubation in microglial culture medium for
an additional 24 h with or without treatment of compound #326
of 10 ␮M. Prior to addition of opsonized zymosan, some cells were
treated with cytochalasin d (1 ␮g/ml) for 30 min in the presence
or absence of compound #326. To obtain opsonized zymosan par-
ticles, Alexa Fluor 488-zymosan (Molecular Probes) was incubated
with opsonizing reagent (Molecular Probes) per the manufacturer’s
instruction. Cells were treated with opsonized zymosan particles
(10 particles per cell) for 1 h, and then washed with ice-cold PBS.
Cells were then stained with 0.05% trypan blue (in PBS) for 3 min
to quench the fluorescence of un-ingested particles. The trypan
blue solution was removed and the cells were then washed with
PBS twice. Cells were fixed in 4% paraformaldehyde (in PBS). The
amount of opsonized zymosan particles was measured by fluores-
cent microscopy. We observed >1300 cells for each condition in
randomly chosen fields and the percentage of cells with >2 fluores-
cence particles was determined. Three independent experiments
were performed for each condition.
2.7. Analysis for Aˇ uptake and Aˇ degradation
RAW264.7 cells were seeded in six-well plates at 3 × 105 cells
per well in microglial culture medium. After overnight seeding, the
medium was replaced with 1 ml of serum-free DMEM containing
penicillin and streptomycin. Prior to cell treatment, a 100× stock
solution of FITC-labeled A␤ was prepared by dissolving the com-
pound in 2 mmol/l of NaOH; the dissolved peptide was diluted to
a concentration of 10 ␮mol/l in PBS. To measure A␤ uptake, cells
were treated with FITC-labeled A␤1–42 (200 nmol/l) for 24 h fol-
lowed by trypsinization (0.05%) and centrifugation as described
previously [19]. Cells were then resuspended in PBS and subjected
to flow cytometry. A flow cytometer (FACScan; BD Biosciences) set
to read 10,000 cells per measure was used to analyse A␤ uptake as
an indication of A␤ phagocytosis. Phagocytosis activity is presented
as the percentage of mean FITC intensity of drug-free controls.
For the A␤ degradation assay, cells were re-seeded in 24-well
plates following two washes with cold PBS after 24 h of A␤ treat-
ments (0 h) as described above. Cell lysates were collected after a
6 h or 24 h chase and were subjected to 16.5% tricine SDS-PAGE fol-
lowed by Western blot analysis. The amount of A␤ was quantified
using Image J and percent changes from the original A␤ levels (0 h)
are presented.
2.11. Statistical analysis
Data are presented as the mean standard error of the mean
(SEM). Statistical analyses were performed using one-way ANOVA
and Bonferroni’s post-hoc analysis. A value of p < 0.05 was consid-
ered to be statistically significant.
3. Results
3.1. Anti-inflammatory effects of novel thiourea derivatives on
microglial activation
The effects of the newly synthesized thiourea derivatives on
microglial immune responses, including expression levels of iNOS
and COX-2, were examined by a fluorescent cell-based ELISA
screening platform using RAW264.7 cells, a microglia-like cell line.
To validate the cell-based ELISA, dose response curves for iNOS
and COX-2 were examined using primary microglia and RAW 264.7
cells (Fig. 1). Various doses of LPS treatments, ranging from 10 to
500 ng/ml, were used. For comparison, some samples were also
subjected to Western blot analysis. The data show that a signifi-
cant up-regulation of iNOS expression was detected in RAW264.7
cells at doses of ≥10 pg/ml (p < 0.01) and ≥100 pg/ml (p < 0.01) using
cell-based ELISA and Western blot analysis, respectively (Fig. 1A).
LPS-induced iNOS expression showed no further increase in cells
treated with LPS at ≥10 ng/ml by either method. These results
indicate that the cell-based ELISA showed a slightly higher sen-
sitivity than Western blot analysis in the detection of LPS-induced
iNOS expression. Response curves for the same doses were exam-
ined in primary microglia using the cell-based ELISA. Results from
primary microglial cultures show that a significant up-regulation
of iNOS expression was observed at ≥10 ng/ml of LPS treatments
(p < 0.01); in addition, LPS-induced iNOS expressions showed no
further increase in cells treated with LPS at ≥100 ng/ml. These
data suggest that the cell-based ELISA using RAW264.7 cells may
have a greater sensitivity for detecting changes in iNOS expres-
sion compared with ELISA using primary microglia. A significant
up-regulation of COX-2 expression was observed in cells treated
with LPS concentrations ≥1 ng/ml (p < 0.01) by both methods and
2.8. Effects of compound #326 on secretion of IL-6
RAW264.7 cells were seeded overnight at 3 × 105 cells per well
in 24-well plates followed by pretreatments with the compound
#326 at concentrations of 1, 10, 20, and 50 ␮M for 1 h in serum-
free DMEM containing penicillin and streptomycin. Cell cultures
were then treated with LPS (10 ng/ml) for an additional 24 h. The
culture media were collected and subjected to the measurement of
IL-6 using ELISA kit (Invitrogen) per the manufacturer’s instruction.
Three independent experiments were performed for each condi-
tion.
2.9. Trypan blue staining for the effects of compound #326 on cell
viability
For an alternative assay to measure cell viability, RAW264.7 cells
were seeded overnight at 3 × 105 cells per well in 4-well cham-
ber slides (Lab-Tek) followed by an additional 24-h incubation in
serum-free DMEM containing penicillin and streptomycin with or
without treatment of compound #326 of 50 ␮M. After washing with
ice-cold PBS, the cells were stained with 0.05% trypan blue (in PBS)
for 5 min. After washing with PBS, cells with positive staining were
observed by microscopy. We observed >1400 cells for each con-
dition in randomly chosen fields and the data are presented as a

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J.-H. Chern et al. / Chemico-Biological Interactions 188 (2010) 228–236
Our results show that most of the thiourea compounds, except
#482 and #487, significantly reduced LPS-induced expression of
iNOS (Fig. 2A) at a concentration of 10 ␮M, whereas in the anti-
inflammatory category, only IBU had significant effects. There were
11 thiourea compounds that showed significant suppression of
COX-2 levels at a concentration of 50 ␮M, and only DM from the
group of anti-inflammatory drugs had a significant effect (Fig. 2B).
Taken together, nine compounds showed significant effects on both
iNOS and COX-2 expression. Among the thiourea compounds, treat-
ment with compound #326, #449, #457, or #458 resulted in at least
a 50% of suppression of both iNOS and COX-2 expression.
To examine whether the anti-inflammatory effects of these
compounds were due to cytotoxicity, primary microglia and
RAW264.7 cells were subjected to cell viability analysis that was
evaluated by an MTT assay as described in the methods section.
The results show that the individual compounds, including all of
the tested thiourea derivatives, l-NMA, IBU, and DM, at concen-
trations of 50 ␮M had no significant effects as assessed by the
MTT assay in either the primary microglial culture or in RAW264.7
cells (data not shown). This suggests that all of the tested com-
pounds in the present study have no effect on cell viability. Thus,
the anti-inflammatory effects of these compounds cannot simply
be explained by a total reduction in cellular functions, and the
inhibitory mechanisms underlying the effects of these compounds
remain to be explored.
3.2. Effects of the thiourea derivatives on Aˇ uptake
A␤ phagocytosis is an important cellular function in AD patho-
genesis, and this phagocytosis may reduce amyloid levels in
the brain and prevent neurons from further damage by limit-
ing the exposure to neurotoxic substances. To screen the potency
of the compounds as modulators of microglial A␤ phagocytosis,
RAW264.7 cells were treated with the various drugs and were sub-
jected to an analysis of A␤ uptake by flow cytometry, as described
previously [19]. Our results show that ten compounds signifi-
cantly increased A␤ uptake: the effect of compounds #326, #449,
#450, #457, #458, #462, #484, and #485 reached a significance
of p < 0.001, while that of compounds #416 and #462 reached
p < 0.01 (Fig. 3). The three most potent compounds, #326, #450, and
#485, enhanced A␤ uptake by 3.1 0.2, 2.8 0.2, and 4.3 0.3 fold,
respectively. Of note, the anti-inflammatory drugs had no effect on
A␤ uptake.
To select the most potent compound that can achieve a favor-
able combination of functions in modulating microglial activation,
the potency of the compounds for enhancing A␤ phagocytic activ-
ity and for inhibiting LPS-induced immune responses, including
iNOS and COX-2 expression, was compared (Fig. 4). The potency
of the compounds in the inhibition of LPS-induced iNOS and COX-
2 expression is presented as an immune inhibition index. The
immune inhibition index was calculated as follows: 100%/(average
inhibitory % of iNOS and COX-2). A higher score on the immune
inhibition index indicated a stronger inhibition of the expres-
sion of iNOS and COX-2. A␤ phagocytic activity was measured
by determining the extent of A␤ uptake compared with that of
compound-free controls using flow cytometry. The results showed
that compounds #326, #449, #457, and #458 had both immune
inhibition indices >2 (indicating >50% inhibition of both iNOS and
COX-2 expression) and phagocytic activities >2 (indicating >200%
A␤ uptake compared to the controls). Among those compounds,
#326 showed the highest total score of the two functions (phago-
cytic activity = 3.1 and immune inhibition index = 2.5), suggesting
that compound #326 may be the most potent compound for pro-
moting A␤ phagocytosis and for inhibiting microglial immune
responses. The in vitro efficacy of compound #326 on microglial
functions was subjected to further investigation.
Fig. 1. Dose responses curves for LPS-induced expression of iNOS and COX-2 for
validating the use of the cell-based ELISA. The LPS-induced expressions of iNOS and
COX-2 were measured by cell-based ELISA and Western blot analysis using either
primary microglia or RAW264.7 cells. Cells were equally seeded at 1 × 105 cells
per 24-well plate for all experiments. Expression levels are presented as a % change
compared with expression levels in the non-treated controls (mean SEM). Expres-
sion levels measured by the cell-based ELISA were normalized with cell number (as
measured by DAPI staining in the corresponding samples). For Western blot anal-
ysis, expression levels were normalized by ␤-actin. ELISA-primary and ELISA-raw
denote the use of primary microglia and RAW264.7 cells in the cell-based ELISA,
respectively. Western-raw denotes that RAW264.7 cells were used in Western blot
for comparison.
in both cell types. There was no further increase in COX-2 expres-
sion when cells were treated with LPS concentrations ≥10 ng/ml.
Althoughthe cell-basedELISAusing RAW264.7cells tended toshow
lower expression levels of COX-2 in cells treated with LPS concen-
trations ≥1 ng/ml compared with COX-2 expression levels using
another method or cell type, it appeared that the cell-based ELISA
using RAW264.7 cells had the best linearity in detecting signifi-
cant changes of LPS-induced COX-2 expression between 0.1 and
10 ng/ml of LPS treatments. All of the methods tested in this study
appear to have comparable capabilities for detecting changes in
LPS-induced microglial immune responses, but the cell-based ELISA
using RAW264.7 cells is much simpler (with respect to the prepa-
ration) and more cost-effective compared with the methods of
Western blot analysis and primary microglial cultures.
In the present study, 20 newly synthesized compounds and
three well-known anti-inflammatory drugs, NG-methyl-l-arginine
(l-NMA), ibuprofen (IBU), and dextromethorphan (DM), were
examined at concentrations of 10 ng/ml LPS using cell-based ELISA.

J.-H. Chern et al. / Chemico-Biological Interactions 188 (2010) 228–236
233
Fig. 2. Inhibition of immune responses by thiourea derivatives. Cell-based ELISA was used to evaluate the potency of compounds in the inhibition of immune responses.
Expression levels are presented as the % of LPS (10 ng/ml) treated controls (mean SEM). Concentrations of the compound were 10 and 50 ␮M for iNOS and COX-2, respectively.
The series # for each compound and the three well-known anti-inflammatory drugs, NG-methyl-l-arginine (l-NMA), ibuprofen (IBU), and dextromethorphan (DM), that were
used are listed on the x-axis. The results show that most of the thiourea compounds, except #482 and #487, significantly reduced LPS-induced expression of iNOS (p < 0.001
except #484, p < 0.01). Only IBU in the group of anti-inflammatory drugs had a significant effect on iNOS expression (p < 0.01). For COX-2, compounds #326, #356, #371, #416,
#449, #450, #457, #458, #482, #483, and #487 at concentrations of 50 ␮M showed significant effects (p < 0.001), whereas only DM from the group of anti-inflammatory
drugs reached significance (p < 0.001). Among the thiourea compounds, #326, #449, #457, and #458 showed at least 50% suppression of both iNOS and COX-2.
3.3. Modulation of microglial innate immunity by compound
#326
ues of compound #326 for iNOS and COX-2 are estimated at
>0.55 ␮M and >35 ␮M, respectively. In addition, compound #326
suppressed the LPS-induced secretion of IL-6 in RAW264.7 cells
in a dose-dependent manner. LPS-induced secretion levels of IL-
6 were 23.9 0.6, 22.8 0.9 (non-significant), 16.8 1.4 (p < 0.01),
15.6 0.5 (p < 0.001), and 12.5 0.6 ng/ml (p < 0.001) when cells
were treated without or with compound #326 at concentrations
of 1, 10, 20, and 50 ␮M, respectively (significance denotes the
comparisons between the samples with and without treatment of
compound #326). The basal levels of IL-6 secretion in cells without
LPS treatment were not affected by compound #326 treatments at
all concentrations, ranging from 1 to 50 ␮M, and were undetectable
by ELISA.
To examine the dose response of the anti-inflammatory activ-
ity for compound #326, RAW264.7 cells were pretreated with
various doses of compound #326 followed by an additional 24 h
of LPS treatment (10 ng/ml). Samples were subjected to Western
blot analysis as shown in Fig. 5A. The data show that treat-
ment with compound #326 significantly reduced LPS-induced
iNOS and COX-2 expression in a dose-dependent manner. The
lowest doses that cause a significant suppression of iNOS and
COX-2 were 1 ␮M (30 9% of the controls) and 50 ␮M (41 20%
of the controls), respectively (Fig. 5B). The predicted IC50 val-

234
J.-H. Chern et al. / Chemico-Biological Interactions 188 (2010) 228–236
Fig. 3. Effects of thiourea derivatives on A␤ phagocytosis. RAW264.7 cells were pretreated with compounds and anti-inflammatory drugs followed by incubation with
fluorescently labeled A␤ for 24 h. A␤ uptake was measured by flow cytometry, which serves as an indicator for A␤ phagocytosis. Data are presented as fold changes in
fluorescence intensity compared with the control without compound treatment (mean SEM). The significance of the effects demonstrated by compound #326, #449, #450,
#457, #458, #462, #484, and #485 reached p < 0.001, whereas for #416 and #462 the significance reached p < 0.01. l-NMA, IBU, and DM, had no effect on A␤ uptake.
As shown by the MTT assay, treatments of compound #326
for 24 h had no influence on cell viability for RAW264.7 cells
(110 3.4 and 114.4 0.4% of the controls for compound #326 at
10 and 50 ␮M, respectively) compared with controls (100 5.9%).
Similarly, an alternative assay for cell viability using trypan blue
also showed no difference in the percentage of live cells between
RAW264.7 cells treated with 50 ␮M of compound #326 for 24 h
(97.7 0.2%) and those of the non-treated controls (97.3 0.2%).
As mentioned above, compound #326 may promote A␤
phagocytosis. Our results also showed that treatment with com-
Fig. 4. Functional analysis of the potency of microglial immunity modulation: com-
pound #326 as a lead compound. To select the most potent compound that can
achieve a favorable combination of functions in the modulation of microglial acti-
vation, the potency of the compounds for enhancing A␤ phagocytic activity and for
inhibiting LPS-induced immune responses was analyzed. A␤ phagocytic activity was
Fig. 5. Efficacy of compound #326 in suppressing microglial immune responses in
vitro. RAW264.7 cells were seeded overnight at 3 × 105 cells per well in 24-well
plates followed by pretreatment with various doses of compound #326 (ranging
from 0.1 to 100 ␮M) for 1 h followed by LPS treatment (10 ng/ml) for an additional
measured by the extent of A␤ uptake compared with compound-free controls using
24 h. Samples were subjected to Western blot analysis for iNOS and COX-2 expres-
flow cytometry. The inhibition of LPS-induced expression of iNOS and COX-2 is pre-
sion. Treatment with compound #326 significantly reduced LPS-induced iNOS and
sented as immune inhibition index. The immune inhibition index was calculated
COX-2 expression in a dose-dependent manner. The effective doses of compound
as follows: 100%/average inhibitory % for both iNOS and COX-2. Compound #326
#326 that demonstrated a significant suppression for iNOS and COX-2 were ≥1 ␮M
exerted the highest total score of the two cellular functions, phagocytic activity = 3.1
and immune inhibition index = 2.5.
and ≥50 ␮M, respectively. *p < 0.01 compared with control.

J.-H. Chern et al. / Chemico-Biological Interactions 188 (2010) 228–236
235
in this disease. Control of microglial activation, therefore, is a
potential prophylactic and/or interventional strategy for AD treat-
ment. It has been proposed that a favorable combination of a
reduced microglia-mediated neuro-inflammation and an enhanced
microglial phagocytic activity may be essential for AD therapy [33].
LPS has been widely used to study the innate immunity of microglia,
and its receptors, Toll-like receptors are known to contribute to
the induction of microglial activation to promote AD pathogene-
sis [34]. Thus, we took advantage of the use of LPS in our study.
Here, we have synthesized a series of novel compounds derived
from thiourea and found that some of these compounds may
pharmacologically modulate microglial activation in the desired
combinatorial manner. Among the compounds tested, compound
#326 appeared to be the most potent in promoting A␤ phago-
cytosis and in inhibiting the LPS-induced expression of iNOS and
COX-2. Furthermore, compound #326 suppressed the LPS-induced
secretion of IL-6, an important pro-inflammatory cytokine in exac-
erbating cognitive deficits [35]. Our data suggest that compound
#326 may possess the desired properties for the regulation of
microglial activation.
In contrast to many proposed drugs for AD that are prone to
deactivating microglia, the beneficial effects of compound #326
may not simply be due to complete blockade of cellular func-
tions, as evidenced by the MTT assay and the trypan blue assay.
In this study, treatments of compound #326 had no influence on
cell viability. Compound #326 appeared to modulate, rather than
deactivate, microglial function via an unknown mechanism and,
therefore, makes it possible to fine-tune microglial activation and
ensure its beneficial effects on AD treatment.
It has been known that multiple receptors, including scavenger
receptors, Fc␥Rs, ␣(6)␤(1)-integrin, and integrin-associated pro-
tein/CD47, are involved in the phagocytic machinery of microglia
by interacting with A␤ [36–39], while other receptors may indi-
rectly modulate microglial A␤ phagocytosis [40–42]. Conceivably,
the underlying mechanism of the observed effects of compound
#326 on A␤ phagocytosis may be involved in the modulation of one
or more of the receptor systems. Indeed, our results suggest that
treatment with compound #326 enhanced Fc␥ receptor-dependent
phagocytosis in RAW264.7 cells as indicated by an opsonized
zymosan assay, and the effects can be significantly inhibited by
the treatment of cytochalasin d. Intriguingly, while enhanced A␤
phagocytosis increased A␤ levels in cells treated with compound
#326 after A␤ treatment, the accumulated A␤ was degraded by
cells to levels comparable with control counterparts after 24 h of
chase (although there was a difference at 6 h of chase). This data
suggests that the accumulated A␤ levels induced by the treatment
with compound #326 were not affecting the efficiency of A␤ degra-
dation after 24 h. However, the direct impact of compound #326 on
microglial A␤ phagocytytic machinery, including A␤ retention and
clearance, and the underlying mechanism are under investigation.
Of note, the A␤ measured in the present study may be a mixture of
different orders of structure, although monomers were the major
species observed under the denaturing condition.
In conclusion, the unique combination of dual cellular func-
tions provided by compound #326 not only counteracts excess
oxidative stress, but also promotes the clearance of a neurotoxic
substance. If this remains true in vivo, the compound could serve as
both a prophylactic and as an intervention for early and advanced
stages of AD. As mentioned above, the effective dose for suppress-
ing the microglial immune responses was in the low ␮M range.
Thus, there is room for improvement in compound potency in vitro.
The present study revealed that the effects of this class of thiourea
compounds were very sensitive to variations in the chain length
of the alkyl linker. It is also interesting to note that the hydropho-
bic aryl group largely influenced the dual function effects of the
thiourea compounds. Compound #326, with a chain length of five
Fig. 6. Effects of compound #326 on the degradation of accumulated A␤. RAW264.7
cells were pretreated with 10 ␮M of compound #326 followed by treatment with
0.2 ␮M of A␤ for an additional 24 h. Cells were trypsinized and reseeded followed
by chase for 6 h or 24 h in the absence of compound #326 and A␤. Cell lysates were
subjected to tricine SDS-PAGE and the amount of A␤ (the 4 kDa monomer) was
quantified by Western blot analysis. The percent change of A␤ in cell lysates at two
chase time points, as compared with the original A␤ levels immediately after 24 h
treatment (0 h), are presented. At 0 h, the total A␤ in cells pretreated with compound
#326 was approximately 249 24% of that of the 0 h control (p < 0.01). After 6 h
of chase, the total A␤ content in cells treated with compound #326 was reduced
to164 33% of the 0 h control (approximately 66 13% of its original level at 0 h),
which was higher than that of the counterpart controls (22 6%, p < 0.01). After 24 h
of chase, the total A␤ content for compound #326-treated cells decreased further to
54 12% of the 0 h control (approximately 22 5% of its original level at 0 h) and was
not significantly different from those in the control counterparts (26 5%, p = 0.08).
pound #326 enhanced Fc␥ receptor-dependent phagocytosis in
RAW264.7 cells as indicated by an opsonized zymosan assay
(100 4.7 and 181.9 5.4% for the non-treated controls and
the cells treated with compound #326 of 10 ␮M, respectively,
p < 0.001). Intriguingly, cytochalasin d (1 ␮g/ml) significantly inhib-
ited opsonized zymosan phagocytosis in RAW264.7 cells (24.2 5.1
and 28.8 1.6% of controls for the cells in the presence and absence
of compound #326, respectively). It is also pathologically impor-
tant to know whether the accumulated A␤ remains un-degraded
inside the cells after treatment. Thus, RAW264.7 cells were pre-
treated with compound #326 (10 ␮M) for 1 h followed by treatment
with 0.2 ␮M of A␤ for an additional 24 h. After washing, cells were
trypsinized and reseeded followed by a chase for 6 h or 24 h in the
absence of compound #326. Cell lysates were subjected to tricine
SDS-PAGE and the amount of A␤ (approximately 4 kDa) was quan-
tified using Western blot analysis. The percent change from the
original A␤ levels immediately after the 24 h treatment with A␤
(0 h) is presented in Fig. 6. The data show that at 0 h, the total
A␤ in cells pretreated with compound #326 was approximately
249 24% of that in the 0 h controls (p < 0.01). These data are con-
sistent with the theory presented above: that compound #326 may
promote A␤ uptake. After 6 h of chase, the total A␤ content in cells
treated with compound #326 was reduced to164 33% of the 0 h
controls (approximately 66 13% of its original level at 0 h), which
was higher than that of the counterpart controls (22 6%, p < 0.01).
After 24 h of chase, the total A␤ content for compound #326 treated
cells decreased further to 54 12% of the 0 h control (approximately
22 5% of its original level at 0 h) and was not significantly different
from those in the control counterparts (26 5%, p = 0.08).
4. Conclusions
Microglial activation plays an important role in the patho-
genesis of AD, because microglia are inappropriately activated

236
J.-H. Chern et al. / Chemico-Biological Interactions 188 (2010) 228–236
carbons, exhibited the most potent functioning in this class of com-
pounds. The structure–activity relationship also indicates that the
chain length of the alkyl linker and hydrophobic aryl group are crit-
ical for the dual functions, and this evidence may offer a structural
basis for future drug design.
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