Modulation of g-Secretase Reduces
b-Amyloid Deposition in a Transgenic
Mouse Model of Alzheimer’s Disease
Maria Z. Kounnas,4,6,7Anne M. Danks,4,6,8Soan Cheng,4,10Curtis Tyree,4Elizabeth Ackerman,4Xulun Zhang,1,6
Kwangwook Ahn,2,6Phuong Nguyen,4,10Dan Comer,4Long Mao,4Chengzhi Yu,4David Pleynet,4Paul J. Digregorio,4
Gonul Velicelebi,4,9Kenneth A. Stauderman,4,9William T. Comer,4,7William C. Mobley,5Yue-Ming Li,2
Sangram S. Sisodia,1Rudolph E. Tanzi,3and Steven L. Wagner4,10,*
1The Center for Molecular Neurobiology, University of Chicago, Chicago, IL 60637, USA
2Molecular Pharmacology and Chemistry Program, Memorial Sloan Kettering Cancer Center, New York, NY 10065, USA
3Genetics and Aging Research Unit, Massachusetts General Hospital, Department of Neurology, Charlestown, MA 02129, USA
4TorreyPines Therapeutics, Inc., La Jolla, CA 92037, USA
5Department of Neurosciences, University of California, San Diego, La Jolla, CA 92037, USA
6These authors contributed equally to this work
7Present address: Neurogenetic Pharmaceuticals, Inc., San Diego, CA 92121, USA
8Present address: Helicon Therapeutics, Inc., San Diego, CA 92121, USA
9Present address: CalciMedica, Inc., La Jolla, CA 92037, USA
10Present address: Department of Neurosciences, University of California, San Diego, La Jolla, CA 92037, USA
Alzheimer’s disease (AD) is characterized pathologi-
cally by the abundance of senile plaques and neuro-
fibrillary tangles in the brain. We synthesized over
pounds that reduced Ab42levels without inhibiting
epsilon-site cleavage of APP and Notch, the genera-
tion of the APP and Notch intracellular domains,
respectively. These compounds also reduced Ab40
levels while concomitantly elevating levels of Ab38
and Ab37. Immobilization of a potent GSM onto an
agarose matrix quantitatively recovered Pen-2 and
to a lesser degree PS-1 NTFs from cellular extracts.
Moreover, oral administration (once daily) of another
potent GSM to Tg 2576 transgenic AD mice dis-
played dose-responsive lowering of plasma and
brain Ab42; chronic daily administration led to signif-
icant reductions in both diffuse and neuritic plaques.
These effects were observed in the absence of
Notch-related changes (e.g., intestinal proliferation
of goblet cells), which are commonly associated
with repeated exposure to functional gamma-secre-
tase inhibitors (GSIs).
A large number of published studies, including those providing
genetic, biochemical, pathological, and epidemiological evi-
dence, lend significant support to the theory that alterations in
the relative levels of the Ab42and Ab40peptide species, i.e.,
Ab42/40ratio, may play a pivotal role in the pathogenesis of AD
(reviewed in Tanzi and Bertram, 2005). Generation of Ab
peptides requires sequential cleavage of APP by g-secretase-
mediated proteolysis of the b-secretase-generated C-terminal
APP cleavage product known as APP-C99 or b-CTF (Vassar
et al., 2009). Early approaches to therapeutic intervention
focused on lowering total Ab peptide production by inhibiting
cleaving enzyme 1) or g-secretase. ‘‘g-secretase’’ is a heteroge-
neous complex of membrane proteins (Serneels et al., 2009)
which regulate intramembrane proteolysis of APP (Sisodia and
St George-Hyslop, 2002) and a multitude of other substrates
tase-mediated Notch cleavage at the site 3 (S3) or epsilon (3) site
yields a large cytoplasmic peptide (Notch intracellular domain,
NICD) that translocates to the nucleus and which is necessary
for proper cellular differentiation and development (De Strooper
et al., 1999). Typical g-secretase inhibitors (GSIs) prevent catal-
ysis of both the g-sites and 3-sites, resulting in the potential for
adverse effects secondary to inhibition of 3-site proteolysis of
Notch, thereby preventing NICD formation (Kreft et al., 2009;
In view of the potential problems associated with inhibiting
both the g-site and 3-site cleavages of g-secretase, a more
attractive therapeutic alternative is to identify small molecules
that can preferentially lower the levels of the most fibrillogenic
Ab peptide, Ab42, without affecting catalytic activity at the
3-site. This approach has been supported in AD animal models
using an APP/Ab-binding nonsteroidal anti-inflammatory drug
(NSAID)-like compounds to achieve allosteric modulation of
g-secretase activity and attenuate the Ab42/Ab40ratio (Weggen
et al., 2001; Kukar et al., 2008).
In order to identify potent, selective, and orally bioavailable
small molecule modulators of g-secretase, we employed
cell-based assays to screen for compounds that lowered Ab42
levels without inhibiting Notch proteolytic processing (NICD
Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc. 769
generation) or related 3-site g-secretase-mediated cleavages.
For in vivo proof-of-principle studies, the most potent orally
bioavailable compounds were then tested for efficacy in Tg
2576 transgenic mice, which express the ‘‘Swedish mutant’’ of
human APP (APPswe) and overproduce Ab42and Ab40, at levels
leading to neuritic plaques and cerebral amyloid angiopathy
(Hsiao et al., 1996). If successful, this approach could lead to
the development of therapeutic regimens capable of safely inter-
vening in key neuropathologic processes associated with AD.
A chemical library composed of commercially available
compounds was designed using computational tools to provide
broad coverage of chemical space with ‘‘drug-like’’ chemical
properties. The chemical library, comprising ?80,000 com-
pounds, was purchased from a variety of commercial sources
and screened for the ability to suppress extracellular Ab42levels
produced from a Chinese hamster ovary (CHO) cell line stably
overexpressing APP695(referred to asCHO-PZ3 orCHO-APPwt)
Figure 1. Diarylaminothiazoles (Series A) and Dia-
rylureas (Series B) are Potent Modulators of g-Sec-
(A) Chemical structures of key molecules from Series A
and Series B GSMs, including Compound 6, the ethylene
amino derivative of Compound 3 that was immobilized
onto an Affigel matrix and used as an affinity chromatog-
(B) Concentration response curves (CRCs) for lowering of
Ab42levels produced by SH-SY5Y-APP cells. IC50values
were derived using four parameter fit nonlinear regression
analyses. Data are expressed as mean (±SEM) of
percentage of control.
using a monoclonal antibody-based homoge-
neous fluorescence resonance energy transfer
(FRET) high-throughput screening (HTS) assay.
One hit, which had an IC50value of 15 mM for
the inhibition of Ab42, passed all subsequent
screening criteria and several focused chemical
libraries were then designed and synthesized
based on this structure. One compound from
one of the focused libraries led to Compound 1
the original compound in the 2-aminothiazole
series (Series A). Lead optimization efforts led
to Compound 3 and Compound 4 (Figure 1A).
Subsequently, lead evolution efforts led to
Compound 7). All the compounds were subse-
quently tested in vitro for their ability to inhibit
the production of Ab42from a human neuroblas-
toma cell line (SH-SY5Y) stably overexpressing
human APP751 (SH-SY5Y-APP cells). Several
compounds from Series A exhibited impressive
Ab42lowering potencies (low nanomolar IC50 s)
comparable to some of the most potent GSIs
(e.g., BMS-299897, LY-411575, and GSI-953)
in similar cell-based in vitro assay systems (Martone et al.,
2009). This cell-based assay served to generate structure-
activityrelationships (SAR)formodulators ofg-secretaseactivity
and was the key parameter used to evaluate potencies for the
selection and prioritization of compounds pursued in primary
and secondary pharmacological studies, including in vivo effi-
cacy testing (Figure 1B).
Differentiation of GSMs versus GSIs
The Series A GSM, Compound 3, differentially inhibited Ab42and
Ab40with greater potency on reducing Ab42levels but had no
effect on Abtotal peptide levels in either the CHO-APPswe
or SH-SY5Y-APP cell-based assays (Figures 2A and 2B).
This is in contrast to the arylsulfonamide-containing GSI,
BMS-299897,whichequivalently inhibited Ab42,Ab40,andAbtotal
peptide production in these same two cell-based assay systems
(Figures 2C and 2D). To further characterize the effects of GSM
compounds on Ab peptide species (Ab37, Ab38, Ab40,Ab42, and
Abtotal), several different methods were employed to quantitate
these peptides from both in vitro and in vivo samples. GSMs
and GSIs exhibited differential inhibition profiles for several Ab
Modulation of g-Secretase Reduces Amyloid
770 Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc.
peptide variants in CHO-APPswe cells using immunoprecipita-
tion in combination with surface enhanced laser desorption/ioni-
zation time-of-flight mass spectrometry (SELDI-TOF MS)
(Figure 3A). The arylsulfonamide GSI, BMS-299897 reduced
the levels of all Ab peptide variants to baseline levels (exoge-
nously added Ab1-28 was used as an internal standard).
In contrast, three different Series A GSMs (Compound 2, Com-
pound 5, and Compound 3) reduced Ab40levels while increasing
Similar results were observed when comparing the effects of the
arylsulfonamide GSI, BMS-299897, with the Series A GSM
Compound 2, on various Ab peptide variants secreted from Tg
2576-derived primary mixed brain cultures (MBCs) analyzed
using tris-glycine gel electrophoresis in combination with anti-
(Figure 3B). In good agreement with the SELDI-TOF MS experi-
ments depicted in Figure 3A, BMS-299897 reduced the levels of
while the Series A GSM Compound 2 reduced the levels of Ab42
and Ab40while concomitantly increasing the levels of Ab38and
Ab37. We next attempted to extend the observations on the
brain samples from Tg 2576 mice dosed orally with Compound 2
(100 mg/kg for 5 consecutive days) showed a similar profile with
respect to effects on the various Ab peptide variants (decreased
levels of Ab42and Ab40; increased levels of Ab38and Ab37). Inter-
estingly, plasma levels of Ab39 were also reduced in the
Compound 2-treated versus the vehicle-treated mice, as were
the levels of Ab40and Ab42, while the levels of Ab37and Ab38
were increased, in concordance with the in vitro studies as
well as data from the various brain extracts (Figure 3C).
% Veh control
% Veh control
% Veh control
0.11 10 100100010000
% Veh control
Figure 2. Differential Effects of a Series A
GSM, on Levels of Ab42, Ab40, and Abtotal
Concentration-response curves showing effects
of the Series A GSM, Compound 3 (A and B) or
the functional GSI, BMS-299897 (C and D) on
levels of Ab42, Ab40, and Abtotalpeptide variants
produced by CHO-APPswe cells (A and C) or
SH-SY5Y-APP cells (B and D). Data are expressed
as mean (±SEM) of percentage of control.
APP-C99 is cleaved by g-secretase at
two distinct processing sites; one near
the middle of the transmembrane domain
to release Ab peptides (g-cleavage sites)
and another near the membrane-cytosol
interface releasing an intracellular domain
termed AICD (3-cleavage sites). GSMs
not only differ dramatically from GSIs
with respect to their effects on g-secre-
tase processing at g-cleavage sites but
also at 3-cleavage sites (Figure 4). Upon
binding to certain ligands such as Delta
and Jagged, the Notch receptor is
rendered a g-secretase substrate (LaVoie
and Selkoe, 2003) that is cleaved at a site
analogous to the 3-site of APP. This generates the Notch
intracellular domain (NICD), a signaling fragment that translo-
cates to the nucleus and ultimately affects cell fate (Kopan and
Goate, 2000). In order to compare the effects of GSIs and
GSMs on g-secretase-mediated 3-site processing of APP
and Notch, we employed doubly transfected human embryonic
kidney 293 (HEK-APP-NDE_7-4 cells) harboring both APP and
NDE cDNA constructs. The dipeptidic carboxamide GSI,
ester or DAPT (Dovey et al., 2001), inhibited NICD generation
in a dose-dependent manner, at concentrations equivalent to
those required to inhibit Ab peptide generation (?10–30 nM).
In contrast, the Series A GSMs (Compound 5, Compound 3,
and Compound 2) had no detectable effect on NICD formation
in this very same cell-based assay (Figure 4A), even at concen-
trations R1000-fold higher (30 mM) than their IC50’s for inhibiting
To further confirm that GSM compounds can modulate g-sec-
retase cleavages at g-sites without inhibiting cleavage at 3-sites,
a cell-free assay system capable of detecting Ab42, Ab40, and
AICD was employed (Figure 4B; for experimental details, see
Supplemental Information available online). Utilization of this
cell-free assay allowed for a semiquantitative measurement
of AICD formation (3-site activity) and Ab peptide formation
(g-site activity) from identical cell-free preparations. Unlike
DAPT (1 mM), which totally inhibited the formation of AICD and
Ab (Ab data not shown), the Series A GSM Compound 3 had no
effect on AICD levels when tested over a broad range of concen-
strated dose-dependent inhibition of both Ab42and Ab40with
a similar preference for lowering Ab42as previously shown in the
various cell-based assays (see Figure 2). Although Compound 3
Modulation of g-Secretase Reduces Amyloid
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displayed significantly higher Ab42and Ab40IC50 sin this cell-free
assay as compared to the cell-based assays (perhaps due to
increased nonspecific protein binding and/or decreased effec-
tive drug concentrations), it did not affect 3-site activity as evi-
denced bytheunchangedlevels ofAICD,evenatconcentrations
Ab40and Ab42, respectively (Figure 4B).
we employed affinity chromatography using a highly active
Figure 3. Opposing Effects of Series A
GSMs on Levels of Ab42and Ab40Versus
Effects on Levels of Ab38and Ab37Peptide
(A) SELDI-TOF mass spectroscopic analyses of
anti-Ab17–24 mAb (4G8) immunoprecipitates of
conditioned medium from CHO-APPswe cells
treated with either vehicle (top panel), the func-
tional GSI BMS-299897 (1 mM), also referred to
as SIB-3520 (second panel), or the allosteric
GSMs (each at 1 mM), Compound 2, Compound
5, or Compound 3 (bottom three panels, respec-
tively). Equal amounts of synthetic Ab1–28peptide
were spiked into aliquots of vehicle- and the
various compound-treated media samples prior
to performing the immunoprecipitations and was
used as an internal standard.
(B) Immunoblots of anti-Ab1–12(mAb-B436) immu-
medium of Tg 2576 MBCs treated with vehicle,
the functional GSI BMS-299897 (1 mM), also
referred to as SIB-3520, or the indicated concen-
trations of the allosteric GSM, Compound 2.
(C) Immunoblots of anti-Ab1–12(mAb-B436) immu-
noprecipitates (IP/westerns) of CHAPSO-solubi-
lized extracts of plasma or brain pools (left panel)
from either vehicle or Compound 2-treated
(100 mg/kg p.o for 5 consecutive days) Tg 2576
mice; (right panel) SDS-solubilized extracts of
representative individual hemibrains from either
vehicle or Compound 2-treated Tg 2576 mice.
Immunoprecipitates of conditioned medium of
Compound 2 (1 mM) were used as Ab peptide
compound (Compound 6) as a ligand.
We observed a quantitative retention of
Pen-2 and partial retention of PS1-NTFs
X-100/Tween-20 (1.0%/0.2%;v/v) deter-
gent-solubilized cellular lysates to the
Compound 6 affinity column (Figure 5).
There was only minor recovery of the
PS1-CTFs (C-terminal fragments) and
no recovery of APP-CTFs. One possibility
is that under these conditions some PS1-
NTF remains associated with compound
6-bound Pen-2 (perhaps via Pen-2/PS1-
considering the known association of these two essential
subunits (Kim and Sisodia, 2005), under very similar conditions
(Fraering et al., 2004). Another g-secretase subunit, nicastrin,
as well as a control protein, PLC-g, showed no detectable
binding to the Compound 6-derivatized affinity column. Aph-1a
subunits were not probed. Elution with GSM compounds and
competition experiments were not possible due to the core
aqueous solubility of the compounds.
An identical binding pattern to the Compound 6 resin: Pen-2 >
PS1-NTFs >> PS1-CTFs was also observed using the same
cellular extracts when solubilized with another nondenaturing,
Modulation of g-Secretase Reduces Amyloid
772 Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc.
nonionic detergent, dodecyl b-D-maltoside (DDM; 1.0%, v/v)
(Figure S1), but not when using the zwitterionic CHAPSO deter-
turing detergent solutions (DDM and Triton X-100/Tween-20)
manner than would the zwitterionic, nondenaturing CHAPSO
(Li et al., 2000). Steric hindrance due to immobilization of
Compound 6 using a short 10 atom spacer (Affigel 10) may have
prevented access to Pen-2/PS1-NTF when localized within the
fully assembled complex, since affinity capture of CHAPSO-solu-
2009). Considering the ability of the immobilized Compound 6 to
bind to isolated recombinant Pen-2 (Figure 5G) and the ability of
Ab42(as well as Ab40) formation at nanomolar concentrations in
membranes and in a reconstituted g-secretase assay system
utilizing highly purified requisite g-secretase subunits (Figure 6C),
suggest that Series A GSMs essentially shift the preferred g-
cleavage sites, increasing Ab37and Ab38and decreasing Ab42
and Ab40. Together with the lack of inhibition of NICD or AICD
formation (3-site cleavage products),these databegin toillustrate
a mechanism of action distinct from all known subclasses of
GSIs (Kreft et al., 2009) including the ‘‘allosteric GSIs’’ (AGSIs),
coumarin dimer-based noncompetitive inhibitors of g-secretase
et al., 2009). These data also serve to further differentiate these
Series A GSMs from NSAIDs, such as sulindac sulfide, that have
been shown to differentially and independently affect Ab42and
Ab38generation without affecting production of either Ab40or
Ab37(Page et al., 2008).
In Vitro and In Vivo Primary Pharmacology
Comparisons between several different Series A GSMs (data not
shown) demonstrated that Compound 4 had the most favorable
Figure 4. Series A GSMs Do Not Inhibit NICD Formation from Notch
(NDE) or AICD Formation from APP-CTFs
(A) Immunoblots (anti-NICD) of conditioned medium from HEK-APP-NDE cells
treated with the indicated concentrations of either DAPT or the designated
(B) Parallel analysis of AICD production (left panel) and Ab42and Ab40peptide
formation (right panel) from CHO-APPswe membranes treated with either
vehicle(0.2%DMSO),DAPT (1mM),ortheindicated concentrationsoftheallo-
steric GSM, Compound 3. Data are expressed as mean ± SEM (n R 3).
Figure 5. Affinity Chromatography using an
Immobilized Series A GSM Binds Isolated
Pen-2 and Quantitatively Recovers Pen-2
(A–F) Compound 6 specifically retains Pen-2 from
Triton X-100/Tween-20 (1.0%/0.2%)-solubilized
CHO-APPswe whole-cell lysates. Following gentle
agitation of solubilized whole-cell lysates (WCL)
with either the nonmodified Affigel matrix (?) or
Compound 6-immobilized Affigel matrix, flow
thru supernatants (Super) were collected and
with sample dilution buffer containing 1.0% SDS
(Elute). Equivalent aliquots of each fraction were
electrophoresed using 4%–20% gradient gels, im-
(A) anti-Pen-2, (B) anti- PS1-NTF, (C) anti-PS1-
CTF, (D) anti-nicastrin (NCT), (E) anti-APP-CTF,
and (F) anti-phospholipase C-g (PLCg).
(G) Affigel 10 with immobilized Compound 6 (+)
retains purified recombinant Pen-2 and Affigel 10
alone (?) does not. PS-Holo and APP-Holo corre-
spond to PS1-holoprotein and APP-holoprotein,
See also Figure S1.
Modulation of g-Secretase Reduces Amyloid
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PK properties (Tmaxp.o. = 3 ± 0.6 hr; Cl i.v. = 0.2 ± 0.03 l/hr/kg;
t1/2 p.o. = 2.0 ± 0.2 hr; F = 49%) and was therefore selected for
further in vitro and in vivo studies, including single dose, sub-
chronic, and chronic long-term efficacy studies. Using Meso
Scale Ab alloform-specific ELISAs, in vitro characterization of
Compound 4 in MBCs from Tg 2576 mice showed a concentra-
tion-dependent decrease in Ab42 as well as Ab40, markedly
increased levels of Ab38,and no significant change in Abtotal
levels (as defined by Ab1–x, where x is R 24). In contrast,
BMS-299897 induced a dose-dependent decrease in Ab42,
Ab40, Ab38, and Abtotallevels (Figure 6A). To confirm the differen-
tial effect onvarious Abpeptides including Ab37byCompound 4,
MALDI-TOF analysis of conditioned medium of the CHO-
APPswe cells following treatment with Compound 4 was
conducted (Figure 6B). In addition, when using either a HeLa
Figure 6. Compound 4, a Prototypical Series A
GSM that Potently Lowers Levels of Ab42 and
Ab40, Concomitantly Increases Levels of Ab38and
Ab37and Does Not Inhibit g-Secretase-Mediated
Generation of E-Cadherin g-CTFs (E-Cad/CTF-g)
or NICD Generation from Notch (NDE)
(A) CRCs for Compound 4 (left panel) or BMS-299897
(right panel) on various Ab peptide levels in conditioned
medium following treatment (18 hr) of Tg 2576 MBCs.
Data from each specific sandwich ELISA or MesoScale
assay were plotted as percent solvent control (0.2% v/v
DMSO). IC50or EC50values were determined using four
parameter fit nonlinear regression analysis.
(B) MALDI-TOF mass spectrometry of anti-Ab17-24mAb
(4G8) immunoprecipitates of conditioned medium from
CHO-APPswe cells after treatment (18 hr) with either
Compound 4 (1 mM) or vehicle (0.2% v/v DMSO).
(C) Concentration response curves for inhibition of Ab42
and Ab40production in HeLa cell membrane (left panel)
complex (right panel) in vitro g-secretase assays. IC50
values were derived using four parameter fit non-linear
regression analyses. Data are expressed as mean
(±SEM) of percentage of control.
(D) Immunoblots of E-cad g-CTFs and the GAPDH control
protein following treatmentof human A431 cells witheither
DAPT(1mM)or theindicated concentrationsofCompound
4 for 18 hr. Proteolysis of E-cadherin is induced by staur-
osporine (1 mM) treatment of A431 cells for 6 hr.
(F) Immunoblots of NICD and the GAPDH control protein
following treatment of HEK-APP-NDE cells with either
DAPT(1mM)or theindicated concentrationsofCompound
4. DMSO (0.2%v/v) is the vehicle control (Veh) and 0.53
sample volume from vehicle treated cells (1/2 Veh) was
analyzed to quantify relative levels of E-cad g-CTF immu-
noreactivity (E) or NICD immunoreactivity (G) using laser
See also Figure S2.
cell membrane in vitro g-secretase assay
system (Li et al., 2000) or a reconstituted g-sec-
retase assay composed of tandem affinity puri-
(Figure 6C; see also Figure S2 and Supple-
mental Information for experimental details)
(IC50 = 131 nM in the HeLa cell membrane
in vitro assay; IC50= 12 nM in the reconstituted TAP-isolated
protein complex g-secretase assay) and Ab40 production
(IC50= 531 nM in the HeLa cell membrane assay; IC50= 56 nM
in the reconstituted TAP-isolated protein complex g-secretase
assay) and did not inhibit NICD formation from the NDE Notch/
APP cotransfected cell line, in contrast to DAPT (1 mM), which
prevented NICD production entirely (Figures 6F and 6G). In order
to assess the effects of Compound 4 on another known g-secre-
tase substrate, E-cadherin, an assay similar to one described
previously (Marambaud et al., 2002) was employed using
a human epithelial cell line (A431) that endogenously expresses
measureable levels of E-cadherin proteolytic peptide products.
Proteolysis of the full length E-cadherin protein is induced
upon treatment of the cells with staurosporin, followed by gener-
ation of the E-cad CTF-g by g-secretase. Results of A431 cells
Modulation of g-Secretase Reduces Amyloid
774 Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc.
treated with Compound 4 in the E-cadherin g-secretase assay
are shown in Figures 6D and 6E, and demonstrate no inhibition
of g-secretase-mediated proteolysis of E-cadherin peptides at
concentrations up to 1000-fold above the IC50 values for
lowering Ab42 levels in SH-SY5Y-APP (Figure 6A) and NDE
Notch/APP (data not shown) cell-based assays. These results
strongly support the conclusion that Compound 4 behaves like
a prototypical Series A GSM.
Single and Repeat-Dose Efficacy Studies
Prior to testing for efficacy in vivo, brain and plasma concentra-
tions following oral dosing (50 mg/kg) were measured to assess
brain: plasma ratios of Compound 4 in C57Bl/6 mice. Plasma
and brain concentration versus time data from a representative
exposure study following oral dosing with Compound 4 are
shown in Figure 7A,and the mean data from two studies showed
plasma exposure (AUC) at 0–24 hr was 70,475 ng$hr/ml while in
brain was 65,545 ng$hr/ml, giving an average brain: plasma ratio
of 0.93. Compound 4 was then tested for efficacy for lowering
concentrations of brain Ab42 and Ab40 peptides in 3- to
Figure 7. Dose-Dependent Reduction of Ab42
Levels in Plasma and Brain in Tg 2576 Mice
following Oral Administration of the Series A GSM
(A) Time course of plasma and brain exposures of
Compound 4 in female C57Bl/6 mice (2–3 months of
age, n = 6 per group) following once daily oral administra-
tion for 3 consecutive days of Compound 4 (50 mg/kg,
p.o.) in F110 vehicle. Plasma and brains were collected
at the indicated times postdose on day 3 and concentra-
tions of Compound 4 were measured in plasma and brain
extracts by LC-MS/MS. Exposure is given as the inte-
grated area under the curve (AUC) of the compound
concentration versus time curve in ng$hr/ml. Brain/plasma
is the brain:plasma ratio of Compound 4 exposure.
(B) Dose-dependent efficacy of Compound 4 (p.o.) for
lowering Ab42in plasma (left panel) and brain (right panel)
in Tg 2576 mice (3–4 months of age, n = 10/dose)following
3 days of once-daily dosing expressed as a percentage of
vehicle (80% PEG-400 v/v) control.
each dose. Data are mean ± SEM. *p < 0.05 by t test for
brain measurements or ANOVA with Dunnett’s post-hoc
analysis for plasma measurements.
See also Table S1.
female mice (5–100 mg/kg p.o. in a PEG
400:water vehicle) following once daily dosing
for 3 consecutive days with samples being
collected 6 hr after the last dose. This in vivo
experimental paradigm was established based
on data from numerous pharmacodynamic
experiments confirming that 6 hr postdose is
an appropriate collection time to capture
(Figure 7A) and maximal effects for lowering
both the plasma and brain levels of Ab42(data
not shown). Brain and plasma Ab42concentra-
tions were measured by ELISA and compound concentrations
were measuredin drug-treated
Compound 4 elicited dose-ordered lowering of Ab42levels in
both plasma and brain following 3 days of consecutive daily
dosing (Figure 7B). The compound appeared to be more effica-
cious in plasma than in brain due to a combination of possibili-
ties: (1) compound exposures were possibly slightly greater in
plasma than in brain; (2) plasma Ab42turns over much more
rapidly than does brain Ab42(Cirrito et al., 2003); (3) nonspecific
protein binding of Compound 4 in brain may be greater than in
plasma, thus lowering the effective Compound 4 concentration
to a greater extent in brain than in plasma. Figure 7 shows that
following treatment of young female Tg 2576 mice with
increasing doses of Compound 4 (5–100 mg/kg, p.o.), Ab42
levels were significantly lower relative to vehicle-treated mice,
with a minimum effective dose of 25–50 mg/kg, and the extent
of reduction in brain Ab42levels correlated with brain concentra-
tions of Compound 4 (Figure 7C). The brain concentrations of
Compound 4 achieved following these dosing regimens were
in excess of those necessary to reduce Ab42levels based on
animals by LC-MS/MS.
Modulation of g-Secretase Reduces Amyloid
Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc. 775
the IC50 (29 nM) obtained from cell-based assays in MBCs
Compound 4 was further examined for in vivo efficacy
following either one day (50 or 200 mg/kg p.o.) or 14 days
(50 mg/kg p.o.) of once daily administration. Compound 4
produces consistent efficacy in lowering both plasma and brain
levels of Ab42and Ab40peptides when given acutely or for up to
two weeks of oral dosing (Table S1).
Chronic Administration Efficacy Study
We next asked whether chronic administration and presumably
sustained reduction of Ab42and Ab40brain concentrations, of
Compound 4 could affect the extent of pathology (i.e., amyloid
plaques) in the brains of Tg 2576 mice. To examine this, 8 month
old female (pre-plaque-bearing) Tg2576 mice were dosed daily
with an estimated 50 mg/kg/day (p.o.) of Compound 4 for 7
consecutive months. For this purpose Compound 4 was milled
tion of 0.3125 g Compound 4/kg chow and repelleted and the
Figure 8. Chronic Daily Exposure of Tg 2576 Mice
to the Series A GSM Compound 4 for Seven
Consecutive Months is Well Tolerated and Signifi-
cantly Reduces Overall Amyloid Load and Ab
Deposition in Neuritic Plaques
(A) Weekly group averaged body weights of Tg 2576 mice
(8–15 months of age) fed (ad libitum) either normal rodent
chow (Vehicle; n = 20) or rodent chow containing an esti-
mated dose of 50 mg/kg/day of Compound 4 (Compound
4;n = 19) for 29 consecutive weeks. Data are mean ± SEM.
(B) Brain Ab peptide (Ab42, Ab40, and Ab38) concentrations
(measured using Meso Scale triplex kits) in aqueous buffer
soluble (DEA), denaturing detergent-extractable (SDS) and
formic acid-extractable (FA) brain fractions from Tg 2576
mice fed either normal rodent chow or chow containing
Compound 4. Data are expressed as mean (±SEM) of
percentage of control levels in normal chow-fed mice
except for Ab38in FA extract; p = 0.0441).
Switzer silver stained sections showing neuritic plaque
staining in the cerebral cortex and hippocampus from
a normal chow-fed 15-month-old Tg 2576 mouse (left
panel) and a 15-month-old Tg 2576 mouse fed Compound
4 (50 mg/kg/day)-containing chow (right panel) from 8 to
15 months of age.
See also Table S2.
(D) Percentage of cerebral cortical area (left panel) and
hippocampal area (right panel) occupied by neuritic pla-
ques, quantified from four brain levels. Data are expressed
as percent area (mm2) of individual mice from normal
chow-fed (Vehicle) and Compound 4-containing chow-
fed (Compound 4) Tg 2576 mice.
mice (n = 19–20 per group) were allowed to
feed ad lib either Compound 4-containing
(treated) or standard (vehicle) rodent chow.
Compound 4-fed and vehicle-fed mice were
carefully observed and food consumption and
body weights were recorded weekly. Com-
pound 4 was well tolerated based upon behav-
ioral observations and the lack of differences in food
consumption (data not shown) or rate of body weight gain
between the groups throughout the 29 week period of exposure
(Figure 8A). In addition, plasma concentrations of Compound 4
were measured in a subset of treated mice at the end of the
chronic study and were found to be 3.95 ± 0.49 nmol/ml
(n = 12). This level of exposure, which presumably reflects
steady-state plasma concentration under this dosing regimen,
was roughly equalto peak plasma concentrations seen following
three days of once-daily dosing at 25 mg/kg given via oral
gavage. Data from the 3 day dosing paradigm has shown that
this plasma concentration is sufficient to significantly reduce
levels of brain Ab42in Tg 2576 mice (Figure 7C).
Evaluation of the gastrointestinal tract and semiquantitative
analysis of GI goblet cell densities in both the small and
large intestine of treated and untreated mice showed no treat-
ment-related differences between vehicle- and Compound
4-fed mice (Table S2). GI lesions, such as those previously re-
ported after repeated exposure to GSI’s (Searfoss et al., 2003;
Modulation of g-Secretase Reduces Amyloid
776 Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc.
Wong et al., 2004; Hyde et al., 2006) are typically associated
with inflammation, erosion and goblet cell hyperplasia, which
was not evidenced after chronic Compound 4 treatment. These
data strongly support the conclusion that allosteric modulation
of g-secretase is sufficient to significantly reduce Ab42-depen-
dent pathological endpoints without induction of gastrointestinal
Serial extraction of one brain hemisphere for each study
animal was done to capture soluble (DEA-extractions), deter-
gent-soluble (SDS), and formic acid-extracted (FA) Ab peptides.
Biochemical analyses of these extracts using the Meso Scale Ab
triplex kit revealed that the levels of all three Ab peptides
measured (Ab38, Ab40, Ab42) were dramatically and significantly
reduced in all three brain fractions of Compound 4-treated
mice, relative to control mice. Figure 8B shows percentage of
each Ab peptide, relative to control levels, in each brain extract
to control mice, resulted in 24% ± 4% (mean ± SEM) concentra-
tion of Ab38(p < 0.0001), 27% ± 4% of Ab40(p < 0.0001) and
24% ± 3% of Ab42(p < 0.0001) concentrations. In the SDS
extracts, Compound 4-treated mice had 47% ± 7% of Ab38
(p = 0.0016), 41% ± 6% of Ab40(p < 0.0001) and 52% ± 8% of
Ab42(p = 0.0003). In the FA extracts, the Compound 4-treated
mice had 55% ± 11% of Ab38(p = 0.0441), 48% ± 8% of Ab40
(p = 0.0014) and 45% ± 7% of Ab42(p = 0.002). Somewhat
surprisingly, levels of Ab38were significantly reduced in each
of the various brain extracts analyzed, in contrast to Compound
4’seffects ofincreasing Ab38inAPPswe-expressing cell cultures
and in brain and plasma with acute or short-term in vivo dosing.
aged plaque-bearing Tg 2576 mice under conditions of excep-
tionally high amyloid loads.
Fixed brain sections were stained with Campbell-Switzer stain
(Campbell et al, 1987) to visualize both diffuse and neuritic
amyloid plaques as depicted in representative photomicro-
graphs of Campbell-Switzer-stained sections from control
and drug-treated mice (Figure 8B). The percentage of cortical
and hippocampal area covered by plaque was measured by
quantitative image analysis through 4 levels of brain and
morphological and statistical comparisons were made between
treated and untreated mice (Figure 8D). While there is noticeable
variability among animals within the treatment groups (which is
entirely expected for this age of animals in this particular AD
mouse model), there is a highly statistically significant difference
between the Compound 4-treated and untreated mice. In
untreated mice, the mean percent of cortex occupied by plaque
was 7.6%, while in the Compound 4-treated mice the mean
percent area was 2.8%, p < 0.0001. In the hippocampus, 1.9%
of the hippocampal area was occupied by plaque in normal
chow-fed animals, while an average of 0.5% of hippocampal
area was occupied by plaque in Compound 4-treated mice,
p=0.0001(Figure8D).Thesehistological data show thatchronic
treatment (29 weeks) with Compound 4, given orally in rodent
chow, substantially inhibited plaque deposition in Tg 2576
mice between the ages of 8 and 15 months as shown by signif-
icantly lower plaque density and amyloid load in both hippo-
campus and cortex.
We have characterized a class of GSM compounds that display
unique pharmacological properties compared to other disease-
modifying therapeutic approaches (e.g., GSI’s) previously tested
or currently undergoing human clinical trials for AD (for review
see Pissarnitski, 2007). Unlike ‘‘substrate-targeting GSMs’’
e.g., R-flurbiprofen or tarenflurbil (Kukar et al., 2008), these
Series A GSMs exhibit potencies and levels of brain penetration
that are far superior to tarenflurbil or other NSAID-like GSMs
such as sulindac sulfide or CHF5074 (Weggen et al., 2001;
Imbimbo et al., 2007). In addition, Series A GSMs are approxi-
mately 1000- to 10,000-fold more potent than tarenflurbil,
sulindac sulfide, or CHF5074, based on in vitro IC50values in
comparable cell-based assays.
The Series A GSMs bind directly to the g-secretase enzyme
complex via Pen-2/PS1-NTFs and, in effect, elicit a truncation
of the major secreted Ab peptides (Ab42and Ab40), without inhib-
iting 3-site proteolysis of APP, Notch, E-cadherin, or LRP-1 (data
tion of Ab42and Ab40and cause a concomitant increase in the
levels of Ab38and Ab37. It is currently not known whether prod-
ucts of g-site cleavages from other g-secretase substrates
also be similarly affected. In any event, selectively affecting the
g-site cleavage products (e.g., Ab, Nb, etc.) from g-secretase-
mediated proteolysis of the numerous g-secretase substrates
would appear to be a prudent therapeutic approach, especially
when weighing the risks of inhibiting 3-site proteolysis. Interest-
ingly, 3-site proteolytic processing by g-secretase has been
much more widely studied than have the g-sites and in an
ever-expanding number of type I membrane proteins, involves
the generation of intracellular domains (ICDs), e.g., AICD,
NICD, and LICD, many of which appear to translocate to the
nucleus (De Strooper et al., 1999; Cao and Sudof, 2001; Waka-
bayashi and De Strooper, 2008).
A few of the functional GSIs previously described, i.e., the
arylsulfonamide- or sulfonamide-containing, alternative-site
binding GSIs, BMS-299897 (Milano et al., 2004), GSI-953 (Bega-
cestat), and BMS-708163 (Martone et al., 2009; Kreft et al.,
2009; Starrett et al., 2009), as well as at least one other
sulfonamide-containing GSI (Best et al., 2007) appear to be
‘‘Notch-sparing.’’ However, the mechanistic basis of this
apparent therapeutic window is poorly understood and the
effects of these ‘‘Notch-sparing’’ GSIs on g-secretase-mediated
3-site proteolysis of APP and other g-secretase substrates, with
the exception of Notch, have not been reported.
In the present study, we analyzed a finite number of g-secre-
tase (3-site-cleaved) substrates including APP (AICD), Notch
(NICD), E-cadherin (E-Cad/CTF-g), and LRP-1 (LICD); and
even when tested at concentrations far above (?1000-fold) the
in vitro IC50’s for lowering the levels of Ab42in cell-based assays,
we detected no inhibition regarding the 3-site proteolysis on any
of these four known substrates of g-secretase. Furthermore, the
lack of inhibition in cell-based in vitro assays on g-secretase-
mediated 3-site proteolysis of Notch (i.e., NICD formation) is
consistent with the lack of effect of Compound 4 on intestinal
goblet cell densities, even after chronic daily administration to
Modulation of g-Secretase Reduces Amyloid
Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc. 777
is not the case for some of the more thoroughly studied GSIs,
including Compound X (Searfoss et al., 2003) and LY-411,575
(Wong et al., 2004; Hyde et al., 2006) that show evidence of
Notch-related toxicity even after much shorter periods of admin-
istration than those employed here.
GSIshavebeenrecentlycategorized into anumberof different
subclasses based on their functional or structural similarities;
peptidic, dipeptidic, or peptidomimetic transition state mimics
or transition state analogs, and alternative binding site car-
boxamides, sulfamides, sulfonamides, sulfones, dibenzaze-
pines, benzodiazepines, etc., (for review see Kreft et al., 2009;
Tomita, 2009). Thus far most, if not all, of the various subclasses
of GSIs appear to interact directly with PS, the catalytic compo-
nent of g-secretase and would therefore be expected to
similarly inhibit the proteolysis of other g-secretase substrates
such as Notch (Lewis et al., 2008). Interestingly, the arylsulfona-
mide-containing GSI, BMS- 299897 (Tian et al., 2002; Anderson
et al., 2005), a noncompetitive g-secretase inhibitor that lowers
the levels of all measureable Ab peptide variants, concomitantly
causes accumulation of both a- and b-CTFs, suggesting a
mechanism consistent with dual inhibition of 3-site and g-site
Three other GSIs, LY450139 (Semagacestat), GSI-953 (Bega-
amide, a sulfonamide, and an arylsulfonamide, respectively)
would all be expected to interact directly with the catalytic
component of g-secretase (PS) and would therefore be antici-
pated to similarly inhibit Notch, APP, and a variety of other
known g-secretase substrates. Interestingly, recent clinical
studies with LY450139 have reported various adverse events
during phase I and phase II that may be interpreted as being
Notch mediated (Fleisher et al., 2008). The other two sulfon-
amide-containing GSIs (GSI-953 and BMS-708163) with an
open IND (investigational new drug) status both have a mecha-
nism of total Ab peptide inhibition described as ‘‘Notch sparing.’’
This classification is based primarily on the relative IC50or EC50
values obtained from a variety of comparable in vitro cell based
two GSIs can preferentially inhibit Ab peptide production from
APP/bCTFs versus inhibiting NICD production from Notch/NDE
with a rather impressive yet highly variable (14-fold for GSI-953
and 26- to 193-fold for BMS-708163) preference for lowering
Ab peptide levels (Martone et al., 2009; Starrett et al., 2009).
However, g-secretase complexes are known to hydrolyze
a large number of type I membrane proteins (Wakabayashi and
De Strooper, 2008), implying possible cooperation in major
membrane protein degradation and signaling pathways. There-
fore, inhibiting an enzymatic complex like g-secretase, which
has been described as the ‘‘proteosome of the membrane’’
(Kopan and Ilagan, 2004), may not be at all beneficial to an AD
population with compromised neuronal catabolism. The amino-
thiazole-containing GSMs described in the present study are not
inhibitors of g-secretase activity and are thus, genuinely ‘‘Notch-
sparing.’’ This is based on numerous in vitro and in vivo
endpoints observed even after prolonged periods of exposure,
throughout which the compound sustained the ability to reduce
Ab42peptide levels and attenuate amyloid deposition without
evidence of Notch-related GI toxicity. Furthermore the in vitro
studies, including affinity chromatography with a biologically
active Series A GSM (Compound 6), strongly suggests
g-secretase (i.e., Pen-2/PS1-NTFs) as the binding target and
support these aminothiazole GSMs as being capable of potently
and preferentially lowering both Ab42and Ab40levels without
inhibiting NICD or AICD formation. In summary, these experi-
ments describe a class of aminothiazole g-secretase modula-
tors, hereafter referred to as AGSMs, which exhibit a unique
pharmacological profile, in vitro and in vivo, that under steady-
state conditions is consistent with a noninhibitory mechanism
of action involving direct interaction with g-secretase. These
types of AGSM compounds warrant further in vitro and in vivo
investigation as a potentially safe and effective therapeutic
approach for the treatment and/or prevention of AD and perhaps
other related neurodegenerative proteinopathies (Ghaemma-
ghami et al., 2010).
Stable Cell Lines and Cell-Based Assays for Measuring Extracellular
Levels of Ab42and Ab40
The CHO-PZ3 (CHO-APPwt) cell line was derived by transfecting a Chinese
hamster ovary (CHO) cell line with a plasmid expressing wild-type human
APP695cDNA and selecting for stable expression of human APP and human
Ab. The CHO-APPswe cell line was derived by transfecting the same CHO
cell line with a plasmid expressing the ‘‘Swedish’’ (swe) double mutant
(670/671 K/M to N/L) cDNA of human APP695; the SH-SY5Y-APP cell line
was derived by transfecting a human neuroblastoma (SH-SY5Y) cell line with
a plasmid expressing wild-type human APP751cDNA. In each case, the levels
monoclonal antibody (mAb)-based homogeneous FRET high-throughput
screen (HTS) or a comparable HTS sandwich ELISA assay. Cytotoxicity was
determined using an Alamar Blue cell-based assay at the same compound
concentrations used to determine compound potency and efficacy up to
a maximum concentration of 30 mM. For complete experimental details, see
Cell-Based Assays for Extracellular Ab42and Ab40in Tg 2576 Mixed
Brain Cultures (MBCs)
Neuronal cultures were prepared from brains of Tg 2576 embryos (E17) and
were plated onto 96-well tissue culture dishes. Cells were grown for 6 days
and then treated in triplicate wells for 18 hr with compound at 10 concentra-
tions with 3-fold dilution steps. Vehicle (PEG-400:H2O 80:20 v/v) and
BMS-299897 were included as negative and positive controls, respectively.
For complete experimental details, see Supplemental Information.
Ab38, Ab40, Ab42, and AbtotalSandwich ELISAs
The Ab38peptide was quantified using either the Meso Scale Ab38,40,42triplex
kit or the Meso Scale Ab38kit alone with the Meso Scale Sector Imager 6000.
Ab40, Ab42, and Abtotallevels were also measured using mAb-specific sand-
wich ELISA assays.
Ciphergen chips prepared according to manufacturer-recommended proto-
cols from GSI- or GSM-treated CHO-APPswe conditioned medium were
analyzed in the SELDI mass spectrometer (Ciphergen). MALDI-TOF mass
spectrometry of anti-Ab peptide immunoprecipitates of conditioned medium
from compound-treated CHO-APPswe cells was performed by Dr. Rong
Wang (Mount Sinai School of Medicine, New York, NY) as described previ-
ously (Wagner and Munoz, 1999).
Modulation of g-Secretase Reduces Amyloid
778 Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc.
Cell-Based Assays for Measuring Notch and E-Cadherin Proteolytic
Doubly transfected human embryonic kidney 293 (HEK-APP-NDE_7-4 cells)
harboring both APP and Notch (NDE) cDNA constructs (A gift of Dr. Edward
H. Koo, University of California, San Diego) were treated with compound over-
night, conditioned medium was collected and cell lysates were prepared. Cell
lysates were analyzed for levels of NICD by western blot, and immunopositive
protein bands were quantified by laser scanning densitometry. As a means of
verifying compound activity, the conditioned medium was concomitantly
analyzed for levels of secreted Ab40by sandwich ELISA, as described above.
Measurement of inhibition of E-cadherin proteolytic processing was similar to
that described previously (Marambaud et al., 2002).
Affinity Chromatography and Immunoblotting
Compound 6 was coupled to Affigel 10 agarose for affinity chromatography.
Whole-cell lysates (WCL) were prepared from CHO-APPswe cells and were
incubated with 100 ml of resin for 2 hr at 4?C. Bound proteins were eluted
with 1% SDS sample dilution buffer and were separated on 4%–20% SDS
polyacrylamide gels, transferred to nitrocellulose filters, and probed with
a panel of antibodies. For complete experimental details, see Supplemental
Brain and Plasma Compound Concentration Measurements
Blood and brains were collected at the indicated interval (after the last dose),
and quantitative measurements of compound concentrations in the corre-
sponding plasma and brain extracts were made by LC-MS/MS (Tandem
Labs, West Trenton, NJ).
Male Sprague-Dawley rats (?300 g) were instrumented with jugular vein or
jugular and femoral vein catheters and allowed several days to recover then
fasted overnight and compounds were injected into the femoral vein catheter
at 2 mg/kg or administered by oral gavage at 10 mg/kg (n = 3–4/group).
Compound concentrations in plasma were measured by LC-MS/MS (Tandem
Labs, West Trenton, NJ). Pharmacokinetic parameters were calculated from
noncompartmental modeling using WinNonlin software (Pharsight Corp.).
In Vivo Efficacy Testing
All procedures conducted on animals were approved by TorreyPines Thera-
peutics’ IACUC and conform to current animal welfare guidelines. Briefly,
3- to 4-month-old female Tg 2576 mice were used for short term (%14 days)
efficacy studies (generally n = 10/group), and 8-month-old female Tg 2576
mice were used for the chronic efficacy study (n = 19–20/group). Dosing
was done by daily oral gavage in an 80% PEG 400 (v/v) vehicle for the
short-term studies and compound was milled into the rodent chow (0.3125 g
Compound 4/kg chow; Harlan Teklad 8664) for oral delivery in the chronic
study. For complete experimental details, see Supplemental Information.
Chronic Study GI Evaluation
Slides containing tissue from the small and large intestine were trimmed, pro-
for goblet cell numbers and density, and results were recorded separately for
small and large intestines. Goblet cell density was graded on a 3 point scale
with 1 having the least and 3 having the greatest goblet cell density. Evaluation
of goblet cell densities in the intestinal tracts of Compound 4-treated and
untreated Tg 2576 mice was performed in a blinded fashion by Midwest
ToxPath Sciences, Inc., Chesterfield, MO.
For complete experimental details, see Supplemental Information. Briefly,
brain hemispheres were processed and stained with the Campbell-Switzer
stain (Campbell et al., 1987) to visualize both diffuse and neuritic amyloid pla-
ques at NeuroScience Associates, Knoxville, TN. Four coronal levels were
used for image analysis to determine the percent area of the brain section
occupied by plaques. Image analysis of plaques in the cortex and hippo-
campus was done using NIH Image/Image J. An area of interest (cortex or
hippocampus) in the tissue sections was traced and was converted into two
other images, one used for obtaining the area (mm2) of the region of interest,
and the second for performing a particle analysis to obtain individual plaque
areas and total plaque number. The area of each plaque and the total number
were evaluated blind as to treatment.
Values are mean ± standard error of the mean (SEM) and expressed as the %
of the mean of vehicle-treated control groups. Differences between treatment
and control groups were analyzed by one-way analysis of variance and by
Dunnett’s t test when a significant (p < 0.05) F-value is attained. Correlation
analyses are performed using Pearson Product Moment correlation analysis
and IC50and ED50values estimated with non-linear regression (GraphPad
Supplemental Information includes two figures, two tables, and Supplemental
Experimental Procedures and can be found with this article online at doi:10.
The authors wish to thank Nicolas Patch for technical support, Pat Baskin for
critical review of the manuscript, and Eisai, Co. Ltd. and the NIH grant RO1
AG026660 (to Y.-M.L.) and the John Adler Foundation (to S.S.S.) for financial
support. M.Z.K. and W.T.C. are employees of Neurogenetic Pharmaceuticals,
Inc. (NGP), and W.T.C.,R.E.T., and S.L.W. are shareholders and cofoundersof
Accepted: August 10, 2010
Published: September 8, 2010
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780 Neuron 67, 769–780, September 9, 2010 ª2010 Elsevier Inc.