A small molecule targeting the multifactorial nature of Alzheimer's disease

Article abstract of DOI:10.1002/anie.200700256

(Figure Presented) Sights set on a cure: Memoquin (1) affects several mechanisms relevant to Alzheimer's disease (AD): the formation of reactive oxygen species, the processing and aggregation of amyloid β (Aβ) peptides, and acetylcholinesterase activity.

Full text of DOI:10.1002/anie.200700256

Angewandte
Chemie
DOI: 10.1002/anie.200700256
Neurochemistry
A Small Molecule Targeting the Multifactorial Nature of Alzheimerꢀs
Disease**
Andrea Cavalli, Maria Laura Bolognesi, Simona Capsoni, Vincenza Andrisano,
Manuela Bartolini, Elisa Margotti, Antonino Cattaneo, Maurizio Recanatini, and
Carlo Melchiorre*
Alzheimerꢀs disease (AD) is a complex multifactorial syn-
drome unlikely to arise from a single causal factor, but due to
a number of different but related biological alterations that
contribute to its pathogenesis.[1] None of the presently
marketed drugs, although valuable in improving cognitive,
behavioral, and functional impairments, alter AD progres-
sion,[2] and the goal of an effective disease-modifying treat-
ment is still unmet. Nowadays, mechanism-based drug-design
approaches are mainly directed toward two proteins, amy-
loid b (Ab) and tau. Ab is the core constituent of senile
plaques, one of the key pathological characteristics of AD,
whereas phosphorylated tau is the main component of
neurofibrillary tangles, the other hallmark lesion of AD.[3]
Despite enormous research effort, no new molecules that
interfere with the biochemical pathways of either Ab or tau
have been approved so far for the treatment of AD. This
failure is probably due to the weakness of the classic drug-
discovery approach based on the “one molecule, one target”
paradigm, which might prove inadequate when the molecular
complexity of the disease is addressed. In fact, the multi-
factorial nature of AD and the current lack of an accepted
unitary theory to account for AD neurodegeneration have
formed the basis of a growing consensus that single com-
pounds are required that are able to interact with several
molecular targets in the neurotoxic cascade (the innovative
“one molecule, multiple targets” paradigm).[4,5]
key target points of AD neurodegeneration. Compound 4 was
designed rationally by incorporating a 1,4-benzoquinone
functionality as a radical scavenger into the polyamine
skeleton of a previously reported series of cholinergic
derivatives.[7] A straightforward synthetic strategy was devel-
oped (Scheme 1), and the biological profile of 4 was then
explored in detail by means of both in vitro and in vivo assays
to assess its therapeutic potential for combating AD.
Scheme 1. Synthesis of 4: a) (EtO)2SO2, toluene, reflux, 76%; b) HBr/
CH3COOH, room temperature, 2 h, 98%; c) 1,4-benzoquinone, EtOH,
room temperature, 6 h, 17%. Cbz=C6H5CH2OCO.
Herein, we report on a new drug candidate (memoquin
(4), patent pending) derived from a program[5–9] aimed at
creating multifunctional molecules to interfere with different
The antioxidant properties of 4 were verified initially by
testing in vitro its ability to neutralize free radicals.[10]
Compound 4 was able to decrease the formation of free
radicals with an inhibition percentage of 44.1 Æ 3.7, which is
slightly lower than that shown by trolox ((57.6 Æ 0.9)%).
Moreover, 4 was a good substrate for the enzyme NAD(P)H:-
quinone oxidoreductase 1(NQO1), with apparent Vmax and
KM values quite similar to those of menadione for reduction
by the enzyme (Table 1). These results show that 4 is itself a
fairly good antioxidant, and, more interestingly, that 4 might
be transformed by NQO1from the quinone into the hydro-
quinone. Therefore, we suggest that the oxidized and reduced
forms of 4 can coexist in vivo. The reduced form was
demonstrated to be the actual antioxidant species in the case
of other 1,4-benzoquinone derivatives, such as coenzyme Q
and idebenone.[11] To further validate this molecular mecha-
nism, 4 was also tested against the formation of reactive
oxygen species (ROS) in SH-SY5Y neuroblastoma cells.[12] At
a concentration of 3 mm, 4 did not cause any marked
difference in ROS formation from that in untreated cells
[*] Dr. A. Cavalli,[+] Prof. M. L. Bolognesi,[+] Prof. V. Andrisano,
Dr. M. Bartolini, Prof. M. Recanatini, Prof. C. Melchiorre
Department of Pharmaceutical Sciences
Alma Mater Studiorum, University of Bologna
Via Belmeloro, 6, 40126 Bologna (Italy)
Fax: (+39)051-209-9734
E-mail: carlo.melchiorre@unibo.it
Dr. S. Capsoni, Dr. E. Margotti, Prof. A. Cattaneo
Lay Line Genomics
Via di Castel Romano, 100, 00128 Roma (Italy)
[+] These authors contributed equally to this research.
[**] We thank C. Bergamini, R. Fato, and G. Lenaz for providing data on
NQO1 activity, A. Tarozzi and P. Hrelia for data on intracellular
antioxidant activity, and F. Mancini for data relating to BACE-1. The
MIUR, the University of Bologna, and Lay Line Genomics are
gratefully acknowledged for their financial support.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
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Table 1: Activities of 4 and reference compounds toward molecular
targets investigated.[a]
Target
NQO1
Memoquin (4)
max =3.48 mmminÀ1 mgÀ1
Reference compound
V
; V ;
max =7.29 mmminÀ1 mgÀ1
KM =12.7 mm
KM =1.20 mm[b]
hAChE
IC50 =(1.55Æ0.11) nm;
Ki =(2.60Æ0.48) nm
IC50 =(28.3Æ0.30) mm
IC50 =(23.1Æ4.8) nm;
Ki =(20.5Æ3.3) nM[c]
IC50 @100 mm[c]
Ab(1–40)
aggregation
(hAChE-
induced)
Ab(1–42)
self-aggrega-
tion
IC50 =(5.93Æ0.33) mm
IC50 =(108Æ23) nm
IC50 =(60.3Æ11.2) mm[d]
IC50 =(18Æ2) nm[e]
BACE-1
[a] Reference compounds (see the Supporting Information for chemical
structures): [b] Menadione. [c] Donepezil.[8] [d] Tetracycline. [e] Statine
derivative.
Figure 1. A docking model of 4 to human AChE: The contacts of the
benzylammonium ends of 4 with residues Trp86 and Trp286 at the
catalytic and peripheral sites of the enzyme, respectively, are shown.
Possible favorable contacts of the quinone moiety with the tyrosine
residues Tyr124 and Tyr341 of the gorge are also shown (H bonds,
yellow dashed lines).
((75.5Æ8.0)% for cells treated with 4, (100 Æ 6)% for
untreated cells). Conversely, when the same experiment was
repeated after pretreating the SH-SY5Y cells with sulfora-
phane, a potent inducer of NQO1,[13] 4 produced a remarkable
inhibitory effect on ROS formation relative to that in the
untreated cells ((56.6Æ4.0)% and (104 Æ 11)%, respectively).
NQO1activity is increased in the hippocampal pyramidal
neurons of AD patients and colocalizes closely with AD
pathology, which supports the hypothesis of its role as an
antioxidant system that is upregulated in response to the
oxidative stress of the AD process.[14,15]
Compound 4 is a potent inhibitor of the activity of human
acetylcholinesterase (AChE), with IC50 and Ki values 15 and 8
times lower, respectively, than those obtained for donepezil
(Table 1), the most potent marketed AChE inhibitor
(AChEI).[16] The Lineweaver–Burk plot (see the Supporting
Information) revealed that the inhibition was of a mixed type,
and we interpreted this result as evidence of the interaction of
4 with both the catalytic and the accessory peripheral anionic
site (PAS) of the enzyme. Docking simulations (Figure 1)
confirmed that 4 is able to bind simultaneously both the
catalytic site and the PAS of AChE.
imental conditions (50 mm, Ab/tetracycline 1:1) was (44.9 Æ
12.1)%.
To further explore the antiamyloidogenic profile of 4, we
also investigated its ability to act as an inhibitor of BACE-1,
an enzyme involved in Ab production from the amyloid
precursor protein.[21,22] Compound 4 was found to inhibit
BACE-1activity in a concentration-dependent manner with
an IC50 value of (108 Æ 23) nm, whereas a previously reported
statine-based derivative had an IC50 value of (18 Æ 2) nm[23]
(Table 1).
To test the action of 4 on AD neurodegeneration in vivo,
we used the anti-NGF transgenic mouse (AD11 mouse),
which has been shown to be a comprehensive animal model
for AD.[24,25] The ability of 4 to prevent AD-like neuro-
degeneration was tested at three stages: at 2 months of age,
which corresponds to a very early stage of neurodegenera-
tion; at 6 months of age, when the neurodegeneration is not
established completely; and at 15 months of age, when the
neurodegeneration is full-blown.[26] At all ages, 4 rescued the
cholinergic deficit in the basal forebrain, as quantified by
stereological counts of the number of cholineacetyltransfer-
ase (ChAT)-positive neurons (Figure 3a).
AChE can induce Ab aggregation, and AChEIs that bind
to the PAS can block this proaggregating action.[16,17] Com-
pound 4 showed a dose-dependent inhibitory effect on AChE-
induced Ab(1–40) aggregation (Table 1; Figure 2a). Remark-
ably, none of the marketed AChEIs tested under the same
conditions showed any antiaggregating activity.[16,18]
Besides the ability to inhibit AChE-induced Ab(1–40)
aggregation, which is likely to be relevant in the brain of AD
patients,[19] 4 also inhibited in vitro the self-assembly of Ab(1–
42), which is the most amyloidogenic Ab fragment found in
the AD plaques, in a dose-dependent manner (Table 1;
Figure 2b). When tested at an equimolar concentration
(50 mm) with Ab(1–42), 4 was able to inhibit fibril formation
by (95.5 Æ 0.4)%, whereas the marketed AD drug galant-
amine did not show any significant inhibitory activity under
the same experimental conditions. For comparison, the
inhibitory activity of tetracycline[20] under the same exper-
The initial appearance of Ab in dystrophic neurites of the
hippocampus of AD11 mice at 6 months of age[26] evolves, by
15 months of age, into a frank accumulation of extracellular
Ab deposits.[26,27] The administration of 4 caused a complete
reduction in Ab expression in 6-month-old AD11 mice and a
marked decrease in Ab deposits in 15-month-old AD11 mice
(Figure 3b).
To determine whether 4 was also able to prevent or rescue
tau hyperphosphorylation in the cortex, neurostereological
counts were performed to determine the number of neurons
labeled abnormally by the monoclonal antibody against
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Figure 2. a) Inhibition of AChE-induced Ab(1–40) aggregation by the
thioflavin T method. Compound 4 was used at different concentrations
(10–250 mm) and showed an IC50 value of (28.3Æ0.3) mm. b) Inhibition
of Ab(1–42) self-aggregation by the thioflavin T method. Compound 4
was used at different concentrations (1–50 mm) and showed an
IC50 value of (5.93Æ0.33) mm. For comparison, inhibition by galant-
amine (Gal) and tetracycline (Tetr) is also reported.
phosphotau AT8. Tau hyperphosphorylation was either
completely (at 2 and 6 months of age) or partially (at 15
months of age) prevented by the administration of 4
(Figure 3c).
Compound 4 was tested finally in
different animal models to assess its
Figure 3. a) Amelioration of the cholinergic deficit in AD11 mice. The administration of 4 restores
the number of cholinergic neurons in the basal forebrain (BF) of 2-, 6-, and 15-month-old AD11
ability to rescue the behavioral def-
icits linked to attention and memory.
The oral and intraperitoneal admin-
istration of 4 rescued the behavioral
impairment, as assessed by object-
mice. b) Effect of 4 on Ab accumulation in AD11 mice. At 6 months of age, Ab is localized in
deposits which form clusters in the hippocampus of AD11 mice. These deposits are absent in
control mice. At this age, treatment with 4 decreases the number of Ab-positive clusters. In 15-
month-old AD11 mice, Ab is localized in clusters and in extracellular plaques; these clusters and
plaques are absent in control mice. At this age, treatment with 4 partially decreases the number of
Ab plaques. The graph shows the number of clusters/plaques per histological section at the two
different ages. c) Effect of 4 on the accumulation of hyperphosphorylated tau in AD11 mice. In
AD11 mice, hyperphosphorylated tau accumulates in the somatodendritic compartment. No
accumulation was seen in age-matched wild-type mice or in animals treated with 4. The graph
shows the neurostereological counts used to quantify the difference in the number of neurons that
recognition
tests.
Remarkably,
besides its effectiveness in vivo, 4
showed other promising properties,
such as good oral bioavailability,
efficacy in crossing the blood–brain
barrier, and a favorable safety pro-
file. In addition, it is tolerated well
after prolonged administration. The
expressed phosphorylated tau in the three treatment groups. In all experiments, 4 (7 mgkgÀ1 dayÀ1
was administered intraperitoneally for 15 days to AD11 mice of different ages (n=4 per treatment
)
group). p<0.05 versus wild-type (WT) mice; # p<0.05 versus AD11 mice treated with a placebo.
*
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Communications
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detailed pharmacological characterization of
reported elsewhere.
4 will be
In conclusion, in a setting in which available drugs against
AD appear to be palliative rather than curative, memoquin
(4) is able to confront AD neurodegeneration on a different
level. This compound shows activity against the formation of
ROS and the processing and aggregation of Ab peptides, and
inhibits AChE. It can therefore be considered a breakthrough
drug candidate to face the biological complexity of AD.
Received: January 19, 2007
Published online: April 2, 2007
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Keywords: amyloid b · drug design · neurochemistry ·
neurodegeneration
.
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