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From bitopic inhibitors to multitarget drugs for the future
treatment of Alzheimer’s disease
Running title: From bitopic to multitarget
Daniel I. Pérez, Ana Martínez, Carmen Gil* and Nuria E. Campillo*
Centro de Investigaciones Biológicas (CIB-CSIC). C/ Ramiro de Maeztu 9, 28040 MadridSpain
Correspondence:
Dr. Nuria E. Campillo and Dr. Carmen Gil
Centro de Investigaciones Biológicas (CIB-CSIC)
C/ Ramiro de Maeztu 9, 28040 Madrid- Spain
[email protected]; [email protected]
Abstract
Dementia is one of the main causes of the disease burden in developed regions. According to the World
Health Organization (WHO), it will become the world’s second leading cause of death by the middle of
the century, overtaking cancer. This will have a dramatic impact on medical care, and have important
social and economic implications, unless more effective preventive procedures or treatments become
available. Alzheimer’s disease (AD) is the most common cause of dementia, accounting for
approximately 50–75% of all dementias worldwide, followed by vascular dementia, mixed dementia, and
Lewy body dementia.
Currently, acetylcholinesterase (AChE) inhibitors, such as donepezil, rivastigmine and galantamine are
used to treat mild to moderate AD. An alternative therapy for severe AD is memantine, an antagonist of
the NMDA-subtype of glutamate receptors. However, these drugs provide only temporary symptom
improvement, and do not alter disease progression, except temporarily in some patients.
In recent years different approaches have been developed to provide a more effective treatment for AD.
These approached include the discovery of emerging targets and new drugs aiming at a single target, but
given the complexity of the disease, different targets may need to be engaged simultaneously. New
strategies have explored bitopic inhibitors, for example a single drug that acts on different sites of the
acetylcholinesterase enzyme to produce at least two different activities, and multitarget drugs that act on
multiple therapeutic targets.
In this review, we explore the journey from a bitopic inhibitor strategy to multitarget drugs for the future
treatment of AD.
Keywords: Alzheimer’s disease, bitopic drugs, multitarget, BACE-1, cannabinoids, GSK3β, AChE,
BuChE
1. INTRODUCTION
Dementia primarily affects older people, although there is growing awareness of cases that start before
the age of 65. Exact estimates of the prevalence of dementia depend on the definition and specific
diagnostic threshold used. The syndrome affects approximately 5–8% of individuals over age 65, 15–20%
of individuals over age 75, and 25–50% of individuals over age 85. Alzheimer’s disease (AD) is the most
common dementia, accounting for 50–75% of the total (Table 1), with a greater proportion in the higher
age ranges. The remaining types of dementia account for a much smaller fraction of the total [1].
Table 1. Characteristics of the different dementias.
Dementia
Symtpoms
Neuropathology
Alzheimer’s
disease
- Marked memory loss,
apthy and depression
- Language difficulties
- Cortical amyloid plaques,
neuronal neurofibrillary
tangles
Vascular dementia
- Smooth memory loss,
mood fluctuations
- Physical frailty
- Cerebrovascular disease,
Single infarct in critical
regions
Dementia with
Lewy bodies
- Marked fluctuation in
cognitive ability
- Hallucinations
- Tremor and rigidity
- Cortical Lewy bodies
Frontotemporal
dementia
- Personality changes
- Mood changes
- Language difficulties
- Degeneration of the
frontal and temporal lobes
% of dementia
cases
50 –75%
20 –30%
<5%
5 –10%
AD was identified more than 100 years ago, but research into its symptoms, causes, risk factors and
treatment has gained momentum only in the last 30 years. Although research has revealed a great deal
about AD, much is yet to be discovered about the precise biologic changes that cause this neurological
disorder, why it progresses at different rates among affected individuals, and how the disease can be
prevented, slowed or stopped.
AD is a neurodegenerative disorder characterized by an insidious onset that progresses in a complex,
chronic, multi-factorial way, affecting hippocampus and frontal cortex (Fig. 1). The symptoms that
characterize this disease are severe memory loss, decline in language skills, as well as other cognitive
impairments, and dramatic behavioral changes. It is estimated that AD affects more than 4.5 million
people in the US and 18 million worldwide [2]. The disease is characterized by neuropathological lesions
manifested as protein deposits. They consist of neuritic plaques composed of extracellular deposits of
beta-amyloid peptide (A plaques) and interneuronal tangles formed by neurofibres of coiled filaments of
tau protein [3, 4].
Fig. (1). Main pathogenic hallmarks (red) and some of the current drug targets (blue) in AD. Figure
adapated from references [5] and [6].
To date, the established treatments offer only symptomatic relief, aiming to counterbalance the
neurotransmitter disturbance in the disease [7-9]. The marketed drugs can be grouped into two therapeutic
strategies. The first is aimed at treating a cholinergic deficit. Choline acetyltransferase (ChAT) and
acetylcholinesterase (AChE) are involved in the synthesis and degradation of acetylcholine, respectively.
The observed reduction of this neurotransmitter in AD suggests a selective destruction of cholinergic
neurons. The second strategy is treatment of glutamate-mediated neurotoxicity. Glutamate excitotoxicity
mediated through excessive activation of N-methyl-D-Aspartate (NMDA) receptors is believed to play a
role in the neuronal death observed in AD and other neurodegenerative conditions. Glutamate is the main
excitatory neurotransmitter in the central nervous system and physiological levels of glutamate-receptor
activity are essential for normal brain function [10, 11].
Generally speaking, all these medications are symptomatic therapies that do not cure the disease. There is
a clear need for improved drugs. During the past two decades, drug discovery has mainly focused on the
single-target paradigm (single keys for specific locks), in order to develop a drug with high potency and
high selectivity of action. However, the traditional single-target approach provides limited success to treat
complex and multifactorial diseases [12]. During recent decades, alternative strategies have been explored
that ranged from bitopic drugs acting on different sites of the same target and producing at least two
different activities [13-16] to multitarget drugs acting on different therapeutic targets [17-22]. These
strategies are particulary suitable for complex diseases with multiple pathogenic factors, such as
neurodegenerative disorders, cancers and cardiovascular diseases. The rationale behind these bitopic
inhibitors together with their development of multitarget drugs is described.
The main goal of this review is to describe the evolution from the first dual binding site AChE inhibitors
to molecules where an AChE inhibitor is linked to other active compounds and finally to compounds able
to interact simultaneously with two targets involved in AD. The importance of the multitarget strategy in
complex diseases as neurodegenartive ones, especially Alzheimer disease, will be here described.
2. BITOPIC INHIBITION STRATEGY
Since the cholinergic hypothesis of memory dysfunction was first reported, many efforts have been made
in the discovery and development of different cholinergic drugs for the treatment of cognitive deficits.
However, only AChE inhibitors are clinically approved drugs. Although they failed to address the wide
variety of symptoms that characterize these disorders, the use of AChE inhibitors for AD palliative
treatment lead to the discovery of non-cholinergic functions of the cholinergic enzyme AChE [23, 24].
Among others, AChE may work as a chaperone involved in the conformational change of A [25]. In
fact, it was shown that AChE is able to promote A plaques in the cerebral cortex in transgenic models
[26]. AChE chaperone activity is mediated by Trp 279 located in the entrance to the catalytic gorge, in the
peripheral anionic site (PAS) of AChE [27, 28]. This discovery suggested the design of bitopic inhibitors
that simultaneously modulate the catalytic (CAS) and peripheral sites (PAS) of AChE. The idea behind
these bitopic compounds, also called dual binding site AChE inhibitors, is not only to slightly improve
cognition by targeting the catalytic site of AChE, but also to interfere with disease progression by
decreasing toxicity and/or aggregation of A [29]. In that way, compounds designed specifically to
interact simultaneously with Trp 86 located in the CAS, and with Trp 279 in the PAS were proposed [30]
(Fig. 2). Some years later, these compounds have been shown to induce not only an improvement in
cognitive function but also cause a potent modulation of A pathology in animal models [31]. These data
demonstrate that it is possible, at least in animal models, to modulate A pathology through targeting
AChE [32].
Fig. (2). Cartoon depicting the two binding sites (CAS and PAS) of AChE and their function in the
pathology of AD. The concept of a dual binding site or bitopic AChE inhibitor is also shown.
Many different compounds have been designed and synthesized following this strategy [33]. In general,
the pattern shows in Fig.3 is followed for the design of bitopic inhibitors. The design contains three
different chemical features: a known and small AChE inhibitor to interact with the catalytic site, a linker
of different length and nature located along the gorge of the enzyme, and a chemical framework able to
interact efficiently in the peripheral site, mainly with the Trp 279 (Fig. 3). Common fragments used in
these bitopic ligands to gain interaction with the catalytic site are tacrine, huperzine A, huprine (hybrid
AChE inhibitor comprising chemical features from huperzine A and tacrine) [34], and benzylpiperidine
(one of the privileged scaffolds present in donepezil) [35]. In regard to the linker, different polymethylene
chains have been used to optimize the length for each compound class, although the introduction of an
amide bond increases positive electrostatic interactions along the gorge, increasing the inhibitory potency
of the compounds [36]. Polyamines have also been used frequently as biologically active spacers in these
bitopic compounds [37]. Some hydrophobic interactions may be gained in this part of the molecule by
using 1,4-disubstituted phenyl rings as spacers, such is the case for the dual binding site AChE inhibitor
AP2238 [38]. Finally, the interaction in the peripheral site is usually achieved by taking advantage of the
potential  interaction between Trp 279 and the chemical scaffold present in the ligand. Thus, the
better this  interaction is, the more potency is gained by the inhibitor. The most potent compound
reported until now is a dimer of 6-chlorotacrine and indol moiety, called NP-61, that has a picomolar
inhibition potency on human AChE (IC50 = 70 pM), two orders of magnitude of selectivity regarding
BuChE and great in vitro inhibition of AChE induced A aggregation (IC50 = 2 M) [39]. Chronic oral
treatment with NP-61 in a hAPP transgenic model for three months lead to an increase in cognitive
learning measured by the Morris water maze test, together with a decrease of the plaque load in both
cortex and hippocampus [31]. These results suggest that NP-61 is a potent Aβ modulator able to reverse
the AD-like neurodegenerative phenotype in transgenic mice, indicating that bitopic inhibitors or dual
binding site AChE inhibitors are promising disease-modifying agents for clinical application. In fact,
clinical trials in humans are needed to assess the efficacy of these new drugs in AD patients.
Fig. (3). Structural schematic followed for the design of dual binding site AChE inhibitors.
Chemical structure of AP2238 and NP-61.
In the past decade, improvements in this field were focused on the development of new assays able to
determine binding and inhibition at the peripheral AChE site [40]. Thus, thioflavin T was reported to be a
more sensititve and versatile fluorescent reporter of ligand interactions with the PAS than propidium, the
classical standard reference. Moreover, dual binding site AChE inhibitors have recently emerged as
multitarget compounds by their general additional inhibition of BACE-1. Recently, a study of the
structural motifs conveying this polypharmacological profile [41] pointed to the complementary shape of
some chemical fragments with the big catalytic site of BACE-1. In addition, the synthetic methodology
developed for coupling different AChE inhibitor fragments to other molecules has been applied to the
design of multitarget compounds that simultaneously inhibit not only AChE but also other targets
involved in AD pathology that are described in the following sections. It is also worthwhile to mention
that new structural motifs in the N-terminal region of AChE recently have been identified as involved in
Aβ aggregation, which may open up new directions for bitopic ligand design in the coming years [42].
Finally, the concept of bitopic drugs has been applied recently to a new generation of specific M1
muscarinic acetylcholine receptor (M1 mAChR) agonists[43]. The development of M1 mAChR-selective
agents has been hampered until now by high orthosteric site sequence homology among the five
muscarinic receptor subtypes, and the wide distribution of this receptor family in both the central nervous
system (CNS) and the periphery. In recent months, a second generation of M1 mAChR-specific ligands
able to bind simultaneously to the orthosteric site and the less structurally conserved allosteric site of the
M1 mAChR have emerged[44]. This approach revealed a new future for this class of bitopic cholinergic
drugs[13].
3. MULTITARGET-DRUG STRATEGY
The aim of a multitarget drug is to act selectively on multiple targets of therapeutic interest. Within the
multitarget strategy it is possible to identify two categories of multitarget therapeutics [45], in one of
them, the drug acts on two or more targets that can be within individual signalling pathways or across
pathways within a cell. In the other one, the response of one target to a drug could modulate a different
target.
Different approaches toward multitarget drugs have been used during the last decade to fight against
multifactorial diseases [19, 46, 47] with special emphasis in the neurodegenerative field, since many
involve multiple targets with pathological functions in complex neurotoxic cascades [48-50].
Recent studies in the neurodegenerative field according to this strategy have led to the synthesis of
several chemically diverse structures with dual or multiple biological profiles for the treatment of AD.
The first group of compounds maintained the inhibition of a cholinergic enzyme such as AChE or
butyrylcholinesterase (BuChE), but added different scaffolds to interact with BACE-1 and/or NMDA,
CB2 or histamine H3 receptors [17, 50], while a second group have been designed with the aim of finding
non-cholinergic drugs with combined activities such as metal chelator properties or tau kinase inhibition.
3.1. Drugs targeting both AChE and NMDA receptors
Tacrine was the first drug approved for AD treatment (1993), followed by donepezil (1996), rivastigmine
(2000) and galantamine (2001). Each of these are inhibitors of AChE, and produce an increase of
acetylcholine neurotrasmission in the synaptic cleft, improving cognitive deficits in the early stages of
AD. These AChE inhibitors together with the NMDA receptor antagonist memantine (2003), which
regulates the activity of glutamate, an important neurotransmitter in the brain involved in learning and
memory, represent the only palliative treatments for AD. In AD, excess glutamate can be released from
damaged cells, leading to chronic overexposure to calcium, which can speed up cell damage. Memantine
helps to prevent this destructive chain of events by partially blocking the NMDA receptors.
Considering the efficacy of the multitarget ligand strategy [51], combination of these two therapeutic
approaches could lead to a synergistic pharmacological outcome. However, there are different results in
the literature regarding this synergistic effect. Multiple studies suggest combination therapy with
memantine and an AChE inhibitor in moderate to severe AD produces consistent benefits beyond the
effects of AChE inhibitor treatment alone [52, 53]. Nevertheless, in other trial, memantine did not show
an advantage over placebo in patients with stable AChE inhibitor regimens [54]. Today, an investigation
of the safety and effectiveness of donepezil and memantine combination therapy in patients with AD is
ongoing (www.clinicaltrials.gov, NCT02162251). Additionally, a commercially available fixed-dose
combination of controlled-release memantine and donepezil is on the market after the benefits provided in
thinking, daily functioning, and behavior in people with moderate to severe AD were shown [52, 55, 56].
One of the main drawbacks to combining two drugs in the same formulation is the different
pharmacokinetic, metabolism and bioavailability of the drugs. For this reason the multi-target drug ligand
(MTDLs) design strategy consists on developing a single chemical compound which are able to modulate
multiple targets simultaneously with superior efficacy and safety profiles [6]. Several multitarget ligands
based on AChE inhibitors and NMDA antagonists have been reviewed recently [57]. The first hybrid
molecule, called carbacrine (Fig. 4), was designed using a tacrine derivative (6-chlorotacrine) as the
AChE inhibitor and carvedilol as the NMDA antagonist [58].
Carvedilol presents a privileged structure as a building block in the search for new rationally designed
multitarget drugs for AD treatment. On one hand it has neuroprotective efficacy because of its
modulatory action at NMDA receptors as a low-affinity antagonist [59] and on the other hand because it
is an efficient inhibitor of A formation [60]. The synthesis of the carbacrine hybrid structure was done
employing different linkers/spacers between the tacrine and carbazole in order to improve the interaction
with the catalytic and peripheral anionic sites of AChE (Fig. 4) leading to a single compound with
nanomolar AChE inhibition, efficient NMDA antagonism, and antioxidant and Aaggregation reducing
properties.
Fig. (4). Design of multitarget compounds with cholinergic activity and NMDA receptor
antagonism.
Another example is memagal (Fig. 4) that was designed employing galantamine (AChE inhibitor) and
memantine (NMDA antagonist), taking into account the dual binding approach to reach the catalytic and
peripheral sites of AChE [61]. It was reported that the combination of galantamine and memantine might
provide a more effective treatment for memory impairment in cognitive disorders than either drug used
alone [62]. From the series of compounds synthesized, the optimal spacer between the two molecules
consisted of six methylene groups. Memagal was shown to be a nanomolar potency AChE inhibitor that
also inhibited the induced NMDA neurotoxicity in neuroblastoma cells in the micromolar range. This
neuroprotective effect of galantamine was shown by blocking the α7 and/or α4β2 nicotinic receptors [63].
Dimebon (latrepirdine) was reported to have micromolar inhibition of AChE (IC 50 = 42 µM) and NMDA
receptor antagonism (IC50 = 10 µM) [64]. Although a preliminary clinical trial (www.clinicaltrials.gov,
NCT00377715) reported that dimebon was safe, well-tolerated, and significantly improved the clinical
course of patients with mild-to-moderate Alzheimer's disease [65], a more recent multinational phase III
study showed no difference between the two drug-treated groups and the placebo group [66], probably
because the brain concentration after acute oral administration was lower than necessary to affect AChE
and NMDA [67]. In order to improve these activities, a bivalent ligand strategy was employed to design
new dimebon derivatives. Two pharmacophoric groups were connected with a linker following the
requirements of dual binding sites in AChE. Docking studies were carried out with the new derivatives,
pointing out the importance of the γ-carboline moiety of dimebon for AChE recognition (Fig. 4).
Dimebon derivatives in which the spacer was formed by 6 and 7 methylene groups showed improved
AChE inhibition (<1 µM) while mainting the same NMDA receptor inhibition [68]. Additionally, these
compounds were able to reduce Aβ aggregation at 50 µM between 37–73% whereas dimebon showed
only 10% inhibition.
Other AChE inhibitors and NMDA antagonist compounds have been described in the literature such as
bis-7 tacrine [69, 70] and bivalent β-carbolines [71]. These compounds were tested as NMDA antagonists
after the discovery of their AChE inhibition.
3.2. Drugs targeting both AChE and histamine H 3 receptors
Another drug target combination that can promote a synergistic effect is AChE and histamine H3 receptor
inhibition. Histamine H3 receptor inhibits histamine release in the brain and inhibits the release of other
neurotransmitters such as acetylcholine, glutamate, serotonin, dopamine, etc. via a parallel role as a
heteroreceptor [72]. Thus, blocking the central histamine H3 receptor will raise acetylcholine levels in the
brain. Combining AChE inhibitors and histamine H3 receptor antagonists in a single molecule will
enhance cholinergic neurotransmission in the cortex. There are several H3 receptor antagonists described
in the literature, and the scaffold of compound JNJ-5207852 [73] was employed in the design of
multitarget ligands for AChE inhibition/H3 receptor antagonism [74]. Tacrine displayed nanomolar
affinity not only for AChE but also for histamine N-methyltransferase, therefore, the combination of the
H3 receptor pharmacophore and an acridine-framework was originally proposed in 2002 [75]. The
resulting multitarget drug, namely FUB 833 [75], displays nanomolar inhibition for AChE and for the H 3
receptor (human H3 Ki = 0.33 nM) (Fig. 5).
Fig. (5). Different drug design strategies for simultaneous targeting of AChE and H3 receptors.
New multitarget drugs were designed employing a series of novel non-imidazole H3 ligands from the
chemical manipulation of 1,1’-octa-, -nona-, and -decamethylene-bis-piperidines—H3 antagonists with
tacrine (Fig. 5). Within the compounds synthesized, the tetrahydroaminoacridine hybrid depicted in Fig. 5
stands out as one of the most attractive molecules, synergistically combining nanomolar potency and
selective H3 antagonism (antagonist potency at human histamine H3 receptors expressed in SK-N-MC
cells, human H3 pKB = 8.93) with remarkable AChE activity (pIC50 = 7.57) even more potent than that
shown by the simple molecule of tacrine on rat brain cholinesterase (pIC50 = 6.54) [76].
A new family of dual AChE/H3 receptor inhibitor compounds was recently published. They were
designed based on previous structure activity relationship studies on H 3 ligands [77, 78], which showed
that phenol ethers in the meta position to the tertiary benzyl amine possess high antagonist potency at the
H3 receptor (Fig. 5). Compound 1 [79], showed a nanomolar inhibition of AChE and was a human H3
receptor antagonist with Ki = 76.2 nM.
There are other examples of AChE/H3 receptor inhibitors described in the literature synthesised using
different methodologies such as the use of crystal structure, pharmacophore modelling and docking
techniques that lead to the identification of an AChE inhibitor/H3 receptor antagonist [80], but they were
found to inhibit these targets in a moderate way.
3.3. Drugs targeting AChE and having antioxidant properties
Nowadays it is accepted that oxidative damage can be involved in the course of AD and can act as a risk
factor for its initiation and progression [81]. Therefore, an antioxidant-based therapy could be a helpful
approach to ameliorate the deleterious effects of oxidative stress in this disease. Moreover, the design of
multitarget ligands able to combine cholinergic activities with antioxidant properties has been
successfully tackled by different research groups.
Although the clinical use of tacrine, the first approved drug for AD, has been restricted due to
hepatotoxicity, it is widely used as a starting point in drug discovery programs. Thus several tacrine
hybrids have been described in which the tacrine core is linked to another moiety with interesting
properties [82].
Regarding bifunctional molecules with cholinergic and antioxidant properties, tacrine has been linked
with well-known antioxidant scaffolds such as melatonin [83], clioquinol [84], flavonoids [85], quinone
[86] or ferulic acid [87].
Melatonin is a neurohormone, whose levels decrease during aging, that has been reported to possess
strong antioxidant actions and free radical scavenger properties [88]. The development of melatonintacrine hybrids (Fig. 6a) yields derivatives able to inhibit AChE in the sub-nanomolar to picomolar range
that also have antioxidant properties [83]. As a matter of fact, the new hybrids show better AChE
inhibitory and antioxidant properties than their separate structures, can cross the blood brain barrier
(BBB) and display neuroprotective properties against cell death induced by various toxic insults in a
human neuroblastoma cell line [89].
Fig. (6). Drug design strategies to generate compounds with AChE inhibition and antioxidant
properties.
Modulation of redox metal ions such as Cu and Fe has been proposed as a potential therapeutic strategy
for the treatment of AD as a way to reduce the production of reactive oxygen species (ROS) and oxidative
stress [90]. As clioquinol is a well-known metal chelator able to reduce oxidation and the A burden [91],
it was selected to be combined with the tacrine scaffold to obtain hybrids of tacrine-8-hydroxyquinoline
(Fig. 6a), which have been shown to have not only cholinergic, antioxidant and copper-complexing
properties but also neuroprotective properties in damaged human neuroblastoma cells [84].
The flavonoids have well-recognised properties as free-radical scavengers [92] and also hybrids with
tacrine have been described (Fig. 6a). The resulting molecules have proved to be not only efficient
antioxidants and cholinergic agents, but also potent BACE-1 inhibitors [85].
The design strategy employed for the discovery of memoquin [93] (Fig. 6b) was based on the
incorporation of a 1,4-benzoquinone functionality as a radical scavenger into the polyamide skeleton of a
previously reported cholinergic derivative named caproctamine [94]. Memoquin is able to interfere with
different key target points of AD neurodegeneration such as AChE, oxidative stress, BACE-1 and A
aggregation, showing not only an interesting in vitro profile, but also suitable in vivo activity in animal
models [95]. The memoquin scaffold also has been employed as a template for the design of new
multitarget ligands [86, 96].
The AChE inhibitor AP2238 (Fig. 6c), was chosen to be hybridised with melatonin in order to obtain new
potent cholinergic and antioxidant agents [97]. Additionally, these kinds of compounds have shown
neurogenic properties, which adds significant value for their use in AD treatment.
3.4. Drugs targeting both AChE and BACE-1
Inhibition of AChE activity would alleviate clinical symptoms in short-term therapy, and inhibition of
BACE-1 would have an additive effect by preventing the formation of Aβ and further slowing the
progression of the long-term disease, since BACE-1 catalyzes the first and rate-limiting step of the
biosynthesis of Aβ from the amyloid precursor protein (APP). This therapy likely may result in a
comprehensive suppression of AD in a synergistic manner and obtain higher therapeutic effectiveness
[98].
The first multitarget AChE/BACE-1 inhibitors were published by Piazzi et al. [99]. They reported a new
amidic nonpeptidic derivative family in which the framework of AP2243, a dual binding site
acetylcholinesterase inhibitor, was maintained, whereas the methoxy groups of the coumarin moiety were
alternately substituted by an amidic chain to extend the activity to BACE-1. Therefore, they introduced
halophenylalkylamidic functions in positions 6 or 7 of the coumarin moiety, choosing a dihalophenyl acid
because this moiety emerged as a leitmotif in different BACE-1 inhibitors. Following this rationale, a
new series of potential multi-target compounds was synthesized (Fig. 7). The most potent compound 2
exhibited an IC50 of 99 nM for BACE-1 and 7.16 M for human AChE.
Fig. (7). Dual inhibitors of AChE and BACE-1.
Zhu and co-workers [98] combined pharmacophores of AChE and BACE-1 inhibitors through a suitable
common scaffold to develop a new family of multitarget compounds. As inhibitor of AChE they used the
N-benzylpiperdine pharmacophoric group of donepezil and an isophthalamide fragment derived from a
known BACE-1 inhibitor (3). These fragments are joined through a classic transition state isostere for
aspartyl protease inhibitors such as statine, homostatine (HE), nostratine (HMC) and hydroxyethyl-amine
(HEA) (Fig. 8).
Fig.(8). a) Structure of known BACE-1 and AChE inhibitors. b) Structure of the linkers. c) Dual
inhibitors of AChE and BACE-1.
Of the three families developed based on the common framework, HE, HMC and HEA, the family of HEcontaining compounds are described as strong BACE-1 inhibitors and weak AChE inhibitors, while the
HMC derivatives show increased potency against AChE along with reduced potency against BACE-1.
The last series, with the HEA motif, showed good inhibition of both AChE and BACE-1. Among this
series, compounds 4 and 5 were the most potent dual-acting inhibitors, with AChE IC50 = 1.83 μM and
BACE-1 IC50 = 0.57 μM for 4, and AChE IC50 = 1.35 μM and BACE-1 IC50 = 0.22 μM for 5 (Fig. 8). In
addition, both compounds demonstrated excellent inhibition of A formation in a cell-based assay (4 IC50
= 54.6 nM, 5 IC50 = 98.7 nM) and also displayed antioxidant properties. Preliminary in vivo studies
(intracerebroventicular dosing to APP transgenic mice) revealed A anti-aggregation properties, with
reduced endogenous A 1-40 production in the 23 –29% range.
Bolognesi et al. [100] reported eight monomeric congeners related to the lead compound memoquin that
showed a polypharmacological profile against AD [93]. This new family of memoquin derivatives was
achieved by cutting the dimeric structure of memoquin into two monomers (Fig. 9).
Fig. (9). Design of monovalent ligands from memoquin structure.
Most of the compounds show a worse profile than memoquin in terms of AChE and BACE-1 activity.
The most potent monovalent ligand is compound 6 that shows nanomolar AChE inhibition and
approximately 60% inhibition of BACE-1. Docking studies of 6 with AChE and BACE-1 showed that 6
was able to interact with the catalytic site and at the same time to protrude toward the solvent-exposed
gorge entrance of AChE. Regarding BACE-1, the reported conformation of 6 produces contact with Asp
32, which belongs to the catalytic dyad through hydrogen bond with several subsites of BACE-1.
The group of Rosales-Hernández has recently published a family of hydroxyphenyl derivatives as
multitarget compounds [101]. This new family, derived from an antioxidant hydroxyphenol compound
(5-ASA), was designed by including two pharmacophore groups reported as AChE [102] and BACE-1
[103] inhibitors (Fig. 10). The best two compounds (7 and 8) (Fig. 10) show activity values ranging from
5.74 µM and 3.19 µM regarding AChE, respectively, and 10.1 nM and 15.1 nM at BACE-1, respectively.
They also inhibit Aβ oligomerization and exhibit antioxidant activity.
Fig. (10). Design of multitarget ligands employing the pharmacophore hypothesis for AChE and
BACE-1 inhibitors. Adapted from [100, 101].
Based on a previous work where Viayna et al. developed novel huprine derivatives with inhibitory
activity toward Aβ aggregation and formation [104], the authors recently have published a new family of
rhein-huprine hybrids (Fig. 11) as dual inhibitors of AChE and inhibitors of BACE-1 [105]. These
hybrids are composed of one unit of huprine and one unit of a natural product, rhein, structurally related
with hydroxyanthraquinone derivatives such as emodin and PHF16 (Fig. 11). The most interesting
compound (9) is racemic and acts as an inhibitor of human AChE (IC 50 = 2.39 nM) and BACE-1 (IC50 =
80 nM). In vivo studies in APP-PSI transgenic mice have shown that this compound has a central soluble
Aβ lowering effect, accompanied by an increase in the levels of mature amyloid precursor protein (APP).
Fig. (11). Structure of the multitarget rhein-huprine derivative.
Very recently, Domínguez et al. have reported the existence of a pharmacophore for a multitarget
approach to AD that displays some of the features of the known lead compounds that binds AChE and
BACE-1. [106] The authors propose as pharmacophore the presence of a functional group such as
hydroxyethylether, hydroxyethlylamine, guanidium, etc., that can interact through hydrogen bonds and
ion pair with the Asp dyad of BACE-1. and one or two aromatic moieties that interact through π–stacking
with aromatic residues in both the catalytic site (CAS) and peripheral site of AChE and/or BuChE (Fig.
12a).
Fig. (12). a) Schematic representation of a multitarget pharmacophore. b) Structure and
experimental results for indole-based compounds.
Thanks to this pharmacophore together with an iterative protocol of docking and screening, the authors
have identified three compounds (10-12) with interesting multitarget profiles (Fig. 12b).
3.5. Drugs targeting both AChE and MAO
Ladostigil (TV3326), a dual acetylcholine-butyrylcholinesterase and brain selective monoamine oxidase
(MAO)-A and -B inhibitor (Fig. 13) that has reached phase II clinical trials [107, 108] was designed and
synthesized using the multitarget strategy.
Fig. (13). Structure of ladostigil.
Ladostigil was designed using rasagiline (Azilect), a potent MAO-B inhibitor, and a pharmacophoric
carbamoyl moiety derived from rivastigmine (Fig. 13).
Based on the linking of MAO inhibitors that possess a propargylamine moiety with different kinds of
AChE inhibitors such as donepezil [109-111] or tacrine [112], new hybrids have been developed in recent
years.
3.6. Drugs targeting both AChE and 5-HT4R
During the last year various studies have validated the 5-HT4 serotonin receptor as a valuable target for
AD treatment [113-115]. Recently, Dallemagne et al. published a novel multitarget family (Fig. 14) based
on the combination of the frameworks of two compounds, donepezil, an AchE inhibitor, and RS67333
[116], a 5-HT4 receptor agonist, which displays both AChE inhibitory effects and partial 5-HT4R agonist
activity in vitro with nanomolar potencies. [117, 118] One of the compounds, donecopride, shows in vivo
procognitive and anti-amnesic effects in NMRI mice, and is able to promote the release of sAPP in
C57BL/6J mice in vivo [118].
Fig. (14). Hybrids of AChE and 5- HT4R
3.7. Drugs targeting BACE-1 with metal chelating properties
The aggregation of Aβ is mediated by interaction with metals, as Zn, Cu or Fe. An alteration in the
homeostasis of these metals may contribute to AD pathogenesis. Therefore other interesting approach is
multi-target ligands able to inhibit BACE-1 and chelate metal ions [119]. The resulting 1,3-diphenylurea
derivatives combined the metal chelator LR-90 with a BACE-1 inhibitor (Fig. 15). All of the new
compounds showed the ability to chelate metal and possessed discrete BACE-1 inhibitory properties.
Fig. (15). Hybrids of BACE-1 inhibitors and metal chelators.
3.8. Drugs targeting both BuChE and CB2 receptors
Alzheimer’s disease is characterized by a significant reduction in AChE activity and an increase in
butyrylcholinesterase (BuChE) activity (about 120%). This increase of BuChE activity, especially found
in the hippocampus, has been suggested to be related to the loss of episodic memory. During recent years,
several studies have shown that BuChE can also hydrolyse acetylcholine in the brain and may play a role
in cholinergic transmission [120, 121]. Likewise, it has been reported that BuChE might facilitate the
transformation of an initial benign form of senile plaque to a malignant form associated with AD.
Therefore, BuChE plays an important role in AD progression that makes it an interesting target for
research in the field [122]. Cannabinoid compounds are very attractive candidates that fit into the
multitarget paradigm since they could act on different pharmacological targets within the so-called
endocannabinoid signaling system, but also on other targets not related to this system [122]. It has been
reported that the cannabinoid system is dramatically altered in brains of AD patients [123, 124].
In 2010 a new family of indazole ethers that possess dual activity as both cannabinoid agonists of CB2
and inhibitors of BuChE was registered [125]. Using computational tools, Gonzalez-Naranjo et al.
designed this new multitarget family. The results of pharmacological tests with these compounds revealed
that three of these derivatives behaved as CB2 cannabinoid agonists and simultaneously showed BuChE
inhibition. In particular, compounds 13 and 14 have emerged as promising candidates as novel
cannabinoids that inhibit BuChE by a non-competitive or mixed mechanism, respectively. Additionally,
both molecules showed antioxidant properties (Fig. 16) [126].
Fig. (16). Design of multitarget ligands. CB2 agonist and BuChE inhibitors.
3.9. Drugs targeting BACE-1 and GSK-3
BACE-1 and glycogen synthase kinase (GSK-3) are two enzymes thought to play a role in AD [127].
GSK-3 is upregulated in AD patients' brains and plays a key role in tau hyperphosphorylation [128,
129].
Recently, molecules able to modulate simultaneously BACE-1 and GSK-3 have been designed based on
a fragment-based strategy. By combining the pharmacophoric features responsible for binding to BACE-1
and GSK-3, such as guanidinio and cyclic amide groups, respectively, a 6-amino-4-substituted
triazinone scaffold has emerged as a new dual-target inhibitor with a promising profile in terms of
neuroprotection and neurogenesis in cells (Fig. 17) [130].
Fig. (17). Dual inhibitors BACE-1/GSK-3.
4. Future work and conclusions
Research to develop effective treatments for AD has evolved over the last decade. Anti-AD drug research
started with the traditional single-target approach (one-to-one) where one selective drug acts on one
specific target, for example, tacrine or donepezil as selective drugs for AChE inhibition. The next step
was the development of bitopic drugs, in which one drug acts simultaneously at two binding sites of the
same target. This strategy, where a unique drug takes advantage of interacting with one enzyme (AChE)
at two different blocking sites and producing two different biological activities, may be considered the
basis of the subsequent multitarget drug approach (one-to-two) where one drug acts on two or more
targets.
The single-target strategy (one drug-one target) shows several limitations, specifically in multifactorial
diseases like cancer or neurodegenerative diseases, since complex diseases involve several aspects. This
traditional strategy does not always produce the desired effect on the entire biological system probably
because organisms can modulate a drug's effectiveness through compensatory changes. In this sense, a
multitarget drug can be more efficacious and less vulnerable to a compensatory response since it is more
difficult for a biological system to counteract the effects on several targets at the same time. Another
important issue regards the constraints of druggability due to the need to develop drugs with high affinity
and selectivity. Multitarget drugs usually have a low affinity towards their targets but there is evidence
that low affinity does not necessarily mean low efficiency [131] and low affinity can reduce side effects.
The multitarget strategy has emerged as a very interesting and attractive approach in order to develop
effective drugs for neurodegenerative diseases. Different approaches have been developed in order to
design and discover multitarget drugs for AD. Among these approaches, several have been highlighted
here including the hybrid approach, using two molecules with different target profiles [35, 132],
fragment-based [19, 133], multitargeted pharmacophore [101, 106] and multitargeted QSAR [5]. In
addition, multitarget ligands have been identified by means of library screening against two or more
targets [134]. It is important to mention that the first synthetic molecules that combined cholinergic
activities with other non-cholinergic ones took advantage of the previous bitopic cholinergic
pharmacophore strategy, with tacrine being one of the most used scaffold. More recently, the multitarget
approach has been expanded to several non-cholinergic drugs, combining different activities in one
molecule, without targeting AChE.
Examples of the efficacy of this strategy are NP-61, a bitopic AChE inhibitor that reached clinical trial
phase I, and ladostigil (TV3326), a dual acetylcholine-butyrylcholinesterase and brain selective MAO-A
and -B inhibitor that is in phase II clinical trials [107, 108]. Clinical studies in AD patients will assess the
value of multitarget compounds for AD pharmacological treatment in the near future.
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