Therapeutic Potential of Phosphodiesterase Inhibitors
against Neurodegeneration: The Perspective of the Medicinal Chemist
Increasing human life expectancy prompts the development of novel remedies for cognitive decline: 44 million people worldwide are affected by dementia, and this number is predicted to triple by 2050. Acetylcholinesterase and N-methyl-d-aspartate receptors represent the targets of currently available drugs for Alzheimer’s disease, which are characterized by limited efficacy. Thus, the search for therapeutic agents with alternative or combined mechanisms of action is wide open. Since variations in 3′,5′-cyclic adenosine monophosphate, 3′,5′-cyclic guanosine monophosphate, and/or nitric oxide levels interfere with downstream pathways involved in memory processes, evidence supporting the potential of phosphodiesterase (PDE) inhibitors in contrasting neurodegeneration should be critically considered. For the preparation of this Review, more than 140 scientific papers were retrieved by searching PubMed and Scopus databases. A systematic approach was adopted when overviewing the different PDE isoforms, taking into account details on brain localization, downstream molecular mechanisms, and inhibitors currently under study, according to available in vitro and in vivo data. In the context of drug repurposing, a section focusing on PDE5 was introduced. Original computational studies were performed to rationalize the emerging evidence that suggests the role of PDE5 inhibitors as multi-target agents against neurodegeneration. Moreover, since such compounds must cross the blood–brain barrier and reach inhibitory concentrations in the central nervous system to exert their therapeutic activity, physicochemical parameters were analyzed and discussed. Taken together, literature and computational data suggest that some PDE5 inhibitors, such as tadalafil, represent promising candidates.
Introduction<!>Targeting PDE Isoforms in
the CNS<!>PDE5<!><!>PDE5<!>Other PDE Isoforms<!><!>Other PDE Isoforms<!>Focus on PDE5 Inhibitors
against Neurodegeneration<!>Sildenafil<!>Tadalafil<!>Natural Compounds<!><!>Natural Compounds<!>The Point of View of the Medicinal Chemist:
Multi-Target Approach and Drug-likeness<!><!>The Point of View of the Medicinal Chemist:
Multi-Target Approach and Drug-likeness<!><!>The Point of View of the Medicinal Chemist:
Multi-Target Approach and Drug-likeness<!><!>The Point of View of the Medicinal Chemist:
Multi-Target Approach and Drug-likeness<!>Conclusion<!>Author Contributions<!>
<p>In the context of cognitive
decline, the search for novel efficient
diagnostic tools and treatments is wide open, since the human population
is constantly aging due to increasing life expectancy. In parallel,
continuous improvements in understanding the dynamic processes that
underlie memory consolidation and, on the other side, pathological
cognitive changes in the brain are being achieved.1 According to recent estimates, 44 million people currently
live with dementia worldwide, and this number is predicted to triple
by 2050. At that stage, the annual cost of Alzheimer's disease
(AD) in the USA would exceed US$600 billion.2 AD symptoms affect memory, attention, personality, intellect, and
speech. Its hallmarks consist in amyloid plaques in the brain and
a progressive degeneration of cholinergic innervation of the hippocampus
and cerebral cortex.3 Concerning therapeutic
options, two classes of small molecules are currently available.4 Acetylcholinesterase (AChE) inhibitors
(donepezil, rivastigmine, galantamine) act by enhancing acetylcholine
levels, but unsatisfying efficacy and adverse side effects limit their
use.5 Memantine belongs instead to the
second class of compounds, and it targets N-methyl-d-aspartate (NMDA) receptors, with modest effects on cognition
in moderate/severe AD.3</p><p>A strategy
for developing novel agents to restore memory function
is represented by the inhibition of phosphodiesterase (PDE)
activity with small molecules. PDEs are a family of enzymes encompassing
11 classes, which will be covered in the following section of this
Review, that physiologically act by hydrolyzing cyclic nucleotide-based
second messengers to their corresponding linear derivatives.3 By elevating 3′,5′-cyclic adenosine
monophosphate (cAMP) and 3′,5′-cyclic guanosine monophosphate
(cGMP) and/or influencing nitric oxide (NO) levels, PDE inhibitors
interfere with several pathways that have been reported to be relevant
in learning functions in animal models of impaired cognition.6,7</p><p>In this context, growing evidence suggests that some inhibitors,
especially those interfering with the PDE5 isoform, may play a role
in influencing cognition-related neural activity and be effective
against central nervous system (CNS)-related diseases.8−10 For the preparation of this paper, more than 140 research papers
and reviews were considered. Scientific contributions were retrieved
by searching PubMed (www.ncbi.nlm.nih.gov/pubmed/) and Scopus (www.scopus.com) databases using
keywords such as "phosphodiesterase", "PDE5",
"sildenafil", "tadalafil", natural compounds",
"neurodegeneration", "Alzheimer's disease",
and their combinations. Research papers and reviews published in the
2000–2020 time frame were considered and screened. Particular
attention was dedicated to the contributions reporting preclinical
and clinical data on PDE inhibitors.</p><p>In the first part of this
Review, an overview of the current evidence
on the therapeutic potential of isoform-selective PDE1–11 inhibitors
against neurodegeneration will be presented, providing updated
insights about the small molecules currently under development and
the underlying molecular mechanisms. As anticipated, particular attention
will be dedicated to PDE5 inhibitors, which have been used for decades
in clinical practice with other indications and are known for their
overall safety and good pharmacokinetic properties. In the second
part of the Review, a closer look at this topic from the point of
view of the medicinal chemist will be provided. Chemical aspects of
selected natural and synthetic PDE5 inhibitors will be discussed in
more detail: computational tools, such as molecular docking and pharmacokinetic
properties prediction, will support the exploration of their potential
as multi-target CNS drugs.</p><!><p>Signaling pathways involving cAMP and cGMP are regulated
by several
enzymes. Transmembrane and soluble adenylyl cyclases (ACs) are responsible
for the synthesis of cAMP in brain, while cGMP is synthesized by particulate
guanylyl cyclase (pGC).11,12 On the other hand,
cAMP and cGMP are degraded by PDEs. The interest in PDEs as targets
to ameliorate age-related brain conditions first relies on the hypothesis
that a breakdown in cyclic nucleotide synthesis/degradation may contribute
to the onset of the disease.11 Moreover,
altered cyclic nucleotide signaling has been previously connected
with aging, and this may be caused by reduced AC activity and/or by
a variation in PDEs activity or expression, as observed in animal
models, and brain region-specific alterations in cyclic nucleotide
signaling are thought to contribute to the onset and progression of
AD.13 In particular, a reduction in AC
activity was reported in the hippocampus, temporal, frontal and occipital
cortex, and cerebellum in AD conditions. Similarly, reductions in
pathway effectors were also observed.14,15 These events
are often paralleled by increased PDE activity in cerebrovessels,
putamen, and temporal cortex (especially PDE5).16,17 However, AD-associated alterations in expression/activity are still
debated, with contrasting data being reported and doubts about the
cause–effect relationship between the events. Most importantly,
these alterations appear to be isoform-specific and brain region-specific.
As stated by Kelly in a recent review, "not all studies identify
an upregulation of cAMP-PDE expression or activity in AD patient and
model studies", but preclinical and clinical evidence supporting
the potential of PDE inhibitors is emerging.11 In similar ways, decreased cGMP signaling can be associated with
aging. In particular, the involvement of cGMP in long-term synaptic
activity in several brain regions should be taken into account.18</p><p>Concerning the involved downstream molecular
mechanisms, variations
in cAMP and cGMP signaling regulate the activation of cAMP response
element binding protein (CREB), influencing the transcription of CRE-dependent
genes and, thus, neurogenesis. By activating protein kinase
A, cAMP also promotes CREB phosphorylation and consequently influences
neuronal plasticity.19,20 Besides, it must be pointed out
that NO itself is an effector of PDE activity of primary relevance.21 NO/cGMP signaling modulates synaptic transmission
and plasticity in the hippocampus and cerebral cortex, which play
critical roles in learning and memory.22 The NO/cGMP pathway induces CREB phosphorylation via protein kinase
G, thus enhancing the effects described above connected to the cAMP-related
signaling.23−25 Furthermore, NO activates the CREB pathway through
the interaction with the NO receptor.26 Besides the effects on the CREB downstream, improved neurological
outcomes related to the use of PDE inhibitors also result from a general
NO-mediated enhancement of cerebral blood flow.27 Moreover, it has been observed that physiological concentrations
of NO can also promote anti-apoptotic/pro-survival response against
several neurotoxic insults through combined mechanisms.22 For a comprehensive overview of the role of
NO in memory formation and in the regulation of cerebral blood flow
to ensure adequate blood supply to the brain, the reader is invited
to refer to the contribution by Maher and colleagues.28</p><p>Even if PDE inhibitors are generally prescribed for
peripheral
indications, their influence on intracellular signaling highlighted
above makes them attractive tools for improving neuronal activity.29−31 Among the 11 PDE classes, isoforms 4, 7, and 8 specifically hydrolyze
cAMP, isoforms 5, 6, and 9 are selective for cGMP, while the remaining
enzymes can degrade both of these cyclic nucleotides.1 Details concerning the brain expression of such classes
will be covered in the following paragraphs.</p><!><p>PDE5 exerts its activity by
selectively hydrolyzing cGMP to its corresponding linear derivative.
It represents the pharmacological target of sildenafil and its analogues
(Figure 1) in the treatment
of erectile dysfunction (ED) and pulmonary hypertension (PH).32</p><!><p>Chemical structures of commercially available PDE5 inhibitors.</p><!><p>The discovery of PDE5 localization in several human
tissues paved
the way for the investigation of novel possible therapeutic applications
of selective PDE5 inhibitors. In this context, the fields of cancer,
inflammation, and CNS-related diseases have been explored throughout
the years.10 Gur et al. reviewed the evidence
on the efficacy of PDE5 inhibitors in the latter research area, highlighting
that such molecules have been studied to contrast epileptic disorders,
stroke and cerebral ischemia, depression, and Huntington's
disease.33 More than two decades ago, the
PDE5 inhibitor zaprinast was investigated for its beneficial effects
on memory in the object recognition test. However, cross-reactivity
with other PDE isoforms (1, 9, 10, and 11) limited its use. Thus,
the attention of medicinal chemists is currently focused toward more
selective inhibitors of natural and synthetic origin.</p><p>While
it is now generally accepted that selective PDE5 inhibitors
can promote beneficial effects on cognition and memory in both physiological
and pathological conditions,34 the presence
and localization of PDE5 in the human brain have been debated, especially
considering the discrepancies highlighted with respect to rodent models.
Nevertheless, PDE5 mRNA has been detected in human cortex, hippocampus,
and striatum,35 and its distribution has
been recently reassessed following the observations on the effectiveness
of PDE5 inhibitors in contrasting AD in animal models.34 Interestingly, PDE5 expression is low or absent
in the hippocampus of aged subjects and AD patients but is increased
in the temporal cortex under similar pathologic conditions.36 In fact, mild AD patients show lower cGMP levels
in cerebrospinal fluid (CSF).37 On the
other hand, it must be stressed how, if sildenafil and its analogues
are thought to exert an effect on central PDEs, the compounds should
cross the blood–brain barrier (BBB), reaching the inhibiting
concentration in the target tissue. It has been observed that both
tadalafil and sildenafil may effectively accumulate in the brain at
sufficient levels in animal models.34,38 This aspect
will be discussed more in detail in the following section of this
Review.</p><p>Sabayan et al. described PDE5 inhibitors as "disease-modifying
agents" against AD and examined the molecular pathways through
which the inhibition of such enzymes may play a role in contrasting
the pathogenic process.9 In this context,
the authors highlighted three main mechanisms that involve PDE5 and,
thus, could be targeted by selective inhibitors. The first one is
related to the "neurovascular theory", based on
the fact that microvascular injuries may reduce amyloid clearance
through altered delivery of nutrients to neurons.39 Endothelial dysfunction has been highlighted in AD patients,
even if the cause–effect relationship is not fully understood.40 Here relies the potential of PDE5 inhibitors,
since they act by relaxing the arterial wall and improving endothelial
functioning via the NO-cGMP pathway. By decreasing endothelin-1 and
E-selectin levels, as has been observed in patients,41 they could act as disease-slowing agents. In the same context,
there is evidence showing that a restored cerebral blood flow may
improve glucose utilization and, in general, metabolic functions in
neurons.42 The second mechanism is connected
with the "cholinergic theory" of AD, and it is based
on the mutual relationship between cGMP and acetylcholine in
physiologic and pathologic conditions. In AD, acetylcholine
levels are reduced in specific brain areas which are connected to
memory and cognitive functions impairment. Consequently, two key points
must be taken into consideration. First, cGMP is the second messenger
of acetylcholine. Second, it has been observed that cGMP analogs
can stimulate acetylcholine release.43 This sort of "double connection" suggests that stimulation
of the NO-cGMP pathway induced by PDE5 inhibitors can result in an
increased concentration of acetylcholine released by the nucleus accumbens and, thus, in memory enhancement due to
the effects on cortical neurons.43 The
third mechanism is related to impaired neurogenesis, a mechanism
that underlies AD and other neurodegenerative diseases.9 Increased neurogenesis is considered a
protective factor against AD, and it has been shown that in human
adults this process occurs in olfactory bulbs and hippocampus. This
is a crucial aspect, especially if the fact that neuronal progenitors
in this area are located in close proximity to blood vessels is considered.9,44,45 Moreover, low levels of cGMP
have been connected with decreased neuronal growth. As stated above,
age-related reduction of cGMP may cause limited neurogenesis
and, thus, impaired cognitive functions.46 It has been observed that sildenafil can revert this mechanism by
stimulating progenitor cells' proliferation in the hippocampus.47</p><p>From the biochemical point of view, multiple
mechanisms underlie
the three events promoted by PDE5 inhibitors described above. Sabayan
and colleagues highlighted the role of enhanced CREB phosphorylation
and the glutamate-NO-cGMP pathway: the NO-cGMP-protein kinase G pathway
and overexpression/upregulation of the bcl-2 protein would be responsible
for the anti-apoptotic effects in neurons.9,48</p><p>Concerning cognitive enhancement in humans, evidence on the role
of PDE5 inhibitors is accumulating, even if with contrasting results
in some of the cases. As an example, the cognitive status of ED patients
without neurological or neuropsychiatric diseases treated with
udenafil was tested: mini-mental and frontal assessment scores increased
after administration (33 months), while the improvement in the Seoul
learning test was not significant.49 The
effects of sildenafil and tadalafil on cognitive functions have been
studied more extensively in the past years, and the results are discussed
in another section of this Review, while modest overall effects were
observed for vardenafil.50</p><!><p>PDE1, which is
represented by different subtypes, is expressed in several brain areas
such as the hippocampus, cerebral cortex, thalamus, and striatum 6
and 10.1 Vinpocetine (Figure 2) is a specific PDE1 inhibitor
that was observed to improve memory and ameliorate streptozocin-induced
cognitive dysfunction in rodent models.51,52 Interestingly,
it has also been demonstrated that this compound can improve synaptic
plasticity in a model of fetal alcohol spectrum disorders with impaired
cortical development.53 Vinpocetine also
gave positive results in other preclinical models and in cognitive
tests in humans. However, despite encouraging results on healthy volunteers,
further studies showed that vinpocetine failed to slow the decline
of AD patients.51,52 Vinpocetine is nevertheless present
in the Cognitex formulation, which was reported to have positive effects
in an open label study.54 A possible development
in the field of PDE1 is represented by a novel, selective inhibitor
known as ITI-214. This compound improves memory acquisition, consolidation,
and retrieval in rats, and Phase I clinical studies have been initiated
(ClinicalTrials.gov Identifier: NCT01900522).55,56 Moreover, Dyck and colleagues recently presented a set of selective
PDE1 inhibitors which showed promising results in enhancing memory
in a rat model. Even more importantly, those authors reported the
structure of the small molecule–protein complex.57</p><!><p>Chemical structures of the main inhibitors of other PDE
isoforms.</p><!><p>PDE2 is expressed in the cortex,
amygdala, and hippocampus.1 It hydrolyzes
both cGMP and cAMP, and it is the
target of the inhibitor BAY 60-7550, which was observed to improve
cognitive functions in rodents, and especially in memory-impaired
rats and in a mouse models of AD.58,59 ND-7001 is
another synthetic PDE2 inhibitor which was investigated for treating
cognitive impairment but its development was then discontinued. Although
PDE2 has attracted the interest of several pharmaceutical companies,
no ongoing clinical trials have been reported.3,60</p><p>The dual-substrate enzyme PDE3 is expressed in the cerebellum,
frontal cortex, hypothalamus, and hippocampus.1 Cilostazol (Figure 2) is a selective inhibitor of this isoform which enhances learning
and ameliorates cognitive impairment in wild-type and AD model mice.
As for PDE5 inhibitors, the effects appear to be the result of a combination
of mechanisms of action, comprehending increased blood flow, and stimulation
of the CREB pathway to promote synaptic plasticity.61,62 From the point of view of the clinical investigation as a potential
target in AD treatment, PDE3 has been one of the earliest isoforms
to be considered. In fact, several treatments and retrospective studies
conducted on different populations of AD patients showed overall positive
results, and the potential of PDE3 inhibitors in mild cognitive impairment
(MCI) has also been demonstrated.3 In mild
to moderate AD patients already on donepezil, cilostazol reduced cognitive
decline.63 Another study investigated the
effects of this inhibitor on cognition and regional cerebral blood
flow. Cilostazol contrasted AD-related cognitive decline, but it has
been observed that long-term treatment is required to obtain positive
effects on blood flow.64 In a recent case-control
study, cilostazol was tested as an add-on therapy for patients on
AChE inhibitors, showing again positive results.65 Moreover, a retrospective study observed that daily administration
of cilostazol decreases the risk of incident dementia.66 Nevertheless, side effects of PDE3 inhibitors
must be taken into account: cilostazol may be dangerous for patients
suffering from heart failure and severe hepatic or renal impairment.3</p><p>The cAMP-selective PDE4 isoform is widely
expressed in the CNS,67 and in particular
it is present in four subtypes
and 20 variants.68 More specifically, PDE4D
is more expressed in the CA1 hippocampal region and may be more closely
involved in memory consolidation.69 Rolipram
(Figure 2) and GEBR-7b
target this isoform, while GSK356278 inhibits PDE4B.1 Rolipram has beneficial effects on hippocampal- and cortex-dependent
memory, as demonstrated by several in vivo studies.70 However, its use in humans is limited by side
effects such as severe headache and emesis. In this connection, studies
on the more potent GEBR-7b and on allosteric modulators are emerging.
For example, BPN14770 is a negative allosteric modulator of PDE4D
well tolerated in Phase I studies.34,71,72 Similarly to rolipram, MEM 1414 entered Phase I but
showed emetic effects.73 For other compounds
which underwent or are undergoing clinical studies, such as MK-0952
and HT-0712, only limited and incomplete efficacy information is available.3 In addition to these examples, the FDA-approved
roflumilast (Figure 2), an anti-inflammatory and anti-chronic obstructive pulmonary disease
(COPD) agent, ameliorated impaired verbal learning performance in
healthy adults. Interestingly, from the biochemical point of view,
the activity of PDE4 inhibitors in contrasting AD appears to be amyloid-independent,
since CREB activation represents the main mechanism.3,74 In a very recent contribution, Iraji et al. reported an overview
of multi-target agents interacting with PDE4D as well as with other
targets involved in neurodegeneration, such as cholinesterases,
monoamine oxidase (MAO), and BACE1, which are relevant in AD
onset and progression.75</p><p>PDE6 is
only expressed in the pineal gland in the CNS, and thus
it does not represent a target for contrasting neurodegeneration.3</p><p>The cAMP-selective PDE7 is expressed in
the brain and has been
studied as a target for ameliorating AD, in particular in combination
with the activity of GSK3β. Its inhibition reduced cognitive
impairment and pathological hallmarks in a mouse AD model.76,77 In a very recent contribution, Morales-Garcia et al. reported that
genetic or small-molecule-mediated inhibition of PDE7 promotes neuroprotective
and anti-inflammatory effects in several neurodegenerative disease
models, with a special focus on Parkinson's disease.78</p><p>PDE8 is another cAMP-specific phosphodiesterase
and represents
a potential target for contrasting AD that has been studied rather
recently. While PDE8A expression is generally low in the human brain,
it has been observed that PDE8B concentration increases in the hippocampus
of AD patients. In particular, selective inhibitors such as PF-04957325
are being developed.79,80</p><p>PDE9 preferentially hydrolyzes
cGMP, and its relevance as a target
is testified by the fact that Pfizer and Boehringer entered Phase
II trials with PF-04447943 and BI 409306 inhibitors, respectively.81,82 Safety, tolerability, dose proportionality, and relative bioavailability
of tablet and oral solution formulations of the second compound were
evaluated by Moschetti et al. in healthy human male subjects.82 Moreover, Li et al. investigated the potential
of BAY 73-6691, a PDE9 inhibitor with good selectivity, on in vitro and in vivo models of AD.83 Nevertheless, it must be pointed out that compounds
targeting PDE9 generally have a degree of inhibitory activity on other
isoforms, which may result in undesired side effects.1</p><p>PDE10 is a dual-substrate enzyme that is mostly expressed
in the
striatum. Prickaerts et al. listed the clinical trials that several
synthetic isoform-specific inhibitors underwent. However, given the
limited results and PDE10 localization, pharmaceutical companies are
currently re-evaluating the compounds for Huntington's and
Parkinson's diseases.3</p><p>PDE11
is another non-specific enzyme, and it is the most recently
identified isoform. According to in vivo studies,
PDE11 seems to play a specific role in short-term and social memory.
Even if some small molecules are being developed as inhibitors, no
clinical investigation in this context has been reported.84,85</p><p>As a conclusion of this section of the review, focused on
the inhibitors
targeting PDE isoforms besides PDE5, relevant information on enzymes'
localization and studied compounds is summarized in Table 1. The reader is invited to refer
to the recent review by Argyrousi et al. for more general information
on PDE isoforms, genetic aspects, their localization in the human
body, and their regulation.86</p><!><p>In this section of the Review,
a closer focus on some known PDE5
inhibitors of natural and synthetic origin will be provided.</p><!><p>Developed as a novel tool
against hypertension and angina pectoris, sildenafil was patented
by Pfizer in 1996 and approved by the FDA in 1998 for the treatment
of ED.8 During the following two decades,
many novel investigational therapeutic applications were proposed
for this compound, especially thanks to its overall safety.10 Sildenafil is reported to cross the BBB, and
an indirect evidence of this feature is testified by some central
side effects such as dizziness, headache, and vision changes.33,87,88 Nevertheless, it has to be pointed
out that recent studies highlight a decreasing incidence of dementia
in men in western countries, and better management of vascular conditions
may be among the causes.89 In this context,
sildenafil and PDE5 inhibitors may play a role and be considered as
potential disease-modifying agents against AD that act mainly, but
not only, by improving endothelial dysfunction.9,90</p><p>In more detail, many mechanisms through which sildenafil could contrast
neurodegeneration were proposed through the years, and it was demonstrated
that this compound can simultaneously act at different levels. One
first event with which sildenafil can interfere is neurogenesis,
or the birth of new neuronal cells. Neurogenesis occurs in adult
forebrain regions of the subventricular zone and the dentate
gyrus, and it is crucial for neuronal plasticity and, thus, for memory
functions.91 It generally decreases with
aging due to lowered cGMP production, as discussed in the previous
section. Several reports (reviewed by Uthayathas et al.) show that
sildenafil administration can promote neuronal cell proliferation
by stimulating this pathway in rodent models,8 and these observations were recently confirmed by researchers using
other PDE5 inhibitors.92 Moreover, sildenafil
may enhance neurogenesis through Akt phosphorylation (as reported
for tadalafil), which leads to an increased phosphorylation of the
downstream target GSK3.93 This is a particularly
relevant feature, given the outcome of the pathway on the secretase
cathepsin B.94 Concerning other possibly
involved molecular mechanisms, Orejana et al. observed that sildenafil
decreases BACE1 expression in a SAMP8 mouse model and interferes with
the Cdk5/p25 pathway.94 Memory enhancement
is a crucial aspect, and in this context, the presynaptic and postsynaptic
effects of sildenafil must be dissected. More specifically, presynaptic
PDE5 inhibition increases cGMP concentration, promoting the release
of glutamate and the activation of NMDA receptors. On the other hand,
interference with postsynaptic PDE5 promotes a cascade leading to
increased protein synthesis and synaptogenesis. Overall augmented
activity of cGMP-coupled ion channels may help the early consolidation
of memory.95,96 In this connection, Son et al.
recently demonstrated that sildenafil exerts neuroprotective
effects from mitochondrial toxicity induced by amyloid Î² (AÎ²)
peptide, and that ATP-sensitive potassium channels are involved. The
authors concluded that sildenafil "suppresses the mitochondrial
Ca2+ overload, disruption of mitochondrial functional integrity,
and mitochondria-dependent apoptotic signaling in the HT-22 cells
following Aβ-induced AD-like insult".97</p><p>This mechanistic evidence is paralleled by experimental
results
on the efficacy of sildenafil in counteracting long-term memory deficit
and focus attention in animal AD models and humans96,98,99 and recent data obtained studying other
PDE5 inhibitors.92 Preclinical data support,
to different degrees, the beneficial role of PDE5 inhibitors in promoting
cognitive enhancements in rodents and primates.34,100 In mouse models, sildenafil administration was reported to ameliorate
cognitive impairment and upregulate the brain-derived neurotrophic
factor (BDNF), which contributes to the neuroprotective effects.101,102 The same compound also improved early consolidation processes and
spatial memory (Morris water maze test, MWM).103 Moreover, the sildenafil-sustained NO-cGMP-protein kinase
G pathway appears to be involved in enhancing learning and memory
performance in rodents, and sildenafil administration also improved
novel object recognition in rats with induced cognitive deficits.42,98</p><p>As previously stated, clinical studies on sildenafil and its
effects
on cognitive functions did not always show results in perfect agreement
with in vitro or preclinical data.96 In general, sildenafil was not found to be effective on
short-term memory, divided attention, and psychomotor tasks.
On the other hand, improvements were observed in focused attention,
simple reaction time tasks, and information processing in humans.96,104,105</p><p>Some sildenafil analogues
were recently synthesized with the aim
of simultaneously targeting PDE5 and histone deacetylase, another
enzyme involved in the memory process, and tested in vitro and in vivo in a rodent model. This strategy aims
at a synergistic effect, since PDE5 inhibition promotes CREB phosphorylation
and the recruitment of CREB binding protein (CBP), which has histone
acetylase activity.106 This path was very
recently followed by Rabal et al., who presented a set of compounds
structurally similar to sildenafil and its analogues that target PDE
and histone deacetylase. The compounds, characterized by adequate
ADMET and toxicity profiles, were tested by the authors on a mouse
model of AD.107,108</p><p>Besides AD, sildenafil
has also been tested for its activity on
cognitive symptoms of schizophrenia, and no cognitive changes were
observed when it was co-administered with anti-psychotics.105 In the context of other CNS-related diseases,
the NO-cGMP pathway influences excitatory and inhibitory neurotransmission
in epilepsy. Both pro- and anti-convulsant activities were reported
for sildenafil, depending on the considered animal model and dose.
Thus, the potential of PDE5 inhibition in this context remains rather
unclear.109 More interestingly, psychotropic
effects have been observed for sildenafil in humans with ED-related
depression. Moreover, sildenafil showed promising results in behavior
studies in rats in combination with atropine. It has also been highlighted
that the anti-depressant-like effect of sildenafil may possibly proceed
through the activation of oxytocin.110,111</p><!><p>The repositioning of marketed
drugs is recently becoming a commonly pursued strategy, since the
safety of such agents has already been proved and their further development
should be facilitated. As already stated, this may also be the case
with PDE5 inhibitors, which are generally characterized by good pharmacokinetic
properties and safety profiles. The evolution of clinical applications
of tadalafil has been recently reviewed by N. S. Ahmed.112 Tadalafil can be safely administered orally
on a daily basis, and this makes it a good candidate for repurposing
as a memory enhancer.113 Tadalafil has
been studied for its positive effects on cognitive processes by García-Barroso
et al., who reported the effects of chronic treatment (5 weeks) on
aged mice. According to the freezing behavior test, a measure of learned
fear, tadalafil showed cognitive enhancements. It also promoted improvements
in terms of escape latency. Moreover, those authors investigated the
neuronal changes induced by this PDE5 inhibitor, showing that tadalafil
administration is associated with an increase in the spine density
on apical dendrites, which may be the mechanism underlying memory
improvements. Anti-apoptotic pathways, such as those depending on
Akt, could also be among the mechanisms of tadalafil in preventing
neuronal cell death.34 To assess its pharmacokinetic
properties, the compound was quantified in CSF by liquid chromatography–mass
spectrometry (LC-MS): tadalafil levels in CSF after oral administration
in non-human primates (2.4 mg/kg, given to Macaca fascilurais) were 1 order of magnitude higher than the IC50 for PDE5.1,114 Tadalafil was also previously reported to enhance spatial memory
in the J20 AD mouse model more efficiently than sildenafil. The longer
half-life, stability, and more efficient BBB permeability of the former
may contribute to the observed effects.34</p><p>It is generally accepted that a chronic lack of blood flow
to the deep brain area is common in, and may be related to the onset
of, AD and other dementias.115 A clinical
trial (ClinicalTrials.gov Identifier: NCT02450253) has been initiated
to study the effects of tadalafil on patients with symptomatic small
vessel disease in the brain but lacking the vascular cognitive impairment
diagnosis, which would contribute to the understanding of the role
of PDE5 inhibitors in contrasting the onset and progression of AD.3,115</p><p>Interestingly, it has been reported that tadalafil also possesses
a certain degree of AChE inhibitory activity. Based on this observation
and on the promising in vivo data discussed in this
section, Mao et al. designed and synthesized a class of novel compounds
inspired by the scaffold of tadalafil with dual AChE/PDE5 inhibitory
activity and improved BBB permeability and solubility.116 This strategy was previously pursued by Zhou
et al., who in a more preliminary study developed a class of multi-targeted,
dual AChE/PDE5 inhibitors with good solubility and low cell toxicity.117 More recently, Ni et al. developed a set of
water-soluble, drug-like, and BBB-permeable tadalafil analogues that
showed good performance in ameliorating scopolamine-induced cognitive
impairment. Such compounds were active on both AChE and PDE5 and induced
enhanced CREB phosphorylation.118</p><!><p>Several natural
compounds have been described throughout the years as selective or
non-selective inhibitors of PDE isoforms, and particular attention
was dedicated to natural small molecules selectively inhibiting PDE5.
Such compounds were often identified in plants traditionally used
in folk medicine, mainly (but not only) as natural remedies for ED.119,120 Experimental evidence on the inhibitory effect of natural compounds
from several different chemical classes ranges from in silico to in vivo.121−125 The potential of herbal extracts or isolated natural compounds with
PDE inhibitory activity in contrasting AD progression was reviewed
by Kumar et al. Those authors clearly listed several examples of natural
plants with cAMP-specific, cGMP-specific, or dual inhibitory activity.126 The flavonoid icariin (Figure 3) is a known natural PDE5 inhibitor from Epimedium brevicornum. It represents an outstanding example
from this class, since it has been shown to improve learning and memory
functions in APP/PS1 transgenic mice. This effect likely occurs through
the stimulation of the NO/cGMP signaling pathway, which results in
decreased physiopathological changes in treated animals.127 Natural compounds often act through parallel
mechanisms, and icariin is also an AChE inhibitor, which makes it
an even more attractive candidate for the development of novel AD
treatments.128,129</p><!><p>Chemical structures of some natural PDE5
inhibitors belonging to
the class of flavonoids.</p><!><p>A multi-target mechanism
was also demonstrated for natural and
semi-synthetic isoflavones (Figure 3). This class of molecules, known for their PDE5 inhibitory
activity, showed in vitro anti-AD effects on human
recombinant BACE-1 inhibition assay and increased the activity of
P-gp ATPase, with a possible role in the efflux of AÎ² across
the BBB.122,130</p><p>Several other examples
of natural compounds with multiple mechanisms,
involving PDEs inhibition at different levels, were previously reported. Ginkgo biloba extracts has been shown to produce anti-amyloid
and anti-tau effects and to improve cognitive functions in dementia
models. More specifically, the extract reduced tau phosphorylation
in the C57BL6 mouse model.126,131,132 However, a clinical trial evaluating long-term use of standardized Ginkgo biloba extract did not highlight a decrease in the
risk of progression to AD in comparison with placebo.133</p><p>Concluding, it must be stressed that
PDE inhibition is not the
only mechanism responsible for the neuroprotective activity
of natural molecules, and several other biochemical pathways, even
with direct effects on amyloid aggregation, were proven to be involved.134−136</p><!><p>This Review originated
from the need to provide an updated report
on the state of the art of novel therapeutic options to contrast neurodegeneration
based on PDE inhibition.137 While overviewing
the past two decades of literature, we were prompted to rationalize
and integrate available data using the tools of contemporary drug
discovery. In particular, we must take into account that the interest
in multi-target-directed ligands (MTDLs) is pushing the medicinal
chemist to look beyond the usual "one molecule–one target–one
disease" approach. Growing evidence shows that single drugs
developed and extremely optimized to act on individual molecular targets
are often inadequate or fail due to (unpredicted) side effects. One
of the prominent current aims is based on the so-called "network
pharmacology", where the development of the drug candidate
consists of combining, in a single small molecule, some multi-targeting
properties.138 First, this applies to the
study of non-selective PDE inhibitors (such as theophylline, resveratrol,
and their derivatives), small molecules that act on different isoforms
showing additive interactions and beneficial synergistic effects.29,139,140 In fact, as stated by Maurice
et al., simultaneous inhibition of multiple PDEs leads to the activation
of several signaling pathways, which may be desirable for treating
complex diseases.141 In this context, the
increasing understanding of the role of tissue and subcellular compartmentalization
of cyclic nucleotides and individual PDE isoforms allows the development
of more sophisticated strategies for interacting with such macromolecular
targets.141</p><p>Then, on the side of
isoform-selective inhibitors, the MTDL approach
is being pursued in the study of novel remedies against dementia,
as highlighted in the previous sections of this Review. This represents
the real "core" of the rationale underlying the repurposing
of PDE5 inhibitors such as tadalafil in this context. Recent contributions
in the literature demonstrate the growing interest toward the identification
of small molecules concurrently acting on PDE5 and on a secondary
target involved in AD.142 This strategy
would also aid the development of more targeted and personalized therapeutic
approaches.143 In particular, the combination
of the inhibitory effects on PDE5 and AChE is particularly attractive.
In this context, a direct comparison between three of the most widely
studied and promising PDE5 inhibitors discussed previously is provided
below. Sildenafil, tadalafil, and the natural compound icariin have
been compared basing on their inhibitory activity toward PDE5 and
AChE and according to predicted pharmacokinetic properties,
which are of primary relevance for CNS-acting drugs.</p><p>Sildenafil
is a potent PDE5 inhibitor (IC50 = 5.2 nM)
but lacks effects on AChE.117,144 Tadalafil is again
very effective on PDE5 (IC50 = 2.4 nM), while it inhibits
AChE in the micromolar range (IC50 = 26.2 Î¼M).116,144 On the other hand, icariin is a micromolar inhibitor of PDE5 (IC50 = 5.9 Î¼M), but it is more effective in contrasting
AChE activity (IC50 = 25.0 nM).128,145 Given these values, tadalafil appears as a promising dual-target
candidate. Figure 4A represents the crystal structure of the tadalafil-PDE5 complex,
and the ligand is highlighted in green. Docking studies were carried
out to investigate the interaction pattern of the same small molecule
with AChE, since a crystal structure of the complex is not available.
Recent research works highlighted the reliability and significance
of computational analysis modeling applied to the study of PDE5 inhibitors.146 The in silico experiments
were carried out using AutoDock Vina, and the 3D structures were visualized
with the UCSF Chimera molecular viewer.147,148 A brief description of the experimental protocol is reported in
the following. Protein models were retrieved from the Protein Data
Bank (www.rcsb.org, PDB ID: 1UDU for PDE5 in complex
with tadalafil and PDB ID: 4EY7 for AChE in complex with donepezil). Target and ligands
were prepared for the blind docking experiment performed using AutoDock
Vina. Receptor grid generation was automatically set, thus encompassing
the whole studied protein (blind docking). The number of docking poses
was set to 10, and the other AutoDock Vina parameters were set to
the default. Output data (energies and interaction patterns) were
analyzed and scored using UCSF Chimera, which was also used to produce
the artwork. Values reported in the following are expressed in kcal/mol
and refer to the most favored pose. According to the predicted binding
model, which was calculated by blind docking on the 3D structure of
AChE, tadalafil interacts with the same binding site of donepezil
(computed binding energy = −9.2 kcal/mol). To validate the
protocol and as a positive control, donepezil was re-docked to the
same protein (computed binding energy = −8.8 kcal/mol). A superimposition
of the two compounds is represented in Figure 4B.</p><!><p>Crystal structure of the complex between tadalafil,
depicted in
green, and PDE5 (A). Calculated interaction pattern of tadalafil (green)
with AChE; the originally co-crystalized ligand donepezil is represented
in red (B).</p><!><p>A more detailed representation
of the computed interaction pattern
of tadalafil with AChE, in comparison with that of donepezil, is depicted
in Figure 5. According
to this docking study, the two compounds interact with the macromolecular
target through the same residues. The amino acids that are located
in the binding pocket (<5 Å from the ligand) have been labeled.</p><!><p>Detailed
view of the AChE binding pocket. Calculated interaction
pattern of tadalafil (green) is shown in comparison with the originally
co-crystalized ligand donepezil, represented in red. Interacting residues
(<5 Å) have been labeled.</p><!><p>Besides the enzymatic activity data, the three compounds can be
also compared from the point of view of their pharmacokinetic
properties, which were calculated using Molinspiration Cheminformatics
software (https://www.molinspiration.com). Topological polar surface area (TPSA), logP, volume, molecular
weight, number of rotatable bonds, and numbers of hydrogen bond donors
(nONH) and acceptors (nON) were computed and analyzed. Pajouhesh and
Lenz efficiently determined the chemical and structural features of
a successful CNS drug candidate:149 the
reference values indicated by the authors are reported in the bottom
line of Table 2, together
with the values calculated for the three studied PDE5 inhibitors.</p><!><p>Reference values
for a successful
CNS drug candidate are reported in the bottom line.</p><!><p>According to these drug-likeness
criteria, tadalafil is the most
promising CNS drug candidate of the set: its polar surface area is
close to the ideal limit, and it is the only one not exceeding the
molecular weight and number of hydrogen bond acceptor limits. Sildenafil
has a higher polar surface area, which may limit BBB permeation, it
has a molecular weight above 400, and its high number of heteroatoms
pushes the value of hydrogen bond acceptors above the limit. Icariin
is a very polar molecule and does not respect the limits proposed
by Pajouhesh and Lenz. Another simple predictive rule states that
BBB penetration is likely if the number of nitrogen and oxygen atoms
is smaller than 5 ((N + O) ≤ 5).149 According to this formula, the computed values are 10 for sildenafil,
7 for tadalafil, and 15 for icariin. Thus, even if the desired value
of 5 is exceeded by all the compounds, it can be stated that tadalafil
is closer to this limit, again suggesting a better BBB permeability.</p><!><p>The selective inhibition of brain-expressed
PDEs for contrasting
neurodegeneration and dementia is a strategy that has been pursued
for over two decades. PDE3, a dual-substrate enzyme, attracted a notable
interest. Moreover, in vivo efficacy data on compounds
targeting this isoform are already available, especially concerning
the use of cilostazol. In addition to this, the attention that several
pharmaceutical companies dedicated to the preclinical and clinical
development of PDE4 and PDE9 inhibitors, belonging to different chemical
classes, must be noted. The cGMP-selective PDE5 isoform is probably
the most widely studied in this context, thanks to the availability
of compounds that are already of clinical use and are known for their
overall safety and good pharmacokinetic properties; in vitro and in vivo (preclinical and clinical)
data currently focus on sildenafil, tadalafil, and some compounds
of natural origin.</p><p>The involved molecular mechanisms underlying
the activity of such
inhibitors include CREB phosphorylation via protein kinase A or protein
kinase B, depending on the involved PDE isoform, which would stimulate
neuronal plasticity. Neuroprotective and anti-apoptotic effects in
neurons are otherwise mediated by bcl-2 upregulation and the Akt-GSK3
pathway.</p><p>In conclusion, even if PDE inhibitors show very promising
performances in vitro and in some in vivo disease model,
contrasting results can be retrieved from clinical studies. Clinical
trials with combined therapy appear to be more promising, and the
multi-target approach may represent a favorable option. Taking advantage
of "network pharmacology", compounds with even modest
activity on multiple, crucial targets could be optimized to tune their
activity toward cognitive improvement. In this connection, early evidence
is already emerging: the combination of PDE with AChE or histone deacetylase
inhibitory activity is a promising strategy. The computational results
here presented, together with the overviewed preliminary in
vitro and in vivo evidence, suggest that
tadalafil represents a promising starting point, in the context of
PDE5 inhibitors, for the development of innovative tools to contrast
neurodegeneration.</p><!><p>Conceptualization,
G.R. and A.G.; methodology, M.M.; investigation, G.R. and A.O.; data
curation, A.G.; writing, original draft preparation, G.R., A.O., and
A.G.; writing, review and editing, M.M. and G.Z.; supervision, M.M.
and A.G.; funding acquisition, A.G.</p><!><p>The authors declare no
competing financial interest.</p>