pharmaceuticals
Review A Perspective on Natural and Nature-Inspired Small Molecules Targeting Phosphodiesterase 9 (PDE9): Chances and Challenges against Neurodegeneration
Giovanni Ribaudo * , Maurizio Memo and Alessandra Gianoncelli
Department of Molecular and Translational Medicine, University of Brescia, 25121 Brescia, Italy; [email protected] (M.M.); [email protected] (A.G.) * Correspondence: [email protected]; Tel.: +39-030-3717419
Abstract: As life expectancy increases, dementia affects a growing number of people worldwide. Be- sides current treatments, phosphodiesterase 9 (PDE9) represents an alternative target for developing innovative small molecules to contrast neurodegeneration. PDE inhibition promotes neurotransmitter release, amelioration of microvascular dysfunction, and neuronal plasticity. This review will provide an update on natural and nature-inspired PDE9 inhibitors, with a focus on the structural features of PDE9 that encourage the development of isoform-selective ligands. The expression in the brain, the presence within its structure of a peculiar accessory pocket, the asymmetry between the two subunits composing the protein dimer, and the selectivity towards chiral species make PDE9 a suitable target to develop specific inhibitors. Additionally, the world of natural compounds is an ideal source for identifying novel, possibly asymmetric, scaffolds, and xanthines, flavonoids, neolignans, and their derivatives are currently being studied. In this review, the available literature data were interpreted and clarified, from a structural point of view, taking advantage of molecular modeling: 3D structures of ligand-target complexes were retrieved, or built, and discussed. Citation: Ribaudo, G.; Memo, M.; Gianoncelli, A. A Perspective on Keywords: PDE9; Alzheimer’s disease; natural compounds; cGMP; neurodegeneration; xanthines Natural and Nature-Inspired Small Molecules Targeting Phosphodiesterase 9 (PDE9): Chances and Challenges against 1. Introduction Neurodegeneration. Pharmaceuticals Dementia currently affects 44 million people worldwide, a number that is constantly 2021, 14, 58. https://doi.org/ growing in light of increasing life expectancy. The symptoms of Alzheimer’s disease (AD) 10.3390/ph14010058 consist of memory, attention, personality, intellect, and speech impairments [1]. From a
Received: 24 December 2020 biochemical point of view, amyloid plaques in the brain and degeneration of cholinergic Accepted: 10 January 2021 innervation in the hippocampus and the cerebral cortex are reported to be involved in Published: 13 January 2021 disease onset and progression [2]. Current therapeutic options include acetylcholinesterase (AChE) inhibitors (donepezil, rivastigmine, galantamine) and compounds, such as meman- Publisher’s Note: MDPI stays neu- tine, that target N-methyl-D-aspartate (NMDA) receptors. However, limited efficacy and tral with regard to jurisdictional clai- frequently occurring adverse effects limit their use and prompt the research towards novel ms in published maps and institutio- tools to contrast neurodegeneration [3]. nal affiliations. In this context, phosphodiesterases (PDEs) are attracting growing attention as po- tential targets for small molecules as novel drug candidates [4,5]. PDEs hydrolyze the second messengers 3’,5’-cyclic adenosine monophosphate (cAMP) and 3’,5’-cyclic guano- sine monophosphate (cGMP) with different degrees of selectivity, depending on the enzyme Copyright: © 2021 by the authors. Li- family. PDE4, PDE7, and PDE8 are in fact cAMP-selective, while PDE5, PDE6, and PDE9 censee MDPI, Basel, Switzerland. are cGMP-selective. On the other hand, PDE1, PDE2, PDE3, PDE10, and PDE11 are “dual This article is an open access article substrate” enzymes [6]. In general, PDEs are involved in intracellular signal transduction distributed under the terms and con- ditions of the Creative Commons At- by regulating cAMP and/or cGMP levels. tribution (CC BY) license (https:// PDE5 inhibitors acting on peripheral enzymes, such as sildenafil and its analogues, creativecommons.org/licenses/by/ currently find application in clinic as therapeutic approaches against erectile dysfunction 4.0/). and pulmonary hypertension. Moreover, compounds targeting PDE5 are being studied for
Pharmaceuticals 2021, 14, 58. https://doi.org/10.3390/ph14010058 https://www.mdpi.com/journal/pharmaceuticals Pharmaceuticals 2021, 14, 58 2 of 13
hearth failure, asthma, diabetes, obesity, and central nervous system (CNS) diseases [7–9]. The PDE4-selective inhibitor rolipram was studied as a potential treatment for AD, but its further development failed due to adverse effects [4,7]. Concerning neuroprotection, cognitive enhancing, and the effects against neurodegen- eration, increased cAMP and cGMP levels by PDE inhibition are involved in promoting neurotransmitter release, amelioration of microvascular dysfunction and neuronal plas- ticity [10–12]. Stimulation of cAMP response element binding protein (CREB) and brain derived neurotropic factor (BDNF), together with improved release of nitric oxide (NO), are reported as the main biochemical mechanisms involved [4]. PDEs are expressed in several tissues. Except for PDE11 and for PDE6, which is only present in the pineal gland, all PDE families are expressed, to different extents, in the central nervous system [13]. Most importantly, several studies investigated the variations of PDE isoforms expression levels in the brain of AD patients: limited changes, mainly referred to PDE1, PDE4, PDE9, and PDE10, were noted [14–17]. PDE9 is among the currently most studied innovative targets for developing inhibitors to combat dementia [5,18], in light of its expression in the brain and of its unique structural features that allow the design of molecules selectively interacting with this target, as will be discussed in the following. PDE9 is endowed with high affinity for cGMP (Km = 70 nM) and the highest selectivity over cAMP (Km = 230 µM) among the other isoforms [19,20]. Moreover, it has been found to be highly expressed in brain of AD patients [4]. In particular, this isoform was detected in the amygdala, cerebellum, cortex, hippocampus, hypotha- lamus, midbrain, olfactory bulb, and striatum [21]. More recently, Patel and colleagues investigated the subcellular localization of PDE9 in the brain, highlighting changes during lifespan [22]. This enzyme is also expressed in the gut, kidney and heart [19]. For more general reviews on the inhibitors currently undergoing preclinical and clinical investiga- tions that target PDE subtypes in brain, the reader is invited to consider our previous work and the paper by Prickaerts et al. [3,5]. The present contribution aims at providing an overview of the reports available in the literature concerning natural or nature-inspired PDE9 inhibitors, and a particular focus will be placed on the structural aspects of PDE9 that encourage the development of selective inhibitors, taking advantage of unique conformational features that allow further optimization of the scaffolds of natural compounds. Current review articles regard mainly PDE1–PDE5 inhibitors [23], while no updated overviews were retrieved concerning recent findings about other isoforms, and PDE9 in particular. For the preparation of this review, more than 60 contributions in the literature from the 2000–2020 timeframe were considered and screened. Original research articles were retrieved searching the PubMed (www.ncbi.nlm.nih.gov/pubmed/) and Scopus (www. scopus.com) databases using keywords such as “phosphodiesterase”, “PDE9”, “natural compounds”, “neurodegeneration”, “Alzheimer’s disease” and their combinations. The 3D models of the studied protein and complexes were retrieved from the Protein Data Bank (PDB, www.rcsb.org) and UCSF Chimera software was used to prepare the models [24]. All the 3D models included in this review are original and were produced for the current work.
2. Chemical Classes of Natural Compounds Targeting PDEs The inhibition of phosphodiesterase activity was described for the first time at the end of the 19th century, when the natural alkaloid caffeine was reported to have bronchodilator properties. Some decades later, caffeine was identified as a nonselective PDE inhibitor [3]. Several plant extracts and, more specifically, their bioactive constituents were reported to possess neuroprotective action and to improve cognitive function against dementia and other neurodegenerative disorders [25]. The activity of natural compounds ameliorating AD conditions in preclinical or clinical studies is generally related to a multi-target mecha- nism of action [26], and inhibition of one or more PDE isoforms is often involved [8,27–29]. Natural, semi-synthetic, and synthetic compounds can in fact inhibit PDEs with different degrees of selectivity, as well as act as non-selective inhibitors [30]. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 3 of 13
Pharmaceuticals 2021, 14, 58 target mechanism of action [26], and inhibition of one or more PDE isoforms is often3 of in- 13 volved [8,27–29]. Natural, semi-synthetic, and synthetic compounds can in fact inhibit PDEs with different degrees of selectivity, as well as act as non-selective inhibitors [30]. ItIt mustmust bebe pointedpointed out thatthat thethe researchresearch fieldfield ofof PDEPDE inhibitioninhibition is traditionallytraditionally con- nected with the world ofof natural compounds,compounds, asas itit waswas initiallyinitially focusedfocused onon alkaloidsalkaloids suchsuch as caffeine,caffeine, asas citedcited above,above, oror theophyllinetheophylline andand papaverine.papaverine. However, itit waswas laterlater foundfound thatthat thethe effectseffects ofof suchsuch compoundscompounds on on blood blood vessels vessels were were mostly mostly connected connected with with the the inhibi- inhi- tionbition of of cAMP-selective cAMP-selective PDEs PDEs [23 [23].]. Within Within the the broad broad class class of of alkaloids, alkaloids, chelerythrinechelerythrine andand glaucine were alsoalso studiedstudied asas naturalnatural PDEPDE inhibitorsinhibitors butbut limitedlimited isoformisoform selectivityselectivity andand μ ICIC50 valuesvalues in in the the higher higher µMM range range limited limited their their further further development [31]. [31]. Flavonoids areare among among the the most most widely widely studied studied classes classes of natural of natural PDE PDE inhibitors, inhibitors, even ifeven the if great the majoritygreat majority of contributions of contributions in the literaturein the literature are focused are focused on the evaluation on the evaluation of such compoundsof such compounds against PDE5 against [8,32 –PDE535]. Concerning [8,32–35]. thisConcerning class, hesperidin this class, and naringeninhesperidin wereand reportednaringenin to inducewere reported relaxing to effects induce of relaxing noradrenaline-, effects okadaicof noradrenaline-, acid-, and KCl-precontractedokadaic acid-, and aorta.KCl-precontracted This activity isaorta. mediated This byactivity inhibition is mediated of cGMP by hydrolysis inhibition by of PDEs cGMP [36 hydrolysis,37]. Dioclein by andPDEs epigallocatechin-3-gallate [36,37]. Dioclein and epigallocatechin-3 (EGCG) were also-gallate studied (EGCG) as a vasorelaxant were also studied agents andas a PDEvas- inhibitorsorelaxant interactingagents and withPDE theinhibitors cGMP pathway,interacting but with the compound’sthe cGMP pathway, lack of selectivity but the com- was demonstratedpound’s lack of to selectivity interact potently was demonstrated with PDE1 [to38 ,interact39]. potently with PDE1 [38,39]. Saponins, lignans, polyphenols, organic acids, and coumarins represent other exam- plesples ofof chemicalchemical classesclasses of naturalnatural compoundscompounds known as non-specificnon-specific PDE-inhibitors [[8].8]. TheThe chemicalchemical structuresstructures ofof thethe mainmain citedcited compoundscompounds areare reportedreported inin FigureFigure1 .1.
O O O O O N N H O O N N N N O N ONN O N N O O O O O O Caffeine Theophylline Papaverine Glaucine Chelerythrine
OH OH OH HO HO O OH HO O O O OH O O OH OH O OH O OH O OH OH Naringenin Dioclein Epigallocatechin-3-gallate Figure 1. Chemical structures of some of thethe knownknown naturalnatural phosphodiesterasephosphodiesterase (PDE)(PDE) inhibitors.inhibitors.
3. Natural and Nature-Inspired PDE9 Inhibitors The promising results of in vitrovitro andand inin vivovivo studies,studies, andand thethe growinggrowing interestinterest ofof major pharmaceuticalpharmaceutical companies, encourage medicinalmedicinal chemists to explore the fieldfield ofof selective PDE9 inhibitorsinhibitors asas aa novelnovel approachapproach againstagainst dementiadementia [[18,18,4040].]. SyntheticSynthetic PDE9PDE9 inhibitorsinhibitors areare indeedindeed currentlycurrently being being investigated investigated in in clinical clinical trials trials as as promising promising therapeutic therapeu- optionstic options against against neurodegeneration neurodegeneration [41 ].[41]. On On the the other other hand, hand, xanthines xanthines and and polyphenols, polyphe- asnols, well as aswell lignans, as lignans, are among are among the mostthe most widely widely studied studied natural natural scaffolds scaffolds for for developing develop- inhibitors interacting with PDEs in the CNS. Peculiar structural features of PDE9 isoform ing inhibitors interacting with PDEs in the CNS. Peculiar structural features of PDE9 iso- and chemical aspects of the investigated nature-inspired ligands will be discussed in the form and chemical aspects of the investigated nature-inspired ligands will be discussed following sections. in the following sections. 3.1. Structural Features of PDE9 3.1. Structural Features of PDE9 The chemical structure of PDE inhibitors and their interaction pattern with the enzyme bindingThe pocketchemical have structure been extensively of PDE inhibitors studied, and and their some interaction pharmacophoric pattern with features the en- of PDEzyme ligands binding were pocket highlighted have been [ 42extensively]. Gln453 andstudied, Phe456 and are some the pharmacophoric main residues involved features inof PDE ligand–target ligands were interaction highlighted in the [42]. context Gln453 of and PDE Phe456 inhibition are the and main such residues amino acidsinvolved are conserved within PDE families [43]. Huai et al. clearly resumed the structural features of PDE9, focusing on the study of the crystal structure of the PDEA2 isoform in complex with 3-isobutyl-1-methylxanthine (IBMX), a non-specific PDE binder (Figure2). This
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 4 of 13
in ligand–target interaction in the context of PDE inhibition and such amino acids are con- served within PDE families [43]. Huai et al. clearly resumed the structural features of PDE9, focusing on the study of the crystal structure of the PDEA2 isoform in complex with 3-isobutyl-1-methylxanthine (IBMX), a non-specific PDE binder (Figure 2). This com- pound, in fact, also interacts with PDE4, PDE5, and PDE9, inhibiting these enzymes in the μM range [20]. The catalytic domain of PDE9 is constituted by 16 helices (residues 181–506) and it is very similar to that of PDE4. On the other hand, a structural comparison with PDE5 high- lighted significant differences [44]. Moreover, in crystal structures, PDE9 catalytic do- mains associate in dimers, while they further combine in tetramers in the case of PDE4 [20]. As well as in PDE4 and PDE5, zinc and magnesium are present inside PDE9, even if biochemical studies demonstrated that manganese also activates the protein [19]. Zinc forms coordination bonds with His256, His292, Asp293, Asp402, and two water mole- cules. The second ion is coordinated by Asp293 and five water molecules [20]. These de- tails and the overall 3D structure of PDE9 are depicted in Figure 2. Huang et al. reported a detailed study concerning the biorecognition of PDE9 by a single enantiomeric compound, in light of a peculiar structural asymmetry of the protein. In particular, the authors demonstrated the presence of a slight conformational difference between M-loops of PDE9 dimers and, most importantly, of a small hydrophobic pocket that may be targeted by enantiomerically pure compounds [45]. This feature will be dis- cussed in detail in the following sections of the review. In this connection, it has also been observed that IBMX shows different orientations when interacting with other PDE isoforms. Nevertheless, as anticipated, key interactions, such as stacking between xan- Pharmaceuticals 2021, 14, 58 thine and phenylalanine and hydrogen bond between N7 and glutamine, are maintained.4 of 13 Interestingly, a slightly different binding motif for IBMX was also highlighted within the two monomers of the PDE9 dimer. These two ligands have a rotation difference of 50°: stacking is conserved but IBMX interacts with Phe251, His252, and Asn405 in one mono- mercompound, and with inMet365 fact, also and interacts Phe441 in with the PDE4,other. Moreover, PDE5, and the PDE9, isobutyl inhibiting chain theseadopts enzymes differ- in µ entthe orientationsM range [(Figure20]. 2) [20].
(a ) (c) O H N N
O N N
(b ) (d )
Figure 2. Chemical structure of 3-isobutyl-1-methylxanthine (IBMX) (a). Three-dimensional structure of PDE9 in complex with its inhibitor IBMX, highlighted in yellow (PDB ID: 2HD1): chain A is depicted in grey and chain B in blue (b). Detailed
view of the interaction pattern of IBMX with chain A: zinc is represented in purple and magnesium is reported in green. Interacting residues have been labeled and highlighted in green (c). Detailed view of the interaction pattern of IBMX with chain B: zinc is represented in purple and magnesium is reported in green. Interacting residues have been labeled and highlighted in green (d).
The catalytic domain of PDE9 is constituted by 16 helices (residues 181–506) and it is very similar to that of PDE4. On the other hand, a structural comparison with PDE5 highlighted significant differences [44]. Moreover, in crystal structures, PDE9 catalytic do- mains associate in dimers, while they further combine in tetramers in the case of PDE4 [20]. As well as in PDE4 and PDE5, zinc and magnesium are present inside PDE9, even if biochemical studies demonstrated that manganese also activates the protein [19]. Zinc forms coordination bonds with His256, His292, Asp293, Asp402, and two water molecules. The second ion is coordinated by Asp293 and five water molecules [20]. These details and the overall 3D structure of PDE9 are depicted in Figure2. Huang et al. reported a detailed study concerning the biorecognition of PDE9 by a single enantiomeric compound, in light of a peculiar structural asymmetry of the protein. In particular, the authors demonstrated the presence of a slight conformational difference between M-loops of PDE9 dimers and, most importantly, of a small hydrophobic pocket that may be targeted by enantiomerically pure compounds [45]. This feature will be dis- cussed in detail in the following sections of the review. In this connection, it has also been observed that IBMX shows different orientations when interacting with other PDE isoforms. Nevertheless, as anticipated, key interactions, such as stacking between xanthine and phenylalanine and hydrogen bond between N7 and glutamine, are maintained. In- terestingly, a slightly different binding motif for IBMX was also highlighted within the two monomers of the PDE9 dimer. These two ligands have a rotation difference of 50◦: PharmaceuticalsPharmaceuticals 2021 2021, 14, ,14 x ,FOR x FOR PEER PEER REVIEW REVIEW 5 of5 13of 13
Pharmaceuticals 2021, 14, 58 5 of 13 FigureFigure 2. Chemical2. Chemical structure structure of of3-isobut 3-isobutyl-1-methylxanthineyl-1-methylxanthine (IBMX) (IBMX) (a ).(a Three-dimensional). Three-dimensional structure structure of ofPDE9 PDE9 in incomplex complex withwith its its inhibitor inhibitor IBMX, IBMX, highlighted highlighted in inyellow yellow (PDB (PDB ID: ID: 2HD1): 2HD1): chain chain A isA depictedis depicted in ingrey grey and and chain chain B inB inblue blue (b ).(b Detailed). Detailed viewview of ofthe the interaction interaction pattern pattern of ofIBMX IBMX with with chain chain A: A: zinc zinc is representedis represented in inpurple purple and and magnesium magnesium is reportedis reported in ingreen. green. InteractingInteracting residues residues have have been beenstacking la beledlabeled isand conservedand highlighted highlighted but in IBMX ingreen green interacts(c ).(c Detailed). Detailed with view view Phe251, of ofthe the His252,interaction interaction and pattern Asn405pattern of ofIBMX in IBMX one with monomer with chainchain B: B:zinc zinc is isrepresented represented in inpurple purple and and magnesium magnesium is isrepo reportedrted in ingreen. green. Interacting Interacting re siduesresidues have have been been labeled labeled and and and with Met365 and Phe441 in the other. Moreover, the isobutyl chain adopts different highlightedhighlighted in ingreen green (d ).(d ). orientations (Figure2)[20].
3.2.3.2.3.2. PDE9 PDE9PDE9 Inhibitors InhibitorsInhibitors Undergoing UndergoingUndergoing Clinical ClinicalClinical Studies StudiesStudies TheTheThe growing growinggrowing understanding understanding understanding of of ofthe the individual individual physiologicalphysiological physiological rolerole role and and and tissue tissue tissue localization localiza- localiza- tionoftion specificof ofspecific specific PDE PDE PDE isoforms isoforms isoforms paved paved paved the the the way way way forfor for targetingtargeting targeting more more precisely precisely precisely cyclic cyclic cyclic nucleotide nucleotide nucleotide signalingsignalingsignaling pathways pathwayspathways involved involvedinvolved in in inspecific specificspecific diseases, diseases,diseases, such suchsuch as as as dementia dementiadementia [46]. [ 46[46].]. Several SeveralSeveral synthetic syntheticsynthetic PDE9PDE9PDE9 inhibitors inhibitorsinhibitors are areare under underunder investigation investigationinvestigation in in inthis thisthis context. context.context. Beneficial BeneficialBeneficial effects effectseffects of of ofPDE9 PDE9PDE9 inhibitor inhibitorinhibitor BAYBAYBAY 736691 736691736691 were werewere reported reportedreported in in inobject objectobject recognition recognitionrecognition and andand passive passivepassive avoidance avoidanceavoidance tests teststests in in inanimal animalanimal models,models,models, suggesting suggestingsuggesting its itsits memory memorymemory enhancing enhancingenhancing role rolerole [47]. [47[47].]. Li LiLi et et etal. al.al. studied studiedstudied the thethe effects effectseffects of of ofthis thisthis selectiveselectiveselective compound compoundcompound on onon in in invitro vitrovitro and andand in in invivo vivovivo models modelsmodels of of ofAD AD AD [48]. [ 48[48].]. In InIn this thisthis connection, connection,connection, it itmustit mustmust bebebe noted notednoted that thatthat Pfizer PfizerPfizer and andand Boehringer BoehringerBoehringer entered enteredentered Ph PhasePhasease II IItrialsII trialstrials with withwith PDE9 PDE9-selectivePDE9-selective-selective inhibitors, inhibitors,inhibitors, namednamednamed PF-04447943 PF-04447943PF-04447943 (Figure (Figure(Figure 3)3 )3)and and and BI BI BI 409306, 409306, 409306, respectively respectively respectively [49,50]. [49 [49,50].,50].
(a(a ) ) (b(b ) ) O O
HNHN N N N N N N N N N N N N O O
(c)(c)
FigureFigureFigure 3. 3.Chemical3. ChemicalChemical structure structurestructure of of ofthe thethe PDE9 PDE9PDE9 inhibitor inhibitorinhibitor PF-04447943 PF-04447943PF-04447943 (a ().(aa ).Three-dimensional). Three-dimensionalThree-dimensional structure structurestructure of of of thethethe complex complexcomplex with withwith PDE9 PDE9PDE9 (PDB (PDB(PDB ID: ID:ID: 4E90) 4E90)4E90) (b ().(bb ).Detailed). DetailedDetailed view viewview of of ofthe thethe interaction interactioninteraction pattern patternpattern of of ofPF-04447943 PF-04447943PF-04447943 withwithwith PDE9: PDE9:PDE9: zinc zinczinc is isrepresentedis representedrepresented in in inpurple purplepurple and andand magn magnesiummagnesiumesium is isreportedis reportedreported in in ingr green.een.green. Interacting InteractingInteracting residues residuesresidues havehavehave been beenbeen labeled labeledlabeled and andand highlighted highlightedhighlighted in in ingreen greengreen (c ( ).c(c ).).
MoschettiMoschettiMoschetti et et etal. al. al. reported reported reported the the the results results results of of ofsafety, safety, safety, tolerability, tolerability, andand and bioavailabilitybioavailability bioavailability studies studies studies of ofBI ofBI 409306BI 409306 409306 on on healthyon healthy healthy human human human male male male subjects subjects subjects [50 ].[50]. The[50]. The chemicalThe chemical chemical structures structures structures of this of of compoundthis this com- com- poundandpound of and BAY and of 736691ofBAY BAY 736691 are736691 reported are are reported reported in Figure in inFigure4. Figure 4. 4.
O O O O
HNHN N N N N NHNH N N F F N N N N N N N N F F F F Cl Cl O O BI 409306BI 409306 BAYBAY 736691 736691 FigureFigureFigure 4. 4.Chemical4. ChemicalChemical structures structuresstructures of of ofthe thethe PDE9 PDE9PDE9 inhibitorsinhibitors inhibitors BIBI BI409306409306 409306 andand and BAYBAY BAY 736691. 736691. 736691.
The compounds investigated to date to combat neurodegeneration showed moderate or at least partial specificity for PDE9, as these molecules are also substrates of PDE1 [44]. E2027 is another promising compound developed by Eisai: it is currently being examined
Pharmaceuticals 2021, 14, x FOR PEER REVIEW 6 of 13
The compounds investigated to date to combat neurodegeneration showed moderate Pharmaceuticals 2021, 14, 58 or at least partial specificity for PDE9, as these molecules are also substrates of PDE1 [44].6 of 13 E2027 is another promising compound developed by Eisai: it is currently being examined in clinical trials as it provides neuroprotection in dementia by increasing central cGMP levelsin clinical and improving trials as it neuronal provides plasticity neuroprotection [41,51]. Not in dementia many details by increasing have been central provided cGMP so far,levels and and even improving if some early neuronal reports plasticity in the li [terature41,51]. Not classified many detailsthis compound have been as provideda PDE1 inhibitor,so far, and it is even currently if some referred early reports to as a in“selective the literature PDE9 classifiedinhibitor” this (IC50 compound = 3.5 nM, 1000-fold as a PDE1 selectivityinhibitor, over it is currently other isoforms) referred [52,53]. to as a In “selective animal models, PDE9 inhibitor” E2027 has (IC been50 = shown 3.5 nM, to 1000-fold inhibit theselectivity degradation over of other cGMP, isoforms) increasing [52,53 its]. Inlevels animal in cerebrospinal models, E2027 fluid. has been Improved shown memory to inhibit retainmentthe degradation and amelioration of cGMP, increasing of cognitive its levels impairment in cerebrospinal were among fluid. the Improved observed memory effects [52].retainment A clinical and study amelioration is investigating of cognitive efficacy, impairment safety and were tolerability among the of observed E2027 in effects patients [52 ]. withA clinical dementia study with is investigatingLewis bodies efficacy,(ClinicalTrials.gov safety and Identifier: tolerability NCT03467152). of E2027 in patients with dementia with Lewis bodies (ClinicalTrials.gov Identifier: NCT03467152). 3.3. Studies on Natural and Nature-Inspired Compounds Targeting PDE9 3.3.Several Studies onplant Natural extracts and Nature-Inspiredwere previously Compounds described Targetingto possess PDE9 cGMP-specific PDE in- hibitorySeveral activity, plant providing extracts a were hope previously for a potential described application to possess against cGMP-specific cognitive disorders. PDE in- Temkitthawonhibitory activity, et al. providing reported a a hope list of for flavon a potentialoids endowed application with against this role cognitive [27], and disorders. other compounds,Temkitthawon such et as al. isoquiritigenin, reported a list icariin, of flavonoids anthocyanin, endowed and with sophoflavescenol, this role [27], andwere other re- portedcompounds, afterwards such [4]. as isoquiritigenin, icariin, anthocyanin, and sophoflavescenol, were reportedIn a recent afterwards study, [ 4our]. research group adopted a set of in silico tools to screen a panel of chemicallyIn a recent variegated study, our natural research compounds group adopted (flavonoids, a set of polyphenols, in silico tools organic to screen acids, a panel al- kaloids,of chemically and anthocyanins) variegated natural against compounds PDE isoforms, (flavonoids, including polyphenols, PDE9 (PDB organic ID: 3JSW) acids, [54]. alka- Asloids, a first and step, anthocyanins) compounds against lacking PDE drug-lik isoforms,e pharmacokinetic including PDE9 (PDBfeatures ID: were 3JSW) excluded [54]. As a fromfirst the step, subsequent compounds validated lacking drug-likemulti-docking pharmacokinetic screening. According features were to the excluded results from of this the study,subsequent in terms validated of calculated multi-docking binding energy screening. (ΔG), flavonoids According were to the highlighted results of this as the study, most in promisingterms of calculated class of PDE9 binding inhibitors, energy with (∆G), krau flavonoidsssianone were 2, kraussianone highlighted 3, as osajin, the most and promis- pom- iferining class showing of PDE9 ΔG inhibitors, values < with−11 kcal/mol. kraussianone More 2, kraussianonespecifically, pomiferin 3, osajin, and(ΔG pomiferin = −11.6 − − kcal/mol)showing was∆G valuespredicted < to11 be kcal/mol. selective Morefor PDE9, specifically, as weak pomiferininteractions (∆ withG = other11.6 kcal/molisoforms ) werewas computed. predicted to The be selectivepredicted for interaction PDE9, as weakpattern interactions of pomiferin with with other PDE9 isoforms is reported were com- in puted. The predicted interaction pattern of pomiferin with PDE9 is reported in Figure5. Figure 5.
(a ) (b )
O O
OH
OH O OH
Figure 5. Chemical structure of pomiferin (a). Detailed 3D view of the predicted interaction motif Figure 5. Chemical structure of pomiferin (a). Detailed 3D view of the predicted interaction motif with PDE9: zinc is with PDE9: zinc is represented in purple and magnesium is reported in green. Interacting residues represented in purple and magnesium is reported in green. Interacting residues have been labeled and highlighted in green have been labeled and highlighted in green (b). Interaction model was built based on the compu- (b). Interaction model wastational built based data on reported the computational by Ongaro et data al. [54]. reported by Ongaro et al. [54]. Notably, in the same study, a lack of selectivity was highlighted for caffeine, that Notably, in the same study, a lack of selectivity was highlighted for caffeine, that showed overall poor binding energies towards all the tested PDE isoforms (between −5.8 showed overall poor binding energies towards all the tested PDE isoforms (between −5.8 and −7.3 kcal/mol) [54]. The chemical structures of the natural compounds discussed in and −7.3 kcal/mol) [54]. The chemical structures of the natural compounds discussed in this section are reported in Figure6. this section are reported in Figure 6. On the other hand, caffeine, xanthines and xanthine-inspired compounds were widely investigated as structures for developing PDE9 inhibitors. In particular, the pyrazolopy- rimidinone scaffold appears particularly promising, and its chemical structure closely resembles that of naturally occurring xanthines [45,55]. In this connection, in their in- vestigation for PDE9 inhibitors, Shao et al. reported a compound capable of targeting a small hydrophobic pocket in the protein structure. This peculiar arrangement of the molecule, named 3r, allowed 800-fold selectivity over PDE1 and 35,000-fold selectivity over PDE4 [56]. This specific pocket is constituted by residues Leu420, Leu421, Phe425, Pharmaceuticals 2021, 14, x FOR PEER REVIEW 7 of 13
O R5 OH OH OH O O O O R4 O HO O R6 HO O OH OH O OH R3 R1 OH HO HO OH O R O O Pharmaceuticals 2021, 14, 58 2 7 of 13 OH OH Isoquiritigenin Icariin Anthocyanin Sophoflavescenol Figure 6. Chemical structures of some of the known c-GMP-specific PDE inhibitors. Ala452, and Phe441 (Figure7). Most importantly, this region represents a unique structural featureOn ofthe this other isoform hand, and, caffeine, thus, it couldxanthines be employed and xanthine-inspire for designing compoundsd compounds selectively were widelytargeting investigated PDE9. Huang as structures et al. further for developing investigated PDE9 this inhibitors. aspect andIn particular, found that the this pyra- site, zolopyrimidinonenamed M-pocket, canscaffold be selectively appears targetparticularly by a chiral promising, compound, and namedits chemical (S)-C33 structure (Figure7). Pharmaceuticals 2021, 14, x FOR PEER REVIEW 7 of 13 closelyThis molecule resembles showed that of improvednaturally occurring potency and xanthines isoform [45,55]. selectivity In this when connection, compared in their to its investigationenantiomer (R)-C33,for PDE9 supporting inhibitors, theShao experimental et al. reported hypothesis a compound of the capable authors of [ 45targeting]. a small hydrophobic pocket in the protein structure. This peculiar arrangement of the mol-
ecule, named 3r, allowed 800-foldO selectivity overR5 PDE1 and 35,000-fold selectivity over OH OH OH O PDE4 [56]. ThisO specificO pocketO is constituted by residues Leu420, Leu421, Phe425, Ala452, R4 O HO O and Phe441 (Figure 7). Most importantly, this regionR6 represents a unique structural fea- HO O ture of this isoformOH and,OH Othus, itOH couldR3 be employedR1 for designing compoundsOH selectively HO HO OH O targeting PDE9. Huang et al. further investigatedR2 this aspectO Oand found that this site, OH named M-pocket, can be selectivelyOH target by a chiral compound, named (S)-C33 Isoquiritigenin(Figure 7). This moleculeIcariin showed improvedAnthocyanin potency and isoformSophoflavescenol selectivity when com- Figure Figure6. Chemical 6.paredChemical structures to its structures enantiomer of some of of some the(R)-C33, known of the supporting knownc-GMP-specific c-GMP-specific the PDE experimental inhibitors. PDE inhibitors. hypothesis of the authors [45]. On the other hand, caffeine, xanthines and xanthine-inspired compounds were (awidely ) investigated as structures for developing(b ) PDE9 inhibitors. In particular, the pyra- zolopyrimidinone scaffold appears particularly promising, and its chemical structure closely resembles that of naturally occurring xanthines [45,55]. In this connection, in their O investigation for PDE9 inhibitors, Shao et al. reported a compound capable of targeting a small hydrophobic HNpocket in the protein structure. This peculiar arrangement of the mol- N ecule, named 3r, allowed 800-fold selectivity over PDE1 and 35,000-fold selectivity over * N N N PDE4 [56]. This specificH pocket is constituted by residues Leu420, Leu421, Phe425, Ala452, and Phe441Cl (Figure 7). Most importantly, this region represents a unique structural fea- ture of this isoform and, thus, it could be employed for designing compounds selectively targeting PDE9. Huang et al. further investigated this aspect and found that this site, named M-pocket, can be selectively target by a chiral compound, named (S)-C33 (Figure 7). This molecule showed improved potency and isoform selectivity when com- Figure 7. Chemical structureFigure of (S)-C33/(R)-C33 7. Chemical structure (a). Detailed of (S)-C33/(R)-C33 view of the (a interaction). Detailed patternview of ofthe (S)-C33 interaction with pattern PDE9: theof (S)- pared to its enantiomerC33 with PDE9: (R)-C33, the residues supporting forming th thee experimental specific hydrophobic hypothesis M-pocket of the have authors been labeled and residues forming the specific hydrophobic M-pocket have been labeled and highlighted in green (PDB ID: 4Y8C) (b). [45]. highlighted in green (PDB ID: 4Y8C) (b). As stated above, most of the currently studied nature-inspired PDE9 inhibitors are based (a ) on theAs stated pyrazolopyrimidinone above,(b ) most of the scaffold, currently and st thisudied also nature-inspired holds true for syntheticPDE9 inhibitors compounds are basedundergoing on the clinicalpyrazolopyrimidinone investigation [44 scaffold,]. Moreover, and startingthis also fromholds this true structure, for synthetic Zhang com- et al. poundsprepared undergoing a set of hybrid clinical compounds investigation generated [44]. Moreover, from the combination starting from of thethis pyrazolopy-structure, O Zhangrimidinone et al. prepared scaffold with a set natural, of hybrid antioxidant compounds moieties generated (Figure from8 ). the The combination authors studied of the the HN pyrazolopyrimidinone scaffold with natural, antioxidant moieties (Figure 8). The authors potentialN activity of such molecules against AD in vitro. These “multi-functional” compounds, studied the potential activity of such molecules against AD in vitro. These “multi-func- * N N combiningN PDE inhibitory activity and antioxidant properties, were designed with the aid of H tional”computational compounds, tools, combining such as molecular PDE inhibitory docking activity and molecular and antioxidant dynamics properties, simulations. were Most Cl designedof the synthesized with the aid derivatives of computational showed IC tools,50 values such Pharmaceuticals 2021, 14, x FOR PEER REVIEW 8 of 13 showed a good selectivity profile over PDE1 (between 25- and 40-fold) and PDE8 (be- Pharmaceuticals 2021, 14, x FOR PEER REVIEW 8 of 13 tween 250- and 400-fold). The authors investigated the antioxidant activity of the deriva- tives in the oxygen radical absorbance capacity assay (ORAC-FL), again highlighting the performance of the compounds bearing the cinnamic group as substituent in the scaffold. Eventually,showed a goodthe lack selectivity of toxicity profile on hu overman PDE1 neuroblastoma (between cells25- and was 40-fold) assayed and[57]. PDE8 (be- tween 250- and 400-fold). The authors investigated the antioxidant activity of the deriva- O Pharmaceuticals 2021, 14, 58 tives in the oxygen radical absorbance capacity assay (ORAC-FL), again highlighting8 of 13the HN performance of the compoundsH N bearing the cinnamic group as substituent in the scaffold. N N Eventually, the lackR1 of toxicityN on human neuroblastoma cells was assayed [57]. O Pyrazolopyrimidone scaffold HN H N N N R1 N R1 = O Q O HO Pyrazolopyrimidone scaffold O S S O Q = OH and/or OMe R1 = abO c Q O HO Figure 8. Chemical structures of the modified pyrazoloO pyrimidinone scaffold and of the moieties S S O studiedQ = OH by and/or Zhang OMe et al., which are functional groups inspired by the structures of cinnamic acid (a), lipoic abacid (b), and trolox (c) [57]. c FigureFigureSingh 8. 8.Chemical Chemical et al. generated structures structures ofa of library thethe modifiedmodified of 200 pyrazolopyrimidinonepyrazolo xanthine-basedpyrimidinone small scaffold molecules and of that the moietieswere in- studied by Zhang et al., which are functional groups inspired by the structures of cinnamic acid studiedvestigated by Zhang for their et al., selectivity which are as functional PDE9 ligands, groups inspiredwith respect by the to structures other PDE of cinnamic isoforms. acid More (a), (a), lipoic acid (b), and trolox (c) [57]. lipoicspecifically, acid (b), the and authors trolox (c )[designed57]. several groups of molecules that were modified at the N1, N3, N9, and C8 positions (Figure 9) [44]. Singh et al. generated a library of 200 xanthine-based small molecules that were in- vestigatedO for their selectivity as PDE9 ligands, with respect to other PDE isoforms. More 1 H 7 specifically,5 the authors designed several groups of molecules that were modified at the 6 N 1HN3 9 8 N , N , N , and C8 positions (Figure 9) [44]. O 2 N 4 N HO 9 1 3 H 7 5 HN 6 N 8 O 2 N 4 N FigureFigure 9.9. GeneralGeneralH structurestructure9 of of the the xanthine xanthine scaffold. scaffold. The The positions positions that that were were modified modified in the in abovemen-the tionedabovementioned study3 have study been marked have been with marked an arrow. with an arrow. PositionsPositions NN11 andand NN33 were foundfound toto bebe notnot suitablesuitable forformodifications: modifications: derivatizationderivatization withwithFigure aliphaticaliphatic 9. General fragmentsfragments structure induces inducesof the xanthine lossloss ofof scaffold. interactioninteraction The positions withwith thethe that PDE9PDE9 were pocket.pocket. modified OnOn in thethe the other other hand,hand,abovementioned modificationmodification study withwith have aromaticaromatic been marked fragmentsfragments with an onon arrow. thethe CC8 8 position,position, alongalong withwith derivatizationderivatization onon NN11 andand NN33,, allowedallowed thethe generationgeneration ofof anan extendedextended scaffoldscaffold capablecapable ofof occupyingoccupying thethe wholewholePositions bindingbinding sitesiteN1 andmoremore N efficiently.efficiently.3 were found WithWith to thebethe not aimaim suitable ofof investigatinginvestigating for modifications: isoformisoform selectivity,selectivity, derivatization thethe authorsauthorswith aliphatic performedperformed fragments anan inin silicosilico induces comparative loss of interaction interactioninteraction with analysisanalysis the withPDE9 cAMP-specificcAMP-specific pocket. On the PDE4,PDE4, other 8 PDE7PDE7hand, and modification PDE8. Interestingly, Interestingly, with aromatic bulky bulky fragments modifications modifications on the onC on position,N N1 and1 and C 8along Cinduced8 induced with a derivatizationdifferent a different ori- orientationentationon N1 and of theofN3 the ,ligand allowed ligand when the when interactinggeneration interacting withof withan the extend thecatalytic catalyticed scaffold subunit subunit capable of other of other of PDEs, occupying PDEs, thus thus sug- the suggestinggestingwhole bindinga selective a selective site recognition more recognition efficiently. of of PDE9. PDE9. With Additi the Additionally, aimonally, of investigating the the side-activity side-activity isoform on selectivity, dual specificspecific the PDE1,PDE1,authors PDE2,PDE2, performed PDE3,PDE3, an andand in PDE10silicoPDE10 comparative waswas alsoalso investigatedinvest interactionigated analysis inin thethe samesame with study. cAMP-specificstudy. AsAs discusseddiscussed PDE4, above,above,PDE7 cross-reactivityandcross-reactivity PDE8. Interestingly, with with PDE1 PDE1 bulky is is detrimental detrimenta modifications forl for PDE9 PDE9 on inhibitors,N 1inhibitors, and C8 especiallyinduced especially a indifferent lightin light of itsori- of highitsentation high expression expression of the inligand the in brain thewhen brain [50 interacting]. [50]. The modifiedThe with modified the xanthine-based catalytic xanthine-based subunit compounds of compounds other werePDEs, predictedwere thus pre- sug- todictedgesting be selective to a be selective selective for PDE9, recognition for as PDE9, less specific ofas PDE9.less hydrophobicspecific Additi hydrophobiconally, and the van side-activity derand Waals van der interactions on Waals dual interac- specific were detectedtionsPDE1, were PDE2, in thedetected casePDE3, of in and the the residues PDE10case of wasthe of PDE1. residuesalso invest Again, of igatedPDE1. Tyr424 inAgain, and the Gln453same Tyr424 study. were and confirmedGln453As discussed were to beconfirmedabove, crucial cross-reactivity for to selectivebe crucial binding forwith selective PDE1 [44]. is binding detrimenta [44].l for PDE9 inhibitors, especially in light of its highOn the expression basis of thein the considerations brain [50]. The about modified selective xanthine-based recognition of compounds PDE9 by chiral were com- pre- poundsdicted reportedto be selective above, for Cheng PDE9, et al. as investigated less specific the hydrophobic activity of a classand van of (± der)-torreyunlignans Waals interac- fromtionsTorreya were detected yunnanensis in .the The case authors of the reported residues the of inhibitory PDE1. Again, activity Tyr424 of the and ethanolic Gln453 plant were extractconfirmed against to be PDE9, crucial and for the selective same effectbinding was [44]. not observed on PDE4 and PDE5. (±)- Torreyunlignans represent a peculiar class of 8-90-linked neolignans that were detected in these ethanolic extracts. The isolated compounds, in the form of single enantiomers, were tested against PDE9 and inhibited the enzyme in the µM range. The most promising compound, named 3a in the paper, showed an IC50 value of 5.6 µM. Interestingly, the µ corresponding enantiomer 3b showed similar activity (7.8 M), suggesting that the chiral 1,3-dioxane moiety may have similar orientation within the PDE9 active site [58]. The chemical structure of the two compounds are reported in Figure 10. Pharmaceuticals 2021, 14, x FOR PEER REVIEW 9 of 13 Pharmaceuticals 2021, 14, x FOR PEER REVIEW 9 of 13 On the basis of the considerations about selective recognition of PDE9 by chiral com- poundsOn reported the basis above, of the considerationsCheng et al. investig about atedselective the activityrecognition of a ofclass PDE9 of (±)-torreyun-by chiral com- lignanspounds from reported Torreya above, yunnanensis Cheng. etThe al. authors investig reportedated the the activity inhibitory of a class activity of (±)-torreyun-of the etha- noliclignans plant from extract Torreya against yunnanensis PDE9, and. The the authors same effect reported was not the observed inhibitory on activity PDE4 andof the PDE5. etha- (±)-Torreyunlignansnolic plant extract against represent PDE9, a peculiar and the class same of effect 8-9′-linked was not neolignans observed thaton PDE4 were and detected PDE5. in(±)-Torreyunlignans these ethanolic extracts. represent The aisolated peculiar comp classounds, of 8-9′-linked in the neolignansform of single that enantiomers,were detected μ werein these tested ethanolic against PDE9extracts. and The inhibited isolated the comp enzymeounds, in the in theM range.form of The single most enantiomers, promising μ compound,were tested named against 3a PDE9 in the and paper, inhibited showed the an enzyme IC50 value in the of μ5.6M range.M. Interestingly, The most promising the cor- μ respondingcompound, enantiomer named 3a in3b the showed paper, similar showed activity an IC 50(7.8 value M), of suggesting 5.6 μM. Interestingly, that the chiral the 1,3- cor- dioxane moiety may have similar orientation within the μPDE9 active site [58]. The chemi- Pharmaceuticals 2021, 14, 58 responding enantiomer 3b showed similar activity (7.8 M), suggesting that the chiral9 of 1,3- 13 caldioxane structure moiety of the may two have compounds similar orientation are reported within in Figure the PDE9 10. active site [58]. The chemi- cal structure of the two compounds are reported in Figure 10. O O O O O OO O O OO O O O OO O O O OO O O O O O O O 3a 3b Figure 10. Chemical3a structures of the chiral3b (±)-torreyunlignans [58]. FigureFigure 10.10. Chemical structures ofof thethe chiralchiral ((±)-torreyunlignans±)-torreyunlignans [58]. [58]. To better investigate the findings of the authors on these natural compounds inter- actingToTo with betterbetter PDE9 investigateinvestigate [58], we the theundertook findings findings ofa of thecomputational the authors authors on on thesestudy these natural to natural rationalize compounds compounds the experi- interact- inter- mentalingacting with resultswith PDE9 PDE9 and [58 to], [58], weevaluate undertookwe undertook the interaction a computational a computational pattern studyof the study tosingle rationalize to enantiomers, rationalize the experimental accordingthe experi- toresultsmental the procedure andresults to evaluateand briefly to evaluate thereported interaction the here. interaction patternThe protein pattern of the model single of the was enantiomers,single retrieved enantiomers, accordingfrom theaccording PDB. to the Targetprocedureto the and procedure ligands briefly briefly reportedwere prepared reported here. Thefor here. the protein Theblind protein modeldocking wasmodel experiment retrieved was retrieved performed from the from PDB. using the Target Au-PDB. todockandTarget ligands Vinaand ligands were[59]. preparedReceptor were prepared forgrid the generation for blind the docking blind was docking experimentautomatically experiment performed set, performed encompassing using Autodockusing the Au- wholeVinatodock [protein59 ].Vina Receptor (PDB[59]. ID: gridReceptor 2HD1). generation grid The generationnumber was automatically of dockingwas automatically set, poses encompassing was set set, to ten,encompassing the and whole the protein other the Autodock(PDBwhole ID: protein 2HD1).Vina (PDBparameters The ID: number 2HD1). to default. ofThe docking number Output poses of dockingdata was (energies set poses to ten, was and and set interaction theto ten, other and Autodockpatterns) the other wereVinaAutodock analyzed parameters Vina and toparameters default.scored using Output to default. UCSF data Chimer (energiesOutputa data[24]. and (energies interactionThe values and patterns) reported interaction were here analyzed patterns)are ex- pressedandwere scored analyzed in kcal/mol using and UCSF and scored refer Chimera tousing the [ 24mostUCSF]. The favored Chimer values pose.a reported [24]. The The computed here values are expressed reportedmodels are inhere reported kcal/mol are ex- inandpressed Figure refer in11. to kcal/mol the most and favored refer to pose. the most The computed favored pose. models The arecomputed reported models in Figure are reported11. in Figure 11. (a ) (b ) (a ) (b ) Figure 11. Results of the dockingFigure study11. Results based of on the the docking chemical study structures based of on the the natural chemical PDE9 structures inhibitors of( ±the)-torreyunlignans natural PDE9 in- hibitors (±)-torreyunlignans reported by Cheng et al. [58]. View of the full protein (a) and detailed reported by Cheng et al. [58].Figure View 11. of theResults full proteinof the docking (a) and detailedstudy based view on of the the ch interactionemical structures pattern of the3a (green) natural and PDE93b in- view of the interaction pattern of 3a (green) and 3b (blue): the residues involved in the binding (blue): the residues involvedhibitors in the binding (±)-torreyunlignans have been labeled reported and by highlighted Cheng et al. in [58]. green View (b). of the full protein (a) and detailed haveview been of the labeled interaction and highlighted pattern of 3ain green(green) (b and). 3b (blue): the residues involved in the binding have Thebeen docking labeled and study highlighted demonstrated, in green as (b expected,). that both torreyunlignans interact with The docking study demonstrated, as expected, that both torreyunlignans interact the catalytic subunit of PDE9. Calculated binding energy was found to be −9.0 kcal/mol with the catalytic subunit of PDE9. Calculated binding energy was found to be −9.0 for 3aTheand docking−8.7 kcal/mol study demonstrated, for 3b, confirming as expected, the similar that predicted both torreyunlignans activity observed interact by kcal/mol for 3a and −8.7 kcal/mol for 3b, confirming the similar predicted activity ob- thewith authors the catalyticin vitro ,subunit with 3a ofbeing PDE9. slightly Calculated more efficient binding [58 energy]. As hypothesized was found to by be Cheng −9.0 servedetkcal/mol al. [by58], thefor binding authors3a and motif in−8.7 vitro, iskcal/mol similar with 3a forfor being 1,3-dioxane3b, confirmingslightlybut more ourthe efficient studysimilar highlighted[58]. predicted As hypothesized activity a different ob- byorientationserved Cheng by et the ofal. methoxyphenylauthors[58], binding in vitro, motif groupswith is 3asimilar between being for slightly the 1,3-dioxane two more compounds, efficient but our [58].andstudy 3aAs highlightedwas hypothesized predicted a differenttoby bind Cheng keyorientation et residues al. [58], of Gln453binding methoxyphenyl and motif Phe456 is similar groups in closer for between 1,3-dioxane proximity the two (Figure but compounds, our 11 study). highlighted and 3a was a predicteddifferent toorientation bind key residuesof methoxyphenyl Gln453 and groups Phe456 between in closer the pr twooximity compounds, (Figure 11). and 3a was 4.predicted Conclusions: to bind The key Perspective residues Gln453 of the and Medicinal Phe456 Chemistin closer proximity (Figure 11). Natural compounds and plant extracts endowed with PDE inhibitory potential often also act through a combination of other biochemical mechanisms. In particular, plant- derived molecules showing PDE inhibitory activity that are promising candidates against neurodegeneration also have anti-amyloid and anti-tau effects, or, in other cases, are known to inhibit AChE [25]. This multi-target behavior has been reported for flavonoids [60] and for the components of Ginkgo biloba, Poly tenuifolia, and Acorus calamus extracts [61]. The presence of several mechanisms is perfectly in harmony with the growing interest to- wards the development of multi-target-directed ligands (MTDL) that take advantage of synergistic pharmacological effects [62]. Moreover, depending on their chemical struc- tures, the neuroprotective effect of several natural and nature-inspired compounds, as outlined above, is also connected with their activity against reactive oxygen species (ROS) and inflammation [63–66]. In this connection, it was also reported that natural PDE in- Pharmaceuticals 2021, 14, 58 10 of 13 hibitors, by increasing cGMP levels, reduce the production of IL-6 and TNF-α [23]. On the other hand, it must also be considered that compounds showing promising performances in vitro and in preclinical models sometimes fail in clinical studies. In this connection, combined therapy could represent a solution: PDE9 inhibitors could enhance and support the effects of traditional therapy and pave the way for the development of more efficient therapeutic strategies. It must be also pointed out that some of the synthetic compounds targeting PDE9 studied so far demonstrated detrimental off-target inhibitory activity on other isoform, resulting in undesired side effects [67]. Developing selective inhibitors to be applied in con- trasting neurodegeneration requires considering the structural diversification highlighted in PDE9 [44], and this is made possible thanks to modern tools supporting drug discovery, such as molecular modeling. While PDEs share more than 75% identity when the catalytic domain is considered, PDE9 shows unique structural features. As demonstrated in this review, drug design can take advantage of such uniqueness to identify or build selective molecules on the basis of natural scaffolds. The presence of the peculiar hydrophobic M-pocket described above and of the asymmetry between the two subunits composing the dimer make PDE9 a suitable target to search for inhibitors with good selectivity. To date, xanthines, flavonoids, neolignans, and their derivatives have been studied as natural and nature-inspired compounds targeting PDE9. As chirality seems to play a crucial role in accessing the peculiar pocket identified in this isoform, and based on the structural asymmetry of PDE9, the world of natural compounds appears as an ideal source for identifying novel asymmetric scaffolds. Author Contributions: Conceptualization, G.R. and A.G.; software, G.R.; investigation, G.R. and A.G.; writing—original draft preparation, G.R. and A.G.; writing—review and editing, A.G. and M.M.; funding acquisition, G.R. and A.G. All authors have read and agreed to the published version of the manuscript. Funding: This research was granted by University of Brescia. 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