Natural Occurring Polyphenols as Template for Drug Design. Focus on Serine Proteases

Natural Occurring Polyphenols as Template forDrug Design. Focus on Serine Proteases

Massimiliano Cuccioloni1*, MatteoMozzicafreddo1, Laura Bonfili1, ValentinaCecarini1, Anna Maria Eleuteri1 and MauroAngeletti1

1Department of Molecular, Cellular and Animal Biology, Universityof Camerino, Via Gentile III da Varano, Camerino (MC), Italy*Corresponding author: Massimiliano Cuccioloni,[email protected]

Several major physio-pathological processes,including cancer, inflammatory states and throm-bosis, are all strongly dependent upon the fineregulation of proteolytic enzyme activities, anddramatic are the consequences of unbalancedequilibria between enzymes and their cognateinhibitors. In this perspective, the discovery ofsmall-molecule ligands able to modulate catalyticactivities has a massive therapeutic potential and isa stimulating goal. Numerous recent experimentalevidences revealed that proteolytic enzymes can beopportunely targeted, reporting on small ligandscapable of binding to these biological macromole-cules with drug-like potencies, and primarily withcomparable (or even higher) efficiency with respectto their endogenous binding partner. In particular,natural occurring polyphenols and their derivativesrecently disclosed these intriguing abilities,making them promising templates for drug designand development. In this review, we compared theinhibitory capacities of a set of monomeric po-lyphenols toward serine proteases activity, andfinally summarized the data with an emphasis onthe derivation of a pharmacophore model.

Key words: drug template, enzyme inhibition, natural polyphenols,pharmacophore, serine protease

Received 29 December 2008, revised 30 April 2009 and accepted forpublication 16 May 2009

Strong is the interest in specifically controlling enzymes for thera-peutic purposes, possibly with site-directed small molecules, whichgenerally are 'easier to handle' being orally administrable, but thestructure and the specificity of their catalytic regions make this taska huge challenge. Several studies provide evidences for small mole-cules targeting the catalytic site of enzymes, thus modulating theiractivities. Herein, we will set our focus on a particular class ofphysiologically pivotal enzymes, namely the serine proteases.

Serine proteases, proteolytic enzymes widely present in tissues andbiological fluids (1,2), are established regulators of many normalcellular functions, either as non-specific catalysts of protein degra-dation or highly selective agents controlling physiological events.Their roles in protein digestion, limited proteolysis, mammalianreproduction, the cascade mechanisms of blood coagulation, com-plement activation, kallikrein-kinin system, and fibrinolysis are allwell-documented (3–5), being additionally related to various phasesof malignant transformation and cellular activation (6). They share acommon architecture consisting of two 6-stranded b-barrels (7),containing the catalytic site cleft. Essentially, these structures con-sist of a zymogen activation domain, a substrate recognition regionand a catalytic core (8), this latter principally presenting a serine, ahistidine and an aspartic acid residue (9), namely the catalytic triad,that covers the active site cleft, as a part of an extensive hydrogenbonding network. Structurally, the high degree of conservation ofcatalytic sites of serine proteases uncovered a common binding pat-tern with ligands with a similar architecture.

Crucial equilibria between serine proteases and their specific inhibi-tors normally exist in tissues where these agents are involved,since, once activated, they can pose a major threat for the organ-ism (10–19) if they are not correctly regulated by their cognate ser-ine protease inhibitors, namely the serpins (20–23). Therefore, theidentification of new, possibly natural, serine protease inhibitors tobe used in the treatment of diseases involving serine proteasesover-function is an attracting topic in current medicinal chemistry.

Here, we summarized the available data on the inhibitory potenciesof a number of natural polyphenols toward some major serine pro-teases (Figure 1), by means of binding equilibrium dissociation con-stants (Kd) or, in the absence of direct binding data, half-maximalinhibitory concentrations (IC50) from functional assays. Finally, a sub-set of compounds constituted by the most effective polyphenolinhibitors was analysed in order to rationalize the available struc-tural and functional data in the formulation of a polyphenol-basedmodel that shares all the favourable features contributing toenhance the specificity of the interaction.

Inhibition of Serine Proteases Activityby Polyphenols

Thrombin-like proteasesThrombin, plasmin, urokinase-like plasminogen activator (uPA) andtissue-like plasminogen activator (tPA) are collectively referred toas thrombin-like proteases. These proteases are traditionally

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Chem Biol Drug Des 2009; 74: 1–15

Review Article

ª 2009 John Wiley & Sons A/S

doi: 10.1111/j.1747-0285.2009.00836.x

studied in regard to intravascular fibrinolysis and biological pro-cesses involving cell movement along the extracellular matrix.These tissue-remodelling processes are involved in nervous systemdevelopment, response to injury and degenerative diseases(22,24). Besides their central role in thrombogenesis and fibrinoly-sis, thrombin-like proteases are also involved in tumour cell inva-sion, inflammation, ovocyte maturation and cell migration (25).In an attempt to identify natural occurring antithrombotic agents,a large number of evidences report that a constant intake ofmoderate amounts of polyphenols can exert beneficial vasculareffects, reducing the risk for cardiovascular morbidity and mortal-ity (26).

ThrombinThrombin is involved in blood coagulation at different levels, byconverting soluble fibrinogen to fibrin, activating different factors,which generates more thrombin, stimulates platelets and favoursthe stabilization of the clot (27). Normally, the coagulationcascade is regulated by physiological anticoagulants, such as tis-sue factor pathway inhibitor, the protein C and protein S system,and anti-thrombin (28–31). The lack and ⁄ or the misfunction ofthrombin endogenous inhibitors are causes for hyper-coagulativedisorders.

Thus, several site-directed synthetic thrombin inhibitors (bivalirudin,argatroban and ximelagatran) have been developed, and their rolein the treatment of coronary syndromes (32–34), stroke (35–47), sys-temic embolism (48,49) and the prevention of post-surgery venousthromboembolism (50–55) is widely documented. Nevertheless,since hepato-toxicity is observed in prolonged ⁄ acute administrationof these synthetic compounds (56–58), the identification of non-toxic natural occurring molecules (or derivatives thereof) as co-drugsin the treatment of thrombin-related pathologies is a stimulatingtest for current medicinal chemistry (59–70).

We recently reported that thrombin was competitively inhibited bynatural occurring monomeric polyphenols (Figure 2C) (71,72), showingevidences consistent with the presence of a specific binding site forthis class of molecules on the enzyme. In particular, quercetin and itsglycosylated derivatives, namely rutin and hyperoside, showed affini-ties ranging from sub-micromolar (quercetin, Kd = 0.35 € 0.04 lM) tomillimolar for human thrombin, with these differences at least par-tially depending upon the hindering glycosyl groups on the aglycon.As a comparison with a synthetic site-directed thrombin inhibitor, thedissociation equilibrium constant of the quercetin–thrombin complexwas only 10-fold higher than the value reported for argatroban (73).Ellagic acid also exerted a fairly high inhibition (Kd = 7.6 € 0.5 lM),whereas epicatechin (Kd = 369 € 128 lM) and anthocyanidins, both

Figure 1: Selection of poly-phenols that inhibit serineproteases. Six of the most effica-cious site-directed polyphenolicinhibitors are here visualized

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lacking the carbonyl group in position 4, formed the least stable com-plexes with human thrombin.

Salicin and silybin (this latter having a different chemical structure)reported in vitro inhibition activities in micromolar range

(IC50 = 11.4 and 20.9 lM, respectively) (74) making them, togetherwith quercetin and ellagic acid, promising candidates for syntheticmodification as antithrombotic agents. Conversely, luteolin reporteda lower affinity for thrombin with respect to quercetin, this resultdisclosing the importance of H-bonds formation with polar residues

A

B

C

D

E

Figure 2: Visualization of bind-ing modes of the most effectivepolyphenol to serine protease:hyperoside-elastase (A, 1QNJ.pdb),quercetin-plasmin (B, 1DDJ.pdb),quercetin-thrombin (C, 1EB1.pdb),quercetin-uPA (D, 2VIO.pdb) andsylibin-trypsin (E, 1TRN.pdb). Activesite views of dockings are shownin the left insets (catalytic triadsare highlighted in lighter tone).Right insets thoroughly illustratethe enzyme-ligand interactions (redvector: H-bond acceptor group;green vector: H-bond donor group;hydrophobic repulsion: yellow high-light; hydrophobic interaction: blue-ringed vector). The docking analysiswas performed using a two-stagedocking procedure applying theprogram AutoDock 4.0, whereaslowest-energy complexes werere-analysed according to a moredetailed energetic model. Thecharacterization of ligand bindinginteractions was performed withLigandScout 2.02 (Inte:LigandGmbH).

Dietary Polyphenols Inhibit Serine Proteases

Chem Biol Drug Des 2009; 74: 1–15 3

within the catalytic site of thrombin (71). Additionally, (-)-epigallocate-chin-3-gallate (EGCG), hypericin, rhein, chrysophanol, emodin, phys-cion, aloin, sennoside-A and sennoside-B, b-aescin and colchicine, allbeing relatively hindering with respect to the catalytic site geometryof thrombin, affected its activity only at (low) millimolar range ofconcentration, thus showing no significant effect (74). Moreover, thepreincubation of platelets with resveratrol at physiological plasmaconcentrations prevented their adhesion to fibrinogen after the stimu-lation of these cells with thrombin, thus modulating the activity ofblood platelets in inflammatory process (75). Also quercetin showedan anticlotting effect, but only at higher concentrations, possibly dueto the propensity of binding with other plasma proteins (71).

From a structural viewpoint, all currently available findings agreewith the importance of the carbonyl group in position 4 (accessiblefor forming electrostatic interactions with His57, and additionallypreserving the planarity of the molecule) resulting critical for themagnification of the inhibitory efficacy of the reviewed polyphenolstoward thrombin. Besides, the hydroxylation in position 3 and afavourable accommodation of hydrophobic groups within the cata-lytic pocket additionally enhanced this ability (Figure 2C). Conversely,larger hindering replacing groups on B ring or in position 3, reducedthe ligand accessibility to the narrow catalytic pocket of thrombin,with a global restraining effect on the affinity of the complex.

Plasminogen activatorsThe primary role of uPA and tPA is the conversion of plasminogen toplasmin (76). uPA is also involved in tissue-related proteolysis, and itis critical in the degradation of extracellular matrix and transversionof basement membranes (77), whereas tPA is mainly involved infibrinolysis and thrombolysis (78). uPA activity was reported to bediminished by quercetin (IC50 = 7 lM), EGCG (IC50 = 15 lM) (79),genistein and apigenin (80–84), with all these activities mainly rely-ing upon electrostatic interactions involving the hydroxyl group inposition 7, and the hydrophobic interactions with the A ring uponthe insertion within the S1 site (Figure 2D). The inhibitory effect wasconsidered quite beneficial, because uPA is an excellent pro-factorof malignancy and axillary metastasis (85). In this perspective, Lamyet al. reported a concentration-dependent inhibition of uPA by antho-cyanidins in the low micromolar range, with a consequent involve-ment in the regulation of the motility of glioblastoma cells (86). AlsotPA activity was inhibited by quercetin in kidney and tumour tissue.Therefore, since tPA and uPA activities were reported to beincreased in later phases of tumour progression and in early phasesof tumour invasion, respectively (87,88), quercetin can be regardedas an inhibitor of metastasis at both stages. Additionally, quercetinmay exert a two-level impairment of PA relying upon the inhibitionof protein kinase C (89), whose involvement in cell motility in breastcancer was reported (85,90–92). Less significantly in the perspectiveof a possible drug development, Taitzoglou et al. (93) reported adose-dependent decrease in both human and ovine PA activityinduced by tannic acid only at submolar range.

PlasminPlasmin is a two-chain macromolecule joined by two disulphidebridges (94,95) with a broad specificity protease (96–99), that

cooperates with fibrin to form a clot. The role of plasmin in tumourcell invasion and migration is well documented (100–102), with evi-dences for both direct (by degrading proteins of the extracellularmatrix and activating plasminogen activators) and indirect action (byactivating of matrix metalloproteases). Moreover, plasmin plays animportant function in angiogenesis (103–106), and regulates theproteolytic processing of cell surface molecules such as cadherins,the major cell–cell adhesion proteins (107). We recently discrimi-nated the effect of glycosylation on the antiplasmin activity ofpolyphenols (108). In particular, quercetin showed a 20-fold higheraffinity to plasmin with respect to glycosylated counterpart (rutin)in terms of equilibrium dissociation constant (0.71 and 13 lM,respectively). Furthermore, quercetin (but not its glycosylatedcounterpart) prevented plasmin-incubated BB1 cells from releasingE-cadherin fragment, thus representing a limiting factor for thedecrease in intercellular adhesion and consequent cell motility.

Also Osaky et al. (109) tested some flavonoids purified from B. bals-amifera on plasmin activity. Interestingly, the authors demonstratedthat flavonoids with hydroxyl groups on 3¢ and 4¢ positions wereactive in a plasmin-inhibitory test (IC50 = 1.5 and 2.3 lM), with anefficacy comparable to leupeptin (IC50 = 1.2 lM). Additionally, tannicacid showed a 92% reduction of plasmin activity, but only at 200 lM.

Structurally, also these results emphasized the discriminating roleof the carbonyl in position 4 in the inhibition of plasmin, whereasthe presence of hydroxyl or methoxyl groups in position 3 and 7were responsible for the reported differences in terms of inhibitionconstants (Figure 2B).

Trypsin-like proteasesTrypsin-like proteases are implicated in digestive process andimmune response: primarily, they include trypsin, chymotrypsin, neu-trophil (NE) and pancreatic elastases (PE) and cathepsin G (110).Again, several polyphenols were assessed as potent compounds inthe treatment of digestive diseases and disorders (111).

TrypsinTrypsin is secreted by the pancreas as trypsinogen, eventually acti-vated either by enterokinase, or autocatalytically (112). It is one ofthe most extensively characterized serine proteases, and plays anessential role in both normal physiological processes (food diges-tion, blood coagulation, fibrinolysis, control of blood pressure) (113)and in some important pathological processes (atherosclerosis,inflammation, cancer); an imbalance in the endogenous 'protease–inhibitor system' is pivotal in the progression of physio-pathologicalstates, such as pancreatitis (114,115), and is critical in the develop-ment of pancreatic carcinoma (116). Maliar et al. reported a majorinhibitory effect exerted by multiple hydroxyl-substituted flavonolsand flavones, increasing with the number of hydroxyl-substitution.In particular, the authors highlighted the role of the hydroxyl groupin position 3 in the inhibition process, as confirmed by quercetin,myricetin and acacetin, that were shown to be the most effectivewith IC50 = 10, 15 and 28 lM, respectively (74,117). Additionally,flavones with the 2-3 double bond (apigenin) were more effectivethan flavanones, whereas isoflavones (biochanin A) were less

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effective than flavonoids (acacetin). The data on quercetin and my-ricetin inhibition were in good agreement with previous evidencesby Parellada et al. (118) (quercetin, IC50 = 7.1 € 2; myricetin,IC50 = 10.2 € 2), additionally assessing the inhibitory effect of lute-olin (IC50 = 35.3 € 6). Antitryptic activities of phenolic compoundswere also reported by Jedin�k et al. (74). The authors showed thatsilybin and hypericin exerted the highest inhibition activities(IC50 = 3.7 and 4.5 lM, respectively) on trypsin. Interestingly, com-parable results were observed for both quercetin (IC50 = 15.4 lM)and its glycosylated derivative hyperoside (IC50 = 14.5 lM), withthe galactoside group exerting no apparent hindering effect on thebinding, by virtue of a favourable insertion within the catalyticpocket. In addition, sennosides showed remarkable IC50 values ontrypsin (6.1 lM for sennoside A and 10.6 lM for sennoside B).Significant activities were also observed for some derivatives ofanthraquinones, the highest inhibition being reported by rhein, withan IC50 = 25.2 lM. A moderate inhibition was observed for chry-sophanol IC50 = 78.9 lM and for emodin IC50 = 50.2 lM. In thiscase, the glycosylation of the aglycon skeleton seems to destroyinhibitory activity, since aloin did not show any activity on trypsin.Available data are collectively in agreement with a model whereelectrostatic interactions between polyphenols and trypsin (Fig-ure 2D) strongly contributed to the binding process, as suggestedby the relatively high polarity of trypsin catalytic pocket, and inpart confirmed by the computational method proposed by Checaet al. (119).

ChymotrypsinChymotrypsin, secreted in the pancreas as chymotrypsinogen(120,121), catalyses the hydrolysis of peptide bonds of proteins inthe mammalian gut. Close to the active serine of chymotrypsin is adeeply invaginated apolar pocket, large enough to accommodate asmall planar molecule (122). Uncontrolled chymotrypsin in an estab-lished catalyst of the breakdown of blood brain barrier as reviewedelsewhere (123), with deleterious consequences, including Alzhei-mer's disease (124), multi-infarct dementia (125), and head trauma(126). Unfortunately, limited evidences of the interaction betweenthis protease and polyphenols are currently available. Rawel et al.revealed that caffeic acid and quercetin possess the highest declin-ing effect on the chymotryptic digestion (127). Besides, Jonadetet al. (128) showed that extracts from Alchemilla Vulgaris L. andRibes Nigrum L. had a low inhibitory efficacy on the amidolyticactivity of alpha-chymotrypsin, likely to be attributable to caffeicacid (as reported in Ribes Nigrum L. extract by Ehala et al. (129)).

ElastasePrimarily, elastases hydrolyze the connective tissue protein elastin(130,131). Neutrophil elastase is present in granules of polymorpho-nuclear leukocytes and is fundamental for phagocytosis and defenceagainst invading microorganisms.

Besides its physiological defence function, NE is one of the mostdestructive enzymes in the organism, being able to escape fromregulation by protease inhibitors at inflammatory sites (132). Onceunregulated, this enzyme can alter the function of the lung perme-ability barrier and induces the release of pro-inflammatory cytokines

(133), finally resulting in acute lung injury. Additionally, a role forNE in human diseases was proposed in the degradation of elastinassociated with pulmonary emphysema, skin diseases and arthritis(134). Its inhibition by natural occurring polyphenolic compounds isdefinitely the most widely characterized.

Pancreatic elastase is stored as an inactive zymogen in thepancreas and is secreted into the intestines, where contributes tothe digestive process upon trypsin activation. PE is a single peptidechain of 240 amino acids and four disulfide bridges (135). Bothelastases hydrolyze substrates at peptide bonds where the P1 resi-due is an amino acid residue with a small alkyl side chain (136).

Sartor et al. (137,138) tested both the in vitro and in vivo anti-inflammatory activity of a number of polyphenolic molecules onelastase, finally proposing EGCG as an inhibitor of NE in preventivetreatment of inflammatory states, tumour invasion and ⁄ or neoangio-genesis. In fact, EGCG showed a significant inhibition of NE(IC50 = 0.4 lM). This inhibition of NE resulted only mildly lower thanan elastase endogenous inhibitor, a1-PI, (139,140), whereas EGCGexerted a stronger and more prolonged inhibition with respect bothto some natural and synthetic inhibitors (136,141–143).

Structurally, the presence and the position of some functionalgroups (i.e. hydroxyl groups and ⁄ or the 2-3 double bond) were con-sidered crucial by the authors for the inhibitory capacity of a mole-cule, as also evidenced for two anthocyanidins inhibiting NE in thelow micromolar range. Other polyphenols showed a fairly high inhi-bition of NE: myricetin, morin and pelargonidin, fisetin and querce-tin inhibited NE at concentrations 10-fold higher than thoserequired for EGCG. Besides, molecules presenting none of the iden-tified favourable structural features (taxifolin, luteolin, chrysin andapigenin) had minor or negligible inhibitory effects versus NE. Thesepromising results encouraged other authors (144) to synthesizeb-lactam derivatives of EGCG, obtaining no enhancement ininhibitory effect against elastase (IC50 = 0.5 lM).

Two ellagitannins from Alchemilla xanthochlora and again EGCGwere tested for their inhibitory activity against NE also by Hrennet al. (145) with partially different results: besides reporting a 50-fold higher IC50 by EGCG (IC50 = 25.3 lM) with respect to the dataproposed by Sartor et al., EGCG resulted also relevantly less effi-cient than the ellagitannins (IC50 = 0.9 lM and 2.8 lM), that werecapable of hindering more extensively the catalytic pocket of elas-tase, but as a result of an unspecific binding, as predicted bymolecular docking studies (145).

Moreover, Melzig et al. tested other phenolic compounds from plantson the enzymatic activity of NE (146), with hyperoside resulting themost potent inhibitor (IC50 � 0.3 lM). In this case, the glycosidemoiety of quercetin derivatives was fundamental for the inhibitoryactivity, since IC50 values were strongly dependent upon glycosyla-tion: hyperoside and isoquercitrin inhibited the enzyme more effi-ciently than the pure aglycon, oppositely from rutin and quercitrin,both resulting less active than quercetin. Additionally, the com-pounds with hydroxyl groups in position 3¢ and 4¢ showed a strongerinhibitory activity than flavonols with different hydroxylation pat-terns. Interestingly, the methylation of hydroxyl functions in flavones



(diosmetin) decreased the inhibition activity, whereas naringenin anderiocitrin had a negligible efficacy, exemplifying the crucial role of2-3 double bond in the inhibitory activity against NE. In fact, alsoflavan-3-ol-derivatives and other flavones, despite the favourablehydroxylation pattern, were effective only above 400 lM. Singularly,caffeic acid moderately inhibited the enzyme (IC50 = 93 lM), with itsester derivatives reporting an enhanced inhibitory activity againstneutrophil elastase. Other natural polyphenolic compounds like epi-phyllinic acid or its shikimic acid ester also inhibited the enzymewith an IC50 in micromolar range, again emphasizing the dependenceof the inhibitory activity from hydroxyl moiety.

Collectively, these results, besides confirming the inhibitory attitudeof quercetin toward serine proteases, disclose the (singular) propen-sity of elastases to accommodate large ligands with moderatelyhigh affinity, probably by virtue of a more easily accessible catalyticpocket, or to a possible specific interaction with a wider zonearound the catalytic pocket, inducing an unspecific cloaking of thebinding active site.

Cathepsin GCathepsin G is a cationic serine protease found in the azurophilicgranules of polymorphonuclear leukocytes (146–148), with an enzy-matic activity strictly resembling chymotrypsin (149). It is involvedin the degradation of foreign bodies and injured tissues enclosedby phagosomal vesicles during inflammatory responses. Odbergand Olsson described an enzymatic-independent killing processand suggested that the cathepsin G exerts its antibacterial actionthrough a mechanism involving membrane depolarization. Theyfound that cathepsin G-treated bacteria ceased essential biochemi-cal processes associated with intact cytoplasmic membranes(150,151). Moreover, cathepsin G was reported to mimic NE activ-ity, although less potently (138,152). Unfortunately, limited data oncathepsin G treatment with both natural and synthetic polyphenolsare currently available (138,153,154): only Sartor et al. were suc-cessful in reporting an inhibition of cathepsin G by EGCG (in themM range).

Other serine proteasesProteinase 3 (PR3) is a human polymorphonuclear leukocyte serineprotease, with a highly conserved peptide chain (155). PR3 is impli-cated in modulating the activity of platelets and endothelial cells(156) and of inflammatory mediators, such as complement factor 1inhibitor (157), IL-8 (158), tumour necrosis factor (159,160), IL-1b(159) or transforming growth factor b (161).

The proteolytic activities of PR3 on gelatin and casein were evalu-ated by Pezzato et al. (162) in the presence of increasing concentra-tions of EGCG: the authors reported that the activity was mostlyinhibited by EGCG with an IC50 < 20 lM and 100 lM, respectively.Additionally, the degradation of Matrigel components by PR3 wasclearly hindered by 15 lM EGCG. Conversely, a very marginal effectby EGCG was observed against PR3 synthetic substrate. Together,these results provided evidences of EGCG targeting either PR3regions not involved in substrate binding or its active site, thus act-ing as a non-competitive inhibitor.

Acrosin is a protease with trypsin-like specificity, contained in sper-matozoa. The major role of this protease appears to be the diges-tion of a small oblique tunnel through the zona pellucida (163). Thephysicochemical, enzymatic, structural, and antigenic properties ofacrosin are strikingly similar to those of pancreatic trypsin. In fact,it is specific for the peptide and ester bonds of lysine and arginine(164). Acrosin activity was inhibited by tannic acid in both humanand ovine acrosomal extracts, with an IC50 = 150 lM, as reportedby Taitzoglu et al. (93).

The complement system consists of both soluble and membrane pro-teins, and is activated by 3 distinct pathways either on pathogen sur-face or in plasma. The anti-complement activity of some naturalcompounds was tested against the classical pathway of the comple-ment system by Park et al. (165) and Lee et al. (166). Among thetested compounds, only daphnoretin exhibited significant anti-com-plement activity with an IC50 = 11.4 lM, whereas the other com-pounds (both aglycon and glycosylated) were not active in the assay.

Also, a number of polyphenols, namely epicatechin, afzelin, tiliro-side, myricitrin, kaempferol, quercetin, myricetin, and quercitrin,were tested against classical pathway of complement system (167).Afzelin, quercitrin, and tiliroside showed anti-complement activitywith IC50 values of 258, 440, and 101 lM, respectively, with couma-ric acid contributing to the increase of anti-complement activity. Epi-catechin and myricitrin were completely incapable of inhibitingcomplement activity. The authors confirmed the role played byhydroxyl moiety on B-ring of 5,7-dihydroxyflavone: in particular, theinhibitory efficiencies of the polyphenols tested against anti-comple-ment activity increased inversely to the number of free hydroxyls.Furthermore, kaempferol exhibited a weak anti-complement activitywith an IC50 value of 730 lM, whereas quercetin and myricetinwere inactive in this assay system. These data agreed with theanti-complement properties of apigenin and luteolin and their glyco-sides isolated from Ligustrum vulgare and Phillyrea latifolia (168),and underlined the importance of the substitution of coumaric acid,the number of hydroxyl group on B-ring, and the rhamnose of 5,7-dihydroxyflavone in the anti-complement system.

Methods

Pharmacophore modellingPharmacophore modelling is a predictive technique used in the inter-pretation of the binding modes of small ligands to a macromoleculartarget that is applied with increasing success in computational drugdiscovery, finally representing an interface between computer sci-ence and medicinal chemistry. According to the official IUPACnomenclature, a pharmacophore is an 'ensemble of steric and elec-tronic features that is necessary to ensure the optimal molecularinteractions with a specific biological target structure and to triggeror block its biological response' (169). In other words, it is the larg-est common denominator shared by a set of active molecules.

To be a useful tool for drug design, a pharmacophore model has toprovide valid information for medicinal chemists investigating struc-ture–activity relationships, describing the nature of the functionalgroups involved in ligand–target interactions, as well as the type of

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the non-covalent bonding and intercharge distances. Additionally, thepredictive power of the model should enable the design of novelchemical structures that are not evidently derived by the translationof structural features from one active series to the other.

Herein, we derived a polyphenol-based pharmacophore toward ser-ine proteases both in a structure-based manner, by determining thecomplementarities between the ligand and its binding site on thebasis of molecular docking and experimental evidences, and in aligand-based manner, by flexibly overlaying the subset of most activemolecules to determine those conformations that maximize the num-ber of important chemical features geometrically overlapping.

Bioinformatic analysisSerine protease-polyphenols composite models were generated byanalogy to available crystal structures (170). Ligands were submit-ted to a conformational search of 1000 steps with an energy win-dow for saving the structure of 2 kcal ⁄ mol by means of the MonteCarlo algorithm with MMFFs as the force field. The first conforma-tion was then minimized using the conjugated gradient method untila convergence value of 0.05 kcal ⁄ � mol. Automated docking wasperformed with AUTODOCK 4.0 (171); AUTODOCK TOOLS were usedto identify the torsion angles in the ligands. The regions of interestused by AUTODOCK were defined by considering polyphenols asthe central groups; in particular a grid of 46, 44 and 44 points inthe x, y and z directions was constructed on the centre of mass ofthe compound. A grid spacing of 0.375 � and a distance–dependentfunction of the dielectric constant were used for the energetic mapcalculation. Using the Lamarckian Genetic Algorithm, the dockedcompounds were subjected to 100 runs of the AUTODOCK search inwhich the default values of the other parameters were used. Clus-ter analysis was performed on the results using an RMS toleranceof 1.0 �. Ligands were minimized in enzyme catalytic pocket to alocal minimum by means of a MMFF94 Energy, and aligned to gen-erate a pharmacophore model on the base of polyphenol commonfeatures within LigandScout software 2.0 (172). Pharmacophore de-scriptors, representing chemical feature complementarities to thebinding site of the receptor in the three-dimensional space, includedH-bond donors, H-bond acceptors, hydrophobic, aromatic, positive

ionizable groups, negative ionizable groups. Further enhancement ofthe pharmacophore model was obtained by taking into account theshape and exclusion volumes (steric) constraints.

Conclusion

In the past few years, there was remarkable progress in identifying,characterizing and developing small ligands that can target key bio-logical enzymes, regulating their activity, through the binding bothto the catalytic and ⁄ or allosteric sites. Among these, the natural

Table 1: Comparison of most efficacious natural occurring polyphenolic ligands binding to some major serine proteases

Enzyme Ligand MW (uma) ASA (�2) Binding Affinity (lM) Plasma levels (lM)

Thrombin Quercetin 302.24 350.17 0.35(71) 0.3–7.6(199)Ellagic acid 302.69 296.66 7.6(71) 0.05–0.1(200)Salicin 286.28 313.99 11.4(74) Degraded to salycilic acid(203)

Plasmin Quercetin 302.24 350.17 0.71(108) 0.3–7.6(199)Flavonoid 7 330.24 428.75 1.5(109) n ⁄ aFlavonoid 8 316.24 394.58 2.3(109) n ⁄ a

uPA Quercetin 302.24 350.17 7(79) 0.3–7.6(199)EGCG 458.37 447.23 15(79) 0.13–0.7(200)

Trypsin Silybin 482.44 487.59 3.7(74) 2.3–12(201)Hypericin 504.44 383.91 4.5(74) 0.006(202)Sennoside-A 862.74 626.41 6.1(74) Degraded to rhein(204)

Elastase Hyperoside 464.38 437.62 0.3(146) Degraded to quercetin(199)EGCG 458.37 447.23 0.4(137) 0.13–0.7(199)

Accessible surface area (ASA) was calculated with Hyperchem 7.5 (Hypercube, Inc 2002). Binding affinities are expressed in terms of equilibrium dissociationconstants, and where not available IC50 was used instead.

Figure 3: Three-dimensional representation of a thrombin poly-phenol-based pharmacophore model generated from the alignment ofdifferent catechin-like aglycons [quercetin – superimposed to thepharmacophore-, catechin, epicatechin, luteolin, genistein, flavonoid-7and flavonoid-8 (109)] binding to the catalytic site (red vector: H-bondacceptor group; green vector: H-bond donor group; hydrophobic repul-sion: yellow sphere; hydrophobic interaction: blue-ringed vector). Thepharmacophore was generated with LigandScout 2.02 on the base ofthe composite models retrieved with Autodock 4.0. The crucial hydro-phobic interaction with His57 and the H-bond formed with Ser195within the catalytic core are reported. Receptor binding pocket is rep-resented as aggregated lipophilicity-hydrophobicity surface.



occurring polyphenolic compounds gained increasing attention.Polyphenols are widespread in nature, and the two most importantgroups of dietary phenolics are phenolic acids and flavonoids. Flavo-noids are ubiquitous in plants (173), where they are supposed toexert allelopathic functions, in particular during the processes fol-lowing pathogens attacks (174,175). At non-toxic concentrations inorganisms, they were demonstrated to possess chemopreventiveproperties in numerous epidemiological studies (176–178), to inhibitthe proliferation of tumour cells including breast, prostate, and lungcancer cells in vitro (179–187), and to be antiangiogenic in severalin vitro studies (80,188–193).

Conversely, a number of discordant data is currently available onthe mutagenicity of polyphenols, since tannins were occasionallyreported to exert mutagenic effects (194–197).

Flavonoids share a common structure, based on 2-phenyl-benzo-gamma-pyrane, two benzene rings joined by a third pyranic ring.Although their mechanism of action is not fully defined, monomeric

flavonoids were assessed to act mostly as site-directed small mole-cule inhibitors of serine proteases (198). In particular, previousmolecular docking studies demonstrated that these flavonoids canbe accommodated in specific binding sites found on serine proteases(71) in the proximity of the active site, whereas larger polyphenolswere reported to non-specifically hinder the catalytic pocket serineproteases (145). The reversible effect exerted by flavonoids towardserine proteases (71) confirms that the inhibitory effect is caused (inthe (sub)micromolar range) by a specific binding.

Interestingly, as summarized in Table 1, the monomeric flavonoidshere considered (quercetin in particular) exerted their activity atconcentrations readily achievable in human plasma upon a moder-ate consumption ⁄ administration of fruit, beverages or extracts(199–202), although the metabolic conversion must be taken intoaccount when estimating the bioavailability and efficacy of flavo-noids for pharmacological use (199,203,204). Conversely, higher lev-els of flavonoids can stimulate local conformational changes,denaturation and aggregation of proteins (205).

A

D

B C

Figure 4: Schematization of different physiological pathways involving serine proteases and possible pathological consequences. (A) bloodcoagulation cascade. (B) Digestive processes. (C) Inflammatory response. (D) Plasmin ⁄ plasminogen system. Assessed targets of polyphenolsare marked with light-grey boxes, whereas other serine proteases likely to be inhibited by polyphenols are marked with boxes.

Cuccioloni et al.


Based on the reported experimental evidences, a number of struc-tural requirements for an efficient inhibition of all serine proteasescan be evinced. The phenyl-ring in position 2 of the phenyl-benzo-gamma-pyranic core (rather than in position 3), the keton carbonylin position 4 and hydroxyl functions in 3, 5, 7, and 3¢ (and ⁄ or 5¢)and 4¢ position are critical in the inhibitory activity, with the 2-3double bond further increasing this capacity. Most importantly, elec-trostatic or hydrophobic interactions targeting catalytic residuesgreatly impair enzymatic activity, thus magnifying the inhibitoryeffect of the ligand (Figure 2). Moreover, depending upon the acces-sibility to the catalytic pocket of different serine proteases, the gly-cosylation in position 3, and the higher number of condensed rings(n > 2) generally decreases (or even annihilates) the ligand bindingaffinity. Equally, hindering or polar ⁄ apolar groups can be oppor-tunely inserted in the polyphenolic core, in order to achieve theselective inhibition of a specific serine protease.

In addition, from the alignment of a subset of different catechin-likeaglycons (quercetin, catechin, epicatechin, luteolin, genistein, flavo-noid-7 and flavonoid-8 (109), correlation factor = 0.85) to the serineproteases catalytic site, we derived a pharmacophore model takinginto account the shared features for different polyphenols (Figure 3).Besides confirming previous considerations on structure ⁄ activitydualism derived from experimental evidences, our model classifiedthe carbonyl group at position 4 as a H-bond acceptor toward Ser195 acting as the H-bond donor, and generally as a positively ioniz-able group (toward other ionizable residues of the catalytic pocketof serine proteases). The remaining features were two other H-bonddonors ⁄ acceptors, an H-bond donor, a hydrophobic repulsion regionand a hydrophobic interaction region toward His 57. Additionally,planarity provided a significant contribution to binding affinity. Quer-cetin, an attractive natural compound for cancer prevention due toits anti-mutagenic and anti-proliferative effects, and its role in theregulation of cell signalling, cell cycle and apoptosis, all demon-strated both in animal models and in vitro studies (206), showedthe best fit to this pharmacophore.

In conclusion, the derivation of such a flavonoid-based pro-drugcapable of modulating a whole class of proteases deserves particu-lar attention: in fact, since these proteases are mutually involved incomplex cascade mechanisms, such as the blood coagulation cas-cade, digestive processes, and inflammatory response (Figure 4), amulti-target inhibitor of serine proteases is likely to be more suc-cessful in the co-treatment of pathologies, rather than a single-target inhibition that could be probably ineffective.

Moreover, although the bioavailability of flavonoids is still contro-versial (207–211), their activity as specific ligands, provides anotherrole besides their disguising capacity as antioxidants in vivo, inthe presence of very efficient and efficacious enzymatic systems(174,175), in fact, these data propose a potential role for flavonoidsas inhibitors in the regulation of a number of physio-pathologicalprocesses involving serine proteases, and offer a promisingstarting point for the development of specific site-direct syntheticinhibitors, although a structural optimization is required if nano-molar affinity (typical of many drugs) and higher bioavailability aredesired.

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Natural Occurring Polyphenols as Template for Drug Design. Focus on Serine Proteases

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