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

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Chem Biol Drug Des 2009; 74: 1–15

ª 2009 John Wiley & Sons A/S doi: 10.1111/j.1747-0285.2009.00836.x

Review Article

Natural Occurring Polyphenols as Template for Drug Design. Focus on Serine Proteases Massimiliano Cuccioloni1*, Matteo Mozzicafreddo1, Laura Bonfili1, Valentina Cecarini1, Anna Maria Eleuteri1 and Mauro Angeletti1 1

Department of Molecular, Cellular and Animal Biology, University of 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 thrombosis, are all strongly dependent upon the fine regulation of proteolytic enzyme activities, and dramatic are the consequences of unbalanced equilibria between enzymes and their cognate inhibitors. In this perspective, the discovery of small-molecule ligands able to modulate catalytic activities has a massive therapeutic potential and is a stimulating goal. Numerous recent experimental evidences revealed that proteolytic enzymes can be opportunely targeted, reporting on small ligands capable of binding to these biological macromolecules with drug-like potencies, and primarily with comparable (or even higher) efficiency with respect to their endogenous binding partner. In particular, natural occurring polyphenols and their derivatives recently disclosed these intriguing abilities, making them promising templates for drug design and development. In this review, we compared the inhibitory capacities of a set of monomeric polyphenols toward serine proteases activity, and finally summarized the data with an emphasis on the 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 for publication 16 May 2009

Strong is the interest in specifically controlling enzymes for therapeutic purposes, possibly with site-directed small molecules, which generally are 'easier to handle' being orally administrable, but the structure and the specificity of their catalytic regions make this task a huge challenge. Several studies provide evidences for small molecules targeting the catalytic site of enzymes, thus modulating their activities. Herein, we will set our focus on a particular class of physiologically pivotal enzymes, namely the serine proteases.

Serine proteases, proteolytic enzymes widely present in tissues and biological fluids (1,2), are established regulators of many normal cellular functions, either as non-specific catalysts of protein degradation or highly selective agents controlling physiological events. Their roles in protein digestion, limited proteolysis, mammalian reproduction, the cascade mechanisms of blood coagulation, complement activation, kallikrein-kinin system, and fibrinolysis are all well-documented (3–5), being additionally related to various phases of malignant transformation and cellular activation (6). They share a common architecture consisting of two 6-stranded b-barrels (7), containing the catalytic site cleft. Essentially, these structures consist of a zymogen activation domain, a substrate recognition region and a catalytic core (8), this latter principally presenting a serine, a histidine and an aspartic acid residue (9), namely the catalytic triad, that covers the active site cleft, as a part of an extensive hydrogen bonding network. Structurally, the high degree of conservation of catalytic sites of serine proteases uncovered a common binding pattern with ligands with a similar architecture. Crucial equilibria between serine proteases and their specific inhibitors normally exist in tissues where these agents are involved, since, once activated, they can pose a major threat for the organism (10–19) if they are not correctly regulated by their cognate serine protease inhibitors, namely the serpins (20–23). Therefore, the identification of new, possibly natural, serine protease inhibitors to be used in the treatment of diseases involving serine proteases over-function is an attracting topic in current medicinal chemistry. Here, we summarized the available data on the inhibitory potencies of a number of natural polyphenols toward some major serine proteases (Figure 1), by means of binding equilibrium dissociation constants (Kd) or, in the absence of direct binding data, half-maximal inhibitory concentrations (IC50) from functional assays. Finally, a subset of compounds constituted by the most effective polyphenol inhibitors was analysed in order to rationalize the available structural and functional data in the formulation of a polyphenol-based model that shares all the favourable features contributing to enhance the specificity of the interaction.

Inhibition of Serine Proteases Activity by Polyphenols Thrombin-like proteases Thrombin, plasmin, urokinase-like plasminogen activator (uPA) and tissue-like plasminogen activator (tPA) are collectively referred to as thrombin-like proteases. These proteases are traditionally 1

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Figure 1: Selection of polyphenols that inhibit serine proteases. Six of the most efficacious site-directed polyphenolic inhibitors are here visualized studied in regard to intravascular fibrinolysis and biological processes involving cell movement along the extracellular matrix. These tissue-remodelling processes are involved in nervous system development, response to injury and degenerative diseases (22,24). Besides their central role in thrombogenesis and fibrinolysis, thrombin-like proteases are also involved in tumour cell invasion, 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 of moderate amounts of polyphenols can exert beneficial vascular effects, reducing the risk for cardiovascular morbidity and mortality (26).

Thrombin Thrombin is involved in blood coagulation at different levels, by converting soluble fibrinogen to fibrin, activating different factors, which generates more thrombin, stimulates platelets and favours the stabilization of the clot (27). Normally, the coagulation cascade is regulated by physiological anticoagulants, such as tissue factor pathway inhibitor, the protein C and protein S system, and anti-thrombin (28–31). The lack and ⁄ or the misfunction of thrombin endogenous inhibitors are causes for hyper-coagulative disorders. 2

Thus, several site-directed synthetic thrombin inhibitors (bivalirudin, argatroban and ximelagatran) have been developed, and their role in the treatment of coronary syndromes (32–34), stroke (35–47), systemic embolism (48,49) and the prevention of post-surgery venous thromboembolism (50–55) is widely documented. Nevertheless, since hepato-toxicity is observed in prolonged ⁄ acute administration of these synthetic compounds (56–58), the identification of nontoxic natural occurring molecules (or derivatives thereof) as co-drugs in the treatment of thrombin-related pathologies is a stimulating test for current medicinal chemistry (59–70). We recently reported that thrombin was competitively inhibited by natural occurring monomeric polyphenols (Figure 2C) (71,72), showing evidences consistent with the presence of a specific binding site for this class of molecules on the enzyme. In particular, quercetin and its glycosylated derivatives, namely rutin and hyperoside, showed affinities ranging from sub-micromolar (quercetin, Kd = 0.35 € 0.04 lM) to millimolar for human thrombin, with these differences at least partially depending upon the hindering glycosyl groups on the aglycon. As a comparison with a synthetic site-directed thrombin inhibitor, the dissociation equilibrium constant of the quercetin–thrombin complex was 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 Chem Biol Drug Des 2009; 74: 1–15

Dietary Polyphenols Inhibit Serine Proteases A



Figure 2: Visualization of binding modes of the most effective polyphenol to serine protease: hyperoside-elastase (A, 1QNJ.pdb), quercetin-plasmin (B, 1DDJ.pdb), quercetin-thrombin (C, 1EB1.pdb), quercetin-uPA (D, 2VIO.pdb) and sylibin-trypsin (E, 1TRN.pdb). Active site views of dockings are shown in the left insets (catalytic triads are highlighted in lighter tone). Right insets thoroughly illustrate the enzyme-ligand interactions (red vector: H-bond acceptor group; green vector: H-bond donor group; hydrophobic repulsion: yellow highlight; hydrophobic interaction: blueringed vector). The docking analysis was performed using a two-stage docking procedure applying the program AutoDock 4.0, whereas lowest-energy complexes were re-analysed according to a more detailed energetic model. The characterization of ligand binding interactions was performed with LigandScout 2.02 (Inte:Ligand GmbH).



lacking the carbonyl group in position 4, formed the least stable complexes with human thrombin. Salicin and silybin (this latter having a different chemical structure) reported in vitro inhibition activities in micromolar range Chem Biol Drug Des 2009; 74: 1–15

(IC50 = 11.4 and 20.9 lM, respectively) (74) making them, together with quercetin and ellagic acid, promising candidates for synthetic modification as antithrombotic agents. Conversely, luteolin reported a lower affinity for thrombin with respect to quercetin, this result disclosing the importance of H-bonds formation with polar residues 3

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within the catalytic site of thrombin (71). Additionally, (-)-epigallocatechin-3-gallate (EGCG), hypericin, rhein, chrysophanol, emodin, physcion, aloin, sennoside-A and sennoside-B, b-aescin and colchicine, all being relatively hindering with respect to the catalytic site geometry of thrombin, affected its activity only at (low) millimolar range of concentration, thus showing no significant effect (74). Moreover, the preincubation of platelets with resveratrol at physiological plasma concentrations prevented their adhesion to fibrinogen after the stimulation of these cells with thrombin, thus modulating the activity of blood platelets in inflammatory process (75). Also quercetin showed an anticlotting effect, but only at higher concentrations, possibly due to the propensity of binding with other plasma proteins (71). From a structural viewpoint, all currently available findings agree with the importance of the carbonyl group in position 4 (accessible for forming electrostatic interactions with His57, and additionally preserving the planarity of the molecule) resulting critical for the magnification of the inhibitory efficacy of the reviewed polyphenols toward thrombin. Besides, the hydroxylation in position 3 and a favourable accommodation of hydrophobic groups within the catalytic pocket additionally enhanced this ability (Figure 2C). Conversely, larger hindering replacing groups on B ring or in position 3, reduced the ligand accessibility to the narrow catalytic pocket of thrombin, with a global restraining effect on the affinity of the complex.

Plasminogen activators The primary role of uPA and tPA is the conversion of plasminogen to plasmin (76). uPA is also involved in tissue-related proteolysis, and it is critical in the degradation of extracellular matrix and transversion of basement membranes (77), whereas tPA is mainly involved in fibrinolysis and thrombolysis (78). uPA activity was reported to be diminished by quercetin (IC50 = 7 lM), EGCG (IC50 = 15 lM) (79), genistein and apigenin (80–84), with all these activities mainly relying upon electrostatic interactions involving the hydroxyl group in position 7, and the hydrophobic interactions with the A ring upon the insertion within the S1 site (Figure 2D). The inhibitory effect was considered quite beneficial, because uPA is an excellent pro-factor of malignancy and axillary metastasis (85). In this perspective, Lamy et al. reported a concentration-dependent inhibition of uPA by anthocyanidins in the low micromolar range, with a consequent involvement in the regulation of the motility of glioblastoma cells (86). Also tPA activity was inhibited by quercetin in kidney and tumour tissue. Therefore, since tPA and uPA activities were reported to be increased in later phases of tumour progression and in early phases of tumour invasion, respectively (87,88), quercetin can be regarded as an inhibitor of metastasis at both stages. Additionally, quercetin may exert a two-level impairment of PA relying upon the inhibition of protein kinase C (89), whose involvement in cell motility in breast cancer was reported (85,90–92). Less significantly in the perspective of a possible drug development, Taitzoglou et al. (93) reported a dose-dependent decrease in both human and ovine PA activity induced by tannic acid only at submolar range.

Plasmin Plasmin is a two-chain macromolecule joined by two disulphide bridges (94,95) with a broad specificity protease (96–99), that 4

cooperates with fibrin to form a clot. The role of plasmin in tumour cell invasion and migration is well documented (100–102), with evidences for both direct (by degrading proteins of the extracellular matrix and activating plasminogen activators) and indirect action (by activating of matrix metalloproteases). Moreover, plasmin plays an important function in angiogenesis (103–106), and regulates the proteolytic processing of cell surface molecules such as cadherins, the major cell–cell adhesion proteins (107). We recently discriminated the effect of glycosylation on the antiplasmin activity of polyphenols (108). In particular, quercetin showed a 20-fold higher affinity 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 glycosylated counterpart) prevented plasmin-incubated BB1 cells from releasing E-cadherin fragment, thus representing a limiting factor for the decrease in intercellular adhesion and consequent cell motility. Also Osaky et al. (109) tested some flavonoids purified from B. balsamifera on plasmin activity. Interestingly, the authors demonstrated that flavonoids with hydroxyl groups on 3¢ and 4¢ positions were active in a plasmin-inhibitory test (IC50 = 1.5 and 2.3 lM), with an efficacy comparable to leupeptin (IC50 = 1.2 lM). Additionally, tannic acid showed a 92% reduction of plasmin activity, but only at 200 lM. Structurally, also these results emphasized the discriminating role of the carbonyl in position 4 in the inhibition of plasmin, whereas the presence of hydroxyl or methoxyl groups in position 3 and 7 were responsible for the reported differences in terms of inhibition constants (Figure 2B).

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

Trypsin Trypsin is secreted by the pancreas as trypsinogen, eventually activated either by enterokinase, or autocatalytically (112). It is one of the most extensively characterized serine proteases, and plays an essential role in both normal physiological processes (food digestion, 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-pathological states, such as pancreatitis (114,115), and is critical in the development of pancreatic carcinoma (116). Maliar et al. reported a major inhibitory effect exerted by multiple hydroxyl-substituted flavonols and flavones, increasing with the number of hydroxyl-substitution. In particular, the authors highlighted the role of the hydroxyl group in position 3 in the inhibition process, as confirmed by quercetin, myricetin and acacetin, that were shown to be the most effective with IC50 = 10, 15 and 28 lM, respectively (74,117). Additionally, flavones with the 2-3 double bond (apigenin) were more effective than flavanones, whereas isoflavones (biochanin A) were less Chem Biol Drug Des 2009; 74: 1–15

Dietary Polyphenols Inhibit Serine Proteases

effective than flavonoids (acacetin). The data on quercetin and myricetin inhibition were in good agreement with previous evidences by Parellada et al. (118) (quercetin, IC50 = 7.1 € 2; myricetin, IC50 = 10.2 € 2), additionally assessing the inhibitory effect of luteolin (IC50 = 35.3 € 6). Antitryptic activities of phenolic compounds were also reported by Jedink et al. (74). The authors showed that silybin and hypericin exerted the highest inhibition activities (IC50 = 3.7 and 4.5 lM, respectively) on trypsin. Interestingly, comparable results were observed for both quercetin (IC50 = 15.4 lM) and its glycosylated derivative hyperoside (IC50 = 14.5 lM), with the galactoside group exerting no apparent hindering effect on the binding, by virtue of a favourable insertion within the catalytic pocket. In addition, sennosides showed remarkable IC50 values on trypsin (6.1 lM for sennoside A and 10.6 lM for sennoside B). Significant activities were also observed for some derivatives of anthraquinones, the highest inhibition being reported by rhein, with an IC50 = 25.2 lM. A moderate inhibition was observed for chrysophanol IC50 = 78.9 lM and for emodin IC50 = 50.2 lM. In this case, the glycosylation of the aglycon skeleton seems to destroy inhibitory activity, since aloin did not show any activity on trypsin. Available data are collectively in agreement with a model where electrostatic interactions between polyphenols and trypsin (Figure 2D) strongly contributed to the binding process, as suggested by the relatively high polarity of trypsin catalytic pocket, and in part confirmed by the computational method proposed by Checa et al. (119).

Chymotrypsin Chymotrypsin, secreted in the pancreas as chymotrypsinogen (120,121), catalyses the hydrolysis of peptide bonds of proteins in the mammalian gut. Close to the active serine of chymotrypsin is a deeply invaginated apolar pocket, large enough to accommodate a small planar molecule (122). Uncontrolled chymotrypsin in an established catalyst of the breakdown of blood brain barrier as reviewed elsewhere (123), with deleterious consequences, including Alzheimer's disease (124), multi-infarct dementia (125), and head trauma (126). Unfortunately, limited evidences of the interaction between this protease and polyphenols are currently available. Rawel et al. revealed that caffeic acid and quercetin possess the highest declining effect on the chymotryptic digestion (127). Besides, Jonadet et al. (128) showed that extracts from Alchemilla Vulgaris L. and Ribes Nigrum L. had a low inhibitory efficacy on the amidolytic activity of alpha-chymotrypsin, likely to be attributable to caffeic acid (as reported in Ribes Nigrum L. extract by Ehala et al. (129)).

Elastase Primarily, elastases hydrolyze the connective tissue protein elastin (130,131). Neutrophil elastase is present in granules of polymorphonuclear leukocytes and is fundamental for phagocytosis and defence against invading microorganisms. Besides its physiological defence function, NE is one of the most destructive enzymes in the organism, being able to escape from regulation by protease inhibitors at inflammatory sites (132). Once unregulated, this enzyme can alter the function of the lung permeability barrier and induces the release of pro-inflammatory cytokines Chem Biol Drug Des 2009; 74: 1–15

(133), finally resulting in acute lung injury. Additionally, a role for NE in human diseases was proposed in the degradation of elastin associated with pulmonary emphysema, skin diseases and arthritis (134). Its inhibition by natural occurring polyphenolic compounds is definitely the most widely characterized. Pancreatic elastase is stored as an inactive zymogen in the pancreas and is secreted into the intestines, where contributes to the digestive process upon trypsin activation. PE is a single peptide chain of 240 amino acids and four disulfide bridges (135). Both elastases hydrolyze substrates at peptide bonds where the P1 residue 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 antiinflammatory activity of a number of polyphenolic molecules on elastase, finally proposing EGCG as an inhibitor of NE in preventive treatment of inflammatory states, tumour invasion and ⁄ or neoangiogenesis. In fact, EGCG showed a significant inhibition of NE (IC50 = 0.4 lM). This inhibition of NE resulted only mildly lower than an elastase endogenous inhibitor, a1-PI, (139,140), whereas EGCG exerted a stronger and more prolonged inhibition with respect both to some natural and synthetic inhibitors (136,141–143). Structurally, the presence and the position of some functional groups (i.e. hydroxyl groups and ⁄ or the 2-3 double bond) were considered crucial by the authors for the inhibitory capacity of a molecule, as also evidenced for two anthocyanidins inhibiting NE in the low micromolar range. Other polyphenols showed a fairly high inhibition of NE: myricetin, morin and pelargonidin, fisetin and quercetin inhibited NE at concentrations 10-fold higher than those required for EGCG. Besides, molecules presenting none of the identified favourable structural features (taxifolin, luteolin, chrysin and apigenin) had minor or negligible inhibitory effects versus NE. These promising results encouraged other authors (144) to synthesize b-lactam derivatives of EGCG, obtaining no enhancement in inhibitory effect against elastase (IC50 = 0.5 lM). Two ellagitannins from Alchemilla xanthochlora and again EGCG were tested for their inhibitory activity against NE also by Hrenn et al. (145) with partially different results: besides reporting a 50fold higher IC50 by EGCG (IC50 = 25.3 lM) with respect to the data proposed by Sartor et al., EGCG resulted also relevantly less efficient than the ellagitannins (IC50 = 0.9 lM and 2.8 lM), that were capable of hindering more extensively the catalytic pocket of elastase, but as a result of an unspecific binding, as predicted by molecular docking studies (145). Moreover, Melzig et al. tested other phenolic compounds from plants on the enzymatic activity of NE (146), with hyperoside resulting the most potent inhibitor (IC50  0.3 lM). In this case, the glycoside moiety of quercetin derivatives was fundamental for the inhibitory activity, since IC50 values were strongly dependent upon glycosylation: hyperoside and isoquercitrin inhibited the enzyme more efficiently than the pure aglycon, oppositely from rutin and quercitrin, both resulting less active than quercetin. Additionally, the compounds with hydroxyl groups in position 3¢ and 4¢ showed a stronger inhibitory activity than flavonols with different hydroxylation patterns. Interestingly, the methylation of hydroxyl functions in flavones 5

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(diosmetin) decreased the inhibition activity, whereas naringenin and eriocitrin had a negligible efficacy, exemplifying the crucial role of 2-3 double bond in the inhibitory activity against NE. In fact, also flavan-3-ol-derivatives and other flavones, despite the favourable hydroxylation pattern, were effective only above 400 lM. Singularly, caffeic acid moderately inhibited the enzyme (IC50 = 93 lM), with its ester derivatives reporting an enhanced inhibitory activity against neutrophil elastase. Other natural polyphenolic compounds like epiphyllinic acid or its shikimic acid ester also inhibited the enzyme with an IC50 in micromolar range, again emphasizing the dependence of the inhibitory activity from hydroxyl moiety. Collectively, these results, besides confirming the inhibitory attitude of quercetin toward serine proteases, disclose the (singular) propensity of elastases to accommodate large ligands with moderately high affinity, probably by virtue of a more easily accessible catalytic pocket, or to a possible specific interaction with a wider zone around the catalytic pocket, inducing an unspecific cloaking of the binding active site.

Cathepsin G Cathepsin G is a cationic serine protease found in the azurophilic granules of polymorphonuclear leukocytes (146–148), with an enzymatic activity strictly resembling chymotrypsin (149). It is involved in the degradation of foreign bodies and injured tissues enclosed by phagosomal vesicles during inflammatory responses. Odberg and Olsson described an enzymatic-independent killing process and suggested that the cathepsin G exerts its antibacterial action through a mechanism involving membrane depolarization. They found that cathepsin G-treated bacteria ceased essential biochemical processes associated with intact cytoplasmic membranes (150,151). Moreover, cathepsin G was reported to mimic NE activity, although less potently (138,152). Unfortunately, limited data on cathepsin G treatment with both natural and synthetic polyphenols are currently available (138,153,154): only Sartor et al. were successful in reporting an inhibition of cathepsin G by EGCG (in the mM range).

Other serine proteases Proteinase 3 (PR3) is a human polymorphonuclear leukocyte serine protease, with a highly conserved peptide chain (155). PR3 is implicated in modulating the activity of platelets and endothelial cells (156) and of inflammatory mediators, such as complement factor 1 inhibitor (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 evaluated by Pezzato et al. (162) in the presence of increasing concentrations of EGCG: the authors reported that the activity was mostly inhibited by EGCG with an IC50 < 20 lM and 100 lM, respectively. Additionally, the degradation of Matrigel components by PR3 was clearly hindered by 15 lM EGCG. Conversely, a very marginal effect by EGCG was observed against PR3 synthetic substrate. Together, these results provided evidences of EGCG targeting either PR3 regions not involved in substrate binding or its active site, thus acting as a non-competitive inhibitor. 6

Acrosin is a protease with trypsin-like specificity, contained in spermatozoa. The major role of this protease appears to be the digestion of a small oblique tunnel through the zona pellucida (163). The physicochemical, enzymatic, structural, and antigenic properties of acrosin 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 human and ovine acrosomal extracts, with an IC50 = 150 lM, as reported by Taitzoglu et al. (93). The complement system consists of both soluble and membrane proteins, and is activated by 3 distinct pathways either on pathogen surface or in plasma. The anti-complement activity of some natural compounds was tested against the classical pathway of the complement system by Park et al. (165) and Lee et al. (166). Among the tested compounds, only daphnoretin exhibited significant anti-complement activity with an IC50 = 11.4 lM, whereas the other compounds (both aglycon and glycosylated) were not active in the assay. Also, a number of polyphenols, namely epicatechin, afzelin, tiliroside, myricitrin, kaempferol, quercetin, myricetin, and quercitrin, were tested against classical pathway of complement system (167). Afzelin, quercitrin, and tiliroside showed anti-complement activity with IC50 values of 258, 440, and 101 lM, respectively, with coumaric acid contributing to the increase of anti-complement activity. Epicatechin and myricitrin were completely incapable of inhibiting complement activity. The authors confirmed the role played by hydroxyl moiety on B-ring of 5,7-dihydroxyflavone: in particular, the inhibitory efficiencies of the polyphenols tested against anti-complement activity increased inversely to the number of free hydroxyls. Furthermore, kaempferol exhibited a weak anti-complement activity with an IC50 value of 730 lM, whereas quercetin and myricetin were inactive in this assay system. These data agreed with the anti-complement properties of apigenin and luteolin and their glycosides 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 5,7dihydroxyflavone in the anti-complement system.

Methods Pharmacophore modelling Pharmacophore modelling is a predictive technique used in the interpretation of the binding modes of small ligands to a macromolecular target that is applied with increasing success in computational drug discovery, finally representing an interface between computer science and medicinal chemistry. According to the official IUPAC nomenclature, a pharmacophore is an 'ensemble of steric and electronic features that is necessary to ensure the optimal molecular interactions with a specific biological target structure and to trigger or block its biological response' (169). In other words, it is the largest common denominator shared by a set of active molecules. To be a useful tool for drug design, a pharmacophore model has to provide valid information for medicinal chemists investigating structure–activity relationships, describing the nature of the functional groups involved in ligand–target interactions, as well as the type of Chem Biol Drug Des 2009; 74: 1–15

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


MW (uma)

ASA (2)

Binding Affinity (lM)

Plasma levels (lM)


Quercetin Ellagic acid Salicin Quercetin Flavonoid 7 Flavonoid 8 Quercetin EGCG Silybin Hypericin Sennoside-A Hyperoside EGCG

302.24 302.69 286.28 302.24 330.24 316.24 302.24 458.37 482.44 504.44 862.74 464.38 458.37

350.17 296.66 313.99 350.17 428.75 394.58 350.17 447.23 487.59 383.91 626.41 437.62 447.23

0.35(71) 7.6(71) 11.4(74) 0.71(108) 1.5(109) 2.3(109) 7(79) 15(79) 3.7(74) 4.5(74) 6.1(74) 0.3(146) 0.4(137)

0.3–7.6(199) 0.05–0.1(200) Degraded to salycilic acid(203) 0.3–7.6(199) n⁄a n⁄a 0.3–7.6(199) 0.13–0.7(200) 2.3–12(201) 0.006(202) Degraded to rhein(204) Degraded to quercetin(199) 0.13–0.7(199)


uPA Trypsin


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

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

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

Herein, we derived a polyphenol-based pharmacophore toward serine proteases both in a structure-based manner, by determining the complementarities between the ligand and its binding site on the basis of molecular docking and experimental evidences, and in a ligand-based manner, by flexibly overlaying the subset of most active molecules to determine those conformations that maximize the number of important chemical features geometrically overlapping.


Bioinformatic analysis Serine protease-polyphenols composite models were generated by analogy to available crystal structures (170). Ligands were submitted to a conformational search of 1000 steps with an energy window for saving the structure of 2 kcal ⁄ mol by means of the Monte Carlo algorithm with MMFFs as the force field. The first conformation was then minimized using the conjugated gradient method until a convergence value of 0.05 kcal ⁄  mol. Automated docking was performed with AUTODOCK 4.0 (171); AUTODOCK TOOLS were used to identify the torsion angles in the ligands. The regions of interest used by AUTODOCK were defined by considering polyphenols as the central groups; in particular a grid of 46, 44 and 44 points in the x, y and z directions was constructed on the centre of mass of the compound. A grid spacing of 0.375  and a distance–dependent function of the dielectric constant were used for the energetic map calculation. Using the Lamarckian Genetic Algorithm, the docked compounds were subjected to 100 runs of the AUTODOCK search in which the default values of the other parameters were used. Cluster analysis was performed on the results using an RMS tolerance of 1.0 . Ligands were minimized in enzyme catalytic pocket to a local minimum by means of a MMFF94 Energy, and aligned to generate a pharmacophore model on the base of polyphenol common features within LigandScout software 2.0 (172). Pharmacophore descriptors, representing chemical feature complementarities to the binding site of the receptor in the three-dimensional space, included H-bond donors, H-bond acceptors, hydrophobic, aromatic, positive Chem Biol Drug Des 2009; 74: 1–15

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

Figure 3: Three-dimensional representation of a thrombin polyphenol-based pharmacophore model generated from the alignment of different catechin-like aglycons [quercetin – superimposed to the pharmacophore-, catechin, epicatechin, luteolin, genistein, flavonoid-7 and flavonoid-8 (109)] binding to the catalytic site (red vector: H-bond acceptor group; green vector: H-bond donor group; hydrophobic repulsion: yellow sphere; hydrophobic interaction: blue-ringed vector). The pharmacophore was generated with LigandScout 2.02 on the base of the composite models retrieved with Autodock 4.0. The crucial hydrophobic interaction with His57 and the H-bond formed with Ser195 within the catalytic core are reported. Receptor binding pocket is represented as aggregated lipophilicity-hydrophobicity surface. 7

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occurring polyphenolic compounds gained increasing attention. Polyphenols are widespread in nature, and the two most important groups of dietary phenolics are phenolic acids and flavonoids. Flavonoids are ubiquitous in plants (173), where they are supposed to exert allelopathic functions, in particular during the processes following pathogens attacks (174,175). At non-toxic concentrations in organisms, they were demonstrated to possess chemopreventive properties in numerous epidemiological studies (176–178), to inhibit the proliferation of tumour cells including breast, prostate, and lung cancer cells in vitro (179–187), and to be antiangiogenic in several in vitro studies (80,188–193). Conversely, a number of discordant data is currently available on the mutagenicity of polyphenols, since tannins were occasionally reported to exert mutagenic effects (194–197). Flavonoids share a common structure, based on 2-phenyl-benzogamma-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 molecule inhibitors of serine proteases (198). In particular, previous molecular docking studies demonstrated that these flavonoids can be accommodated in specific binding sites found on serine proteases (71) in the proximity of the active site, whereas larger polyphenols were reported to non-specifically hinder the catalytic pocket serine proteases (145). The reversible effect exerted by flavonoids toward serine proteases (71) confirms that the inhibitory effect is caused (in the (sub)micromolar range) by a specific binding. Interestingly, as summarized in Table 1, the monomeric flavonoids here considered (quercetin in particular) exerted their activity at concentrations readily achievable in human plasma upon a moderate consumption ⁄ administration of fruit, beverages or extracts (199–202), although the metabolic conversion must be taken into account when estimating the bioavailability and efficacy of flavonoids for pharmacological use (199,203,204). Conversely, higher levels of flavonoids can stimulate local conformational changes, denaturation and aggregation of proteins (205).




Figure 4: Schematization of different physiological pathways involving serine proteases and possible pathological consequences. (A) blood coagulation cascade. (B) Digestive processes. (C) Inflammatory response. (D) Plasmin ⁄ plasminogen system. Assessed targets of polyphenols are marked with light-grey boxes, whereas other serine proteases likely to be inhibited by polyphenols are marked with boxes. 8

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Based on the reported experimental evidences, a number of structural requirements for an efficient inhibition of all serine proteases can be evinced. The phenyl-ring in position 2 of the phenyl-benzogamma-pyranic core (rather than in position 3), the keton carbonyl in 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 2-3 double bond further increasing this capacity. Most importantly, electrostatic or hydrophobic interactions targeting catalytic residues greatly impair enzymatic activity, thus magnifying the inhibitory effect of the ligand (Figure 2). Moreover, depending upon the accessibility to the catalytic pocket of different serine proteases, the glycosylation in position 3, and the higher number of condensed rings (n > 2) generally decreases (or even annihilates) the ligand binding affinity. Equally, hindering or polar ⁄ apolar groups can be opportunely inserted in the polyphenolic core, in order to achieve the selective inhibition of a specific serine protease. In addition, from the alignment of a subset of different catechin-like aglycons (quercetin, catechin, epicatechin, luteolin, genistein, flavonoid-7 and flavonoid-8 (109), correlation factor = 0.85) to the serine proteases catalytic site, we derived a pharmacophore model taking into account the shared features for different polyphenols (Figure 3). Besides confirming previous considerations on structure ⁄ activity dualism derived from experimental evidences, our model classified the carbonyl group at position 4 as a H-bond acceptor toward Ser 195 acting as the H-bond donor, and generally as a positively ionizable group (toward other ionizable residues of the catalytic pocket of serine proteases). The remaining features were two other H-bond donors ⁄ acceptors, an H-bond donor, a hydrophobic repulsion region and a hydrophobic interaction region toward His 57. Additionally, planarity provided a significant contribution to binding affinity. Quercetin, an attractive natural compound for cancer prevention due to its anti-mutagenic and anti-proliferative effects, and its role in the regulation of cell signalling, cell cycle and apoptosis, all demonstrated both in animal models and in vitro studies (206), showed the best fit to this pharmacophore. In conclusion, the derivation of such a flavonoid-based pro-drug capable of modulating a whole class of proteases deserves particular attention: in fact, since these proteases are mutually involved in complex cascade mechanisms, such as the blood coagulation cascade, digestive processes, and inflammatory response (Figure 4), a multi-target inhibitor of serine proteases is likely to be more successful in the co-treatment of pathologies, rather than a singletarget inhibition that could be probably ineffective. Moreover, although the bioavailability of flavonoids is still controversial (207–211), their activity as specific ligands, provides another role besides their disguising capacity as antioxidants in vivo, in the presence of very efficient and efficacious enzymatic systems (174,175), in fact, these data propose a potential role for flavonoids as inhibitors in the regulation of a number of physio-pathological processes involving serine proteases, and offer a promising starting point for the development of specific site-direct synthetic inhibitors, although a structural optimization is required if nanomolar affinity (typical of many drugs) and higher bioavailability are desired.

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