Plant Phenolics Inhibit Neutrophil Elastase

• Abstract
• Introduction
• Materials and Methods
• Materials and test compounds
• Elastase assays
• Cell culture and ATP assay
• Ligand docking calculations
• Statistical analysis
• Results and Discussion
• Acknowledgements
• References

Abstract

Human neutrophil elastase (HNE) is a serine protease, which is present in its active form in inflamed tissue as well as in psoriatic lesions. In extension of our research on natural compounds as inhibitors of HNE or of its release, several phenolics of different size were tested. The ellagitannins agrimoniin and pedunculagin were the most potent direct HNE inhibitors (IC₅₀ = 0.9 and 2.8 μM, respectively). Ligand docking calculations provided evidence that inhibition may occur in an unspecific manner. Agrimoniin also showed anti-proliferative effects in the ATP assay (IC₅₀ = 3.2 μM), suggesting that this type of tannin could have beneficial effects in the treatment of diseases such as psoriasis. Tests with other phenolics combined with ligand docking experiments revealed that, besides the presence of ortho-dihydroxy groups, a specific lipophilic shape is necessary for an inhibitory activity. The phenolic genistein deserves special interest as an inhibitor of elastase release because its effect was remarkably potent (IC₅₀ = 0.6 μM).

Introduction

Human neutrophil elastase (HNE) is a serine protease which plays an important role in physiological functions of the immune system. Intracellular HNE digests the proteins of invading bacteria, whereas extracellular HNE, secreted by neutrophils, assists neutrophil migration to the site of inflammation by degrading normal skin constituents such as keratin, elastin and different types of collagen [1], [2]. Under normal physiological conditions endogenous inhibitors protect healthy tissue from damage by elastase. However, in inflamed tissue an impaired balance between the enzyme and its natural inhibitors exists resulting in an abnormal turnover of connective tissue proteins. Therefore, this enzyme has been described as an important pathogenic factor in inflammatory diseases such as rheumatoid arthritis or cystic fibrosis [2]. Moreover, active HNE is detected in psoriatic lesions and induces keratinocyte hyperproliferation via the EGFR signalling pathway [3], [4]. Thus, HNE is not only involved in tissue degradation, but is also an important regulator of inflammatory processes. Hence, its inhibition could be an interesting therapeutic approach in diseases like psoriasis.

We here report the investigation of nine natural phenolic compounds of different molecule sizes (for structure see Figs. [1] and [2]) on their direct or release effect on HNE. The ellagitannins agrimoniin (1) and pedunculagin (2), being constituents from Tormentillae rhizoma, were chosen because fractions from this plant extract were recently proven to exhibit anti-elastase activity [5], but no studies were carried out with the isolated compounds. In order to investigate the influence of the molecular size on elastase activity, we compared the effects of the large ellagitannins (1) and (2) with those of smaller molecules such as epigallocatechin gallate (EGCG) (3), a well-known constituent from green tea [6], and ent-epicatechin-(4α-8)-ent-epicatechin (6). The other molecules with a smaller size (4, 5, 7, 8, 9) were studied to continue our recently published structure-activity relationship analysis with derivatives consisting of a cinnamic acid moiety and a lipophilic residue [7]. Ligand docking calculations were undertaken to get insights into the way in which these phenolics directly target HNE. Moreover, the potential anti-proliferative effect of two phenolics (1 and 3) on keratinocytes was evaluated in this study.

Materials and Methods

Materials and test compounds

ent-Epicatechin-(4α-8)-ent-epicatechin was isolated from Byrsonima crassifolia Kunth [8], agrimoniin and pedunculagin from Alchemilla xanthochlora Rothmaler [9]. Resveratrol, 4,3′,5′-triacetoxystilbene (TAS), EGCG, genistein, tyrosol and hydroxytyrosol were provided by DSM Nutritional Products (Kaiseraugst, Switzerland). Purity was evaluated by HPLC/MS or TLC and was > 96 %. Stock solutions were prepared in DMSO, which was restricted to 0.2 %, for the HNE assay. For the proliferation assay compounds were dissolved in ethanol (70 % v/v). Further dilutions were made with phosphate-buffered saline (PBS). The maximal final ethanol concentration did not exceed 1 % and was not toxic to the cells. Enzyme substrate Suc-Ala-Ala-Val-p-nitroaniline and PAF were purchased from Bachem (Bubendorf, Switzerland), cytochalasin B was from Sigma (St. Louis, MO, USA).

Elastase assays

The assay on the direct inhibition of HNE as well as on inhibition of HNE release was performed as previously reported [10]. Both assays are described in detail in the Supporting Information.

In brief, neutrophils were isolated from fresh blood of healthy adult volunteers (description given in Supporting Information), which was done in the morning to avoid basal neutrophil stimulation [10]. This was followed either by incubation with cytochalasin B and platelet-activating factor (PAF) to release elastase and subsequent incubation of the supernatant with the test compound and enzyme substrate (direct HNE inhibition) or by incubation of the neutrophils with a solution of cytochalasin B, stimulant, elastase substrate and the test compound (inhibition of HNE release). In both cases the reaction was stopped with citric acid and the released product was measured photometrically at 405 nm.

Cell culture and ATP assay

The spontaneously transformed human keratinocyte cell line HaCaT [11] was grown in Dulbecco’s modified Eagle’s medium supplemented with 10 % FCS, 1 % L-glutamine and 1 % penicillin/streptomycin (all from Gibco; Eggenstein, Germany) in a humidified atmosphere (5 % CO₂, 37 °C). The cells were grown to subconfluency, washed twice with PBS, resuspended in medium with 1 % FCS and further cultured (10⁴ cells/well in 100 μL) in 96-well microtiter plates (Costar; Cambridge, MA, USA). The compounds were added to the cells at various concentrations. Cells were incubated for 24 h and cell proliferation was determined by the ViaLight Plus cell proliferation and cytotoxicity bioassay kit (BioWhittaker; Verviers, Belgium). This assay is based on the bioluminiscent measurement of the ATP that is present in metabolically active cells. The bioluminiscent method utilises luciferase which catalyses the formation of light from ATP and luciferin. The emitted light intensity is linearly related to the ATP concentration and is measured using a luminometer (Sirius HT-TRF microplate reader; MWG; Ebersberg, Germany). Data are expressed as mean ± S.D. of triplicate measurements.

Ligand docking calculations

Docking calculations were carried out on a Linux Workstation with the program FlexX 1.13 (BioSolveIt), a docking tool designed for the placement of chemical compounds into protein binding sites via matching of complementary interactions like, e. g., H-bonds (FlexX is described in detail in [12], [13]). The X-ray structure of HNE (PDB code: 1HNE) was used [14] and its binding site was determined with the aid of the molecule viewer VMD. All amino acids were included with at least one atom lying within an 8 Å distance from the γ-OH of Ser195, which belongs to the catalytic triad. Binding site hydrogen atoms were added and energy-minimised using What-If [15]. The ligands were sketched by hand, converted to 3D-structures and energy-minimised using the MM+ forcefield of the program Hyperchem as in [16]. For the very large ligand compounds 1 and 2, the selected binding site proved to be too small. Therefore, unrestricted docking to the whole enzyme instead of a preselected binding site was also performed for these compounds.

Statistical analysis

All assays were performed at least in triplicate in at least two experiments. Inhibition rates were calculated relative to the activated cells (cells without inhibitor) in percent. Statistical analysis was performed using Graphpad Prisms 4 software. The results are expressed as the mean values ± SD or in the case of the IC₅₀ values as the mean values ± standard errors.

Results and Discussion

The phenolics 1 - 9 were studied for their effect on the activity of elastase and for their effect on elastase release from PAF-stimulated neutrophils which were isolated from fresh human blood [10], [16]. Agrimoniin (1) and pedunculagin (2) with a high molecular weight and a high number of phenolic groups, had no effect on HNE release, but were strong direct inhibitors (IC₅₀ = 0.9 and 2.8 μM, respectively, see Table [1]). In contrast, EGCG (3) was less active with an IC₅₀ of 25.3 μM. Since similar values were obtained in the elastase inhibition and the elastase release assay, it was concluded that these three compounds had no effect on elastase release (see Table [1]). Docking results revealed that the tannins 1 and 2 are too large to fit into the elastase binding site and cannot form specific interactions with binding site residues. Therefore, inhibition may be due to coverage of the hydrophobic S1 pocket and active site, as shown in Fig. [3], or may occur in an unspecific manner, as supposed from the existence of the numerous phenolic hydroxy groups. Additionally, it can also be speculated that astringent effects of these tannins may participate in the inhibitory effect on HNE, as the relative astringency (RA) of 1 is high [17].

Whereas the ellagitannins 1 and 2 were investigated for their effect on HNE for the first time, the IC₅₀ value of EGCG (3) contrasts previous studies where an IC₅₀ of 0.4 μM was reported [18]. This might be due to different incubation times of the compound with HNE (30 min in this study compared to 60 min). Recently, it was shown that EGCG also inhibits further pro-inflammatory mediators, such as matrix metalloproteinases or COX-2 [6], [18]. This study reveals an additional anti-inflammatory property of EGCG: the inhibition of elastase activity is supposed to limit excessive elastase-triggered tissue erosion in acute and chronic inflammatory reactions.

Other substances did not inhibit elastase but they dose-dependently impaired its release from neutrophils. Resveratrol (4) and genistein (5) were the most active compounds with IC₅₀ values of 12 and 0.5 μM, respectively (see Table [1]). IC₅₀ values were much lower than those published recently (IC₅₀ = 31 and 99 μM); this may be due to the different incubation times used in the assay [19], [20]. Moreover, it cannot be excluded that the physiological status of the cells may also influence the result. Both compounds are tyrosine kinase inhibitors. This common feature might explain that they both inhibit HNE release [3], [19], [21]. The other phenolics showed low effects on HNE release (i. e., < 50 % inhibition at all concentrations tested) and calculation of an IC₅₀ value was impossible. Therefore, the percentage of inhibition of HNE release at a compound concentration of 50 μM is shown (Table [2]). With regard to resveratrol (4) and one of its derivatives, TAS (7), triacetylation virtually abolished the inhibitory property of resveratrol on HNE release. EE (6) reduced HNE activity to 14.4 % at this concentration.

Altogether, none of the phenolics 4, 5, 7 and 8 directly inhibited HNE confirming our recent studies that the structure element of aromatic ortho-dihydroxy groups combined with a lipophilic residue seems to be a prerequisite for an optimal binding within the active site [7].

Surprisingly, compound 9 possessing aromatic ortho-dihydroxy groups and a lipophilic residue failed to inhibit HNE and had a low effect on its release (12.3 % at 50 μM). The corresponding dehydroxy derivative (8) was slightly more active (34.4 % at 50 μM). Therefore, we infer that catecholic structure elements in lipophilic compounds are necessary, but not sufficient to directly target the active site of HNE [7], [22], but a certain shape, such as a bornyl residue, may be an essential structural prerequisite for an inhibitory activity. Ligand docking calculation confirmed this assumption (Table [1]S Supporting Information). Compared to bornyl caffeate (IC₅₀ = 1.6 μM, [23]) a lower number of placements into the active site of HNE was obtained for compounds (4, 5, 7, 8 and 9) [7]. Again, the calculated binding free energies did not significantly correlate with the experimentally determined data. It is well known that ligand docking calculations are much better suited to produce correct binding geometries than binding free energies [24]. Altogether, the ligand docking calculations presented here can be used at best as an additional tool for the identification of elastase inhibitors by visualising their putative binding modes. A more obvious and convincing correlation between experimental and docking data may be desirable. This could be accomplished by utilising computationally more demanding approaches like molecular dynamics calculations in addition to docking [24]. Standards which should be recommended for the respective assay for a better comparison are also necessary considering the great variability of effects that are often reported. Nevertheless, because of its potent effects on HNE, genistein is a most promising compound. Since it exerts many additional biological activities [25], it appears to be suitable both for topical and oral therapeutic use in dermatology.

As mentioned above inhibition of elastase may reduce keratinocyte proliferation via inhibition of epidermal growth factor receptor activation [3], [4]. However, further studies, such as measuring the effect of these phenolics on the HNE induced release of the transforming growth factor alpha (TGF-α) have to be carried out to verify this hypothesis. To investigate whether phenolics also influence proliferation of keratinocytes due to effects on other signalling pathways the most active direct elastase inhibitor (1) as well as (3) were studied in the ATP assay using HaCaT, a transformed human keratinocyte cell line. We used this non-radioactive assay because it was shown that ATP bioluminescence can be a suitable substitute for tritiated thymidine uptake as a measure of cell proliferation [26], [27]. Both compounds inhibited ATP in a concentration-dependent manner. Yet, 1 displayed a significant reduction in ATP synthesis at low concentrations (IC₅₀ = 3.4 μM, see Fig. [4]), whereas EGCG was only active at high concentrations (IC₅₀ = 106 μM, data not shown). Although the ATP assay showed a good correlation with the [³ H]thymidine incorporation [27] it cannot be excluded that reduction of the ATP level may also be due to or influenced by cytotoxic effects. Therefore, these results may be preliminary and the anti-proliferative effect has to be confirmed by further experiments. Nevertheless, these results extend our knowledge on the anti-inflammatory activity of ellagitannin 1 [17]. Its anti-inflammatory effects have already been shown in vivo [17], but, to date, no data exist on the bioavailability and metabolism of ellagitannins after topical application. However, pharmacokinetic parameters of pure EGCG administered topically in form of a hydrophilic ointment to human and mouse skin were already determined [28]. A substantial intradermal uptake of up to 1 - 20 % of the applied dose was observed. Therefore, it might be possible that also agrimoniin (1) displays anti-inflammatory effects, at least in the stratum corneum of inflammatory skin diseases. This may be of relevance, especially in psoriasis, where viable parakeratotic keratinocytes occur up to the outermost surface of the stratum corneum [1].

Acknowledgements

We are grateful to Prof. Rimpler for providing the tannins, to Dr. Marcel Geyer for collecting blood and to all blood donors.

References

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  PubMedSearch in Google Scholar
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Prof. Dr. I. Merfort

Institute for Pharmaceutical Sciences

Department of Pharmaceutical Biology and Biotechnology

University of Freiburg

Stefan-Meier-Str. 19

79104 Freiburg

Germany

Phone: +49-761-203-8373

Fax: +49-761-203-8383

Email: irmgard.merfort@pharmazie.uni-freiburg.de

References

• 1 Wiedow O, Wiese F, Christophers E. Lesional elastase activity in psoriasis - Diagnostic and prognostic-significance.  Arch Dermat Res. 1995;  287 632-5
  PubMedSearch in Google Scholar
• 2 Ohbayashi H. Current synthetic inhibitors of human neutrophil elastase.  Exp Opin Invest Drugs. 2002;  12 65-84
  PubMedSearch in Google Scholar
• 3 Meyer-Hoffert U, Wingertszahn J, Wiedow O. Human leukocyte elastase induces keratinocyte proliferation by epidermal growth factor receptor activation.  J Investig Dermatol. 2004;  123 338-45
  CrossrefPubMedSearch in Google Scholar
• 4 Wiedow O, Meyer-Hoffert U. Neutrophil serine proteases: potential key regulators of cell signalling during inflammation.  J Int Med. 2005;  257 319-28
  PubMedSearch in Google Scholar
• 5 Bos M A, Vennat B, Meunier M T, Pouget M P, Pourrat A, Fialip J. Procyanidins from tormentil: Antioxidant properties towards lipoperoxidation and anti-elastase activity.  Biol Pharm Bull. 1996;  19 146-8
  CrossrefPubMedSearch in Google Scholar
• 6 Shimizu M, Deguchi A, Joe A K, McKoy J F, Moriwaki H, Weinstein I B. EGCG inhibits activation of HER3 and expression of cyclooxygenase-2 in human colon cancer cells.  J Exp Ther Oncol. 2005;  5 69-78
  PubMedSearch in Google Scholar
• 7 Siedle B, Murillo R, Hucke O, Labahn A, Merfort I. Structure activity relationship studies of cinnamic acid derivatives as inhibitors of human neutrophil elastase revealed by ligand docking calculations.  Pharmazie. 2003;  58 337-9
  PubMedSearch in Google Scholar
• 8 Geiss F, Heinrich M, Hunkler D, Rimpler H. Proanthocyanidins with (+)-epicatechin units from Byrsonima crassifolia bark.  Phytochemistry. 1995;  39 635-43
  CrossrefPubMedSearch in Google Scholar
• 9 Geiger C, Rimpler H. Ellagitannins from Tormentillae rhizoma and Alchemillae herba.  Planta Med. 1990;  56 585-6
  PubMedSearch in Google Scholar
• 10 Schorr K, Rott A, Da Costa F B, Merfort I. Optimisation of a human neutrophil elastase assay and investigation of the effect of sesquiterpene lactones.  Biologicals. 2005;  33 175-84
  PubMedSearch in Google Scholar
• 11 Boukamp P, Petrussevska R T, Breitkreutz D, Hornung J, Markham A, Fusenig N E. Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.  J Cell Biol. 1988;  106 761-71
  CrossrefPubMedSearch in Google Scholar
• 12 Kramer B, Rarey M, Lengauer T. Evaluation of the FlexX incremental construction algorithm for protein-ligand dockings.  Proteins. 1999;  37 228-41
  CrossrefPubMedSearch in Google Scholar
• 13 Rarey M, Kramer B, Lengauer T, Klebe G. A fast flexible docking method using an incremental construction algorithm.  J Mol Biol. 1996;  261 470-89
  CrossrefPubMedSearch in Google Scholar
• 14 Navia M A, McKeever B M, Springer J P, Lin T Y, Williams H R, Fluder E M. et al . Structure of human neutrophil elastase in complex with a peptide chloromethyl ketone inhibitor at 1.84-Å resolution.  Proc Natl Acad Sci USA. 1989;  86 7-11
  CrossrefPubMedSearch in Google Scholar
• 15 Vriend G. WHAT IF: a molecular modeling and drug design program.  J Mol Graph. 1990;  8 52-6
  PubMedSearch in Google Scholar
• 16 Siedle B, Cisielski S, Murillo R, Löser B, Castro V, Klaas C A. et al . Sesquiterpene lactones as inhibitors of human neutrophil elastase.  Bioorg Med Chem. 2002;  10 2855-61
  CrossrefPubMedSearch in Google Scholar
• 17 Scholz E. Pflanzliche Gerbstoffe.  Dtsch Apoth Ztg. 1994;  134 17-29
  PubMedSearch in Google Scholar
• 18 Sartor L, Pezzato E, Dell’Aica I, Caniato R, Biggin S, Garbisa S. Inhibition of matrix-proteases by polyphenols: chemical insights for anti-inflammatory and anti-invasion drug design.  Biochem Pharmacol. 2002;  64 229-37
  CrossrefPubMedSearch in Google Scholar
• 19 Rotondo S, Rajtar G, Manarini S, Celardo A, Rotilio D, de Gaetano G. et al . Effect of trans-resveratrol, a natural polyphenolic compound, on human polymorphonuclear leukocyte function.  Brit J Pharmacol. 1998;  123 1691-9
  CrossrefPubMedSearch in Google Scholar
• 20 Tou J S. Differential regulation of phosphatidic acid (PA) formation and degranulation by polyphenolic antioxidants in stimulated human neutrophils.  Faseb J. 2002;  16 A538-9
  PubMedSearch in Google Scholar
• 21 Tuluc F, Garcia A, Bredetean O, Meshki J, Kunapuli S P. Primary granule release from human neutrophils is potentiated by soluble fibrinogen through a mechanism depending on multiple intracellular signaling pathways.  Am J Physiol Cell Physiol. 2004;  287 C1264-72
  CrossrefPubMedSearch in Google Scholar
• 22 Melzig M F, Löser B, Cisielski S. Inhibition of neutrophil elastase activity by phenolic compounds from plants.  Pharmazie. 2001;  56 967-70
  Search in Google Scholar
• 23 Melzig M F, Löser B, Lobitz G O, Tamayo-Castillo G, Merfort I. Inhibition of granulocyte elastase activity by caffeic acid derivatives.  Pharmazie. 1999;  54 712
  PubMedSearch in Google Scholar
• 24 Steinbrecher T, Case D A, Labahn A. A multistep approach to structure-based drug design: studying ligand binding at the human neutrophil elastase.  J Med Chem. 2006;  49 1837-44
  CrossrefPubMedSearch in Google Scholar
• 25 Shen G X, Jeong W S, Hu R, Kong A NT. Regulation of Nrf2, NF-kappaB, and AP-1 signaling pathways by chemopreventive agents.  Antioxid Redox Signal. 2005;  7 1648-63
  CrossrefPubMedSearch in Google Scholar
• 26 Sottong P R, Rosebrock J A, Britz J A, Kramer T R. Measurement of T-lymphocyte responses in whole-blood cultures using newly synthesized DNA and ATP.  Clin Diagn Lab Immunol. 2000;  7 307-11
  CrossrefPubMedSearch in Google Scholar
• 27 Crouch S PM, Kozlowski R, Slater K J, Fletcher J. The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity.  J Immunol Methods. 1993;  160 81-8
  PubMedSearch in Google Scholar
• 28 Dvorakova K, Dorr R T, Valcic S, Timmermann B, Alberts D S. Pharmacokinetics of the green tea derivative, EGCG, by the topical route of administration in mouse and human skin.  Cancer Chemother Pharmacol. 1999;  43 331-5
  CrossrefPubMedSearch in Google Scholar

Prof. Dr. I. Merfort

Institute for Pharmaceutical Sciences

Department of Pharmaceutical Biology and Biotechnology

University of Freiburg

Stefan-Meier-Str. 19

79104 Freiburg

Germany

Phone: +49-761-203-8373

Fax: +49-761-203-8383

Email: irmgard.merfort@pharmazie.uni-freiburg.de

• Supplementary Material