Biomacromolecules 2004, 5, 1633-1636
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Communications Amplification of Inhibitory Activity of Catechin against Disease-Related Enzymes by Conjugation on Poly(E-lysine) Noriko Ihara, Sarah Schmitz, Motoichi Kurisawa, Joo Eun Chung, Hiroshi Uyama,* and Shiro Kobayashi* Department of Materials Chemistry, Graduate School of Engineering, Kyoto University, Kyoto, 615-8510 Japan Received March 25, 2004; Revised Manuscript Received May 28, 2004
A new inhibitor against disease-related enzymes, collagenase, hyaluronidase, and xanthine oxidase, has been developed by the laccase-catalyzed conjugation of catechin on poly(-lysine). The resulting poly(lysine)-catechin conjugate showed greatly improved inhibition effects on activity of these enzymes, whereas the catechin monomer showed very low inhibition activity. The kinetic analysis on the inhibition of collagenase exhibited that the conjugate was a mixed-type inhibitor. The amplified activities might offer high potential as a therapeutic agent for prevention of various enzyme-related diseases. Polypeptides and related artificial poly(amino acid)s have significantly become important due to their specific properties such as biocompatibility and biodegradability. Poly(-lysine) (PL) is produced from culture filtrates from Streptomyces albulus1 and shows high water-solubility and biodegradability.2 Furthermore, PL exhibited good antimicrobial activity against Gram-positive and negative bacteria;3 thus, it is widely used as an additive in the food industry. Green tea catechins, belonging to the group of flavonoids, exhibit biological and pharmacological effects including antioxidant, antimutagenic, anticarcinogenic, antimicrobial, and antiinflammatory properties in numerous human, animal, and in vitro studies.4 These properties might potentially be beneficial in preventing disease and protecting the stability of the genome. Many of these activities have been related to their antioxidant actions.5 They are also known to be potent inhibitors for several enzymes closely related to diseases.6 However, such low-molecular weight flavonoids often do not possess physiological properties enough for biomedical applications. In contrast, a relatively high-molecular fraction of extracted plant polyphenols was reported to exhibit enhanced physiological properties such as antioxidant and anticarcinogenic activity.7 From these perspectives, we have designed not only polymerized flavonoids but also polymeric flavonoid-conjugates in consideration of the amplification of physiological properties of the flavonoids.8-10 Recently, we reported that poly(catechin) as one of the strategic molecular designs was synthesized by peroxidase-catalyzed oxidative coupling and exhibited great improvement in * To whom correspondence should be addressed. Tel: +81-75-383-2460 (H.U.); +81-75-383-2459 (S.K.). Fax: +81-75-383-2461. E-mail: uyama@ mat.polym.kyoto-u.ac.jp (H.U.);
[email protected] (S.K.).
radical scavenging activity, protection effects against lowdensity lipoprotein oxidation, and inhibition effects on xanthine oxidase activity, comparing with a catechin monomer.8 Furthermore, laccase-catalyzed polymerization of rutin produced a water-soluble flavonoid polymer exhibiting much higher scavenging activity of superoxide anion than the rutin monomer.9 Enzymatic conjugation of phenols onto amine-containing polymers has been developed. Tyrosinase catalyzed a coupling of several phenols including catechin with chitosan to produce functional materials based on the biopolymer.11,12 The formation of a Michael-type adduct and/or Schiff base was proposed in the tyrosinase-catalyzed oxidation of catechin with chitosan.12 We have covalently conjugated catechin to poly(allylamine) by a similar enzymatic conjugation technique, and succeeded in conferring antioxidant activity to poly(allylamine) with slightly enhanced antioxidant activity against oxidation of human low-density lipoprotein (LDL), compared with unconjugate catechin.13 Furthermore, the enzymatically synthesized gelatin-catechin conjugate also showed a good antioxidant property against LDL oxidation induced by a free radical.14 Furthermore, the enzymatic conjugation of catechin with amine-containing polymer particles produced the antioxidant microspheres showing good radical scavenging activity.15 In this study, we have developed a new enzyme inhibitor, a PL-catechin conjugate, by the conjugation of flavonoid on a polypeptide (Scheme 1). The synthesized PL-catechin conjugate showed highly amplified inhibition effects against disease-related enzymes, collagenase, hyaluronidase, and xanthine oxidase. To our best knowledge, this is the first example demonstrating high inhibitory activity of a polymeric conjugate of flavonoid for various disease-related enzymes.
10.1021/bm049823x CCC: $27.50 © 2004 American Chemical Society Published on Web 08/04/2004
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Scheme 1
Matrix metalloproteinases (MMPs), typically collagenase and gelatinase, are a family of zinc containing enzymes which degrade and remodel structural proteins in the extracellular matrix (ECM). Since MMPs play an essential role in the homeostasis of ECM, an imbalance in their expression or activity may have important consequences in various pathologies. Thus, MMPs have recently become interesting targets for drug design in the search of novel anticancer, antiarthritis, and other pharmacological agents useful in the management of inflammatory processes.16 Like MMPs, bacterial collagenase from Clostridium histolyticum (ChC) has also been extensively investigated. This enzyme hydrolyzes the triple helical region of collagen under physiological conditions and was reported to be involved in the pathogenicity related to degradation of a connective tissue. So far, several inhibitors of ChC have been developed and the quantitative structure-activity relationship was demonstrated.17 Herein, we examined the ChC inhibitory activity of the PL-catechin conjugate according to the literature method.18 The conjugate was prepared by the laccasecatalyzed reaction of catechin with PL, a slightly modified method for the synthesis of poly(allylamine)-catechin conjugate.13,19 The conjugate showed greatly amplified concentrationdependent inhibition activity against ChC on the basis of
Figure 1. Inhibition activity of catechin and poly(-lysine)-catechin conjugate (catechin content ) 3.4%) against collagenase, n ) 3.
the catechin unit (Figure 1), which is considered to be due to effective multivalent interaction between ChC and the catechin unit in the conjugate.8,10 These data clearly indicate that the conjugation of catechin on PL greatly amplifies the inhibitory activity of catechin against ChC. The type of inhibition by the conjugate was analyzed by Lineweaver-Burk plots in the steady state (Figure 2). The reciprocal plots of the substrate concentration and hydrolysis rate with different concentrations of the conjugate intersected at the left side of the 1/velocity axis above the 1/substrate axis. This result suggests that the conjugate is a mixed-type inhibitor.20 The Ki value is estimated as 23 µM. Hyaluronidase is an enzyme which catalyzes hydrolysis of hyaluronic acid and is often involved in a number of physiological and pathological processes. Potent hyaluronidase inhibitors have antiallergic effects, which may lead to development of new antiallergic agents.21,22 Here, we evaluated antihyaluronidase activity of the conjugate according to the literature.21 The efficient inhibition activity of the conjugate was found (Figure 3), whereas the monomeric catechin showed almost negligible inhibition effect over a range of tested concentrations. No inhibition activity of PL toward hyaluronidase was found. Procyanidine, a catechin oligomer, was reported to exhibit the good inhibition effect against hyaluronidase;18 however, the inhibition activity of procyanidine was inferior to that of the present conjugate.
Figure 2. Lineweaver-Burk plots for the inhibition of collagenase by poly(-lysine)-catechin with different concentration: ([) 0 µM; (9) 10 µM; (2) 20 µM; (b) 50 µM.
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Figure 3. Inhibition activity of catechin and poly(-lysine)-catechin conjugate (catechin content ) 3.4%) against hyaluronidase, n ) 3.
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that the XO inhibitory activity of catechin is greatly amplified by the enzymatic reaction leading to the polymeric flavonoids. In conclusion, a new disease-related enzyme inhibitor, the poly(-lysine)-catechin conjugate, was developed. The conjugate showed great amplification of inhibitory activity against collagenase, hyaluronidase, and xanthine oxidase on the basis of the catechin unit, compared to intact catechin. We believe that the present conjugate may be useful for a therapeutic agent to offer protection against a wide range of enzyme-related diseases including cardiovascular diseases, cancer invasion and metastasis, arthritis, atherogenesis, and gout. Bioavailability and in vivo and in vitro toxicity of the conjugate should be examined for the application of therapic agents. Moreover, we preliminarily found that the PLcatechin conjugate exhibited good antimicrobial activity, comparable with unmodified PL. Further investigation including applications of the present conjugate and related polymeric flavonoids for biomedical purposes is under way in our laboratory. Acknowledgment. This work was supported by Program for the Promotion of Basic Research Activities for Innovative Bioscience. We acknowledge the gift of PL and laccase from Chisso Co. and Novozymes Japan Ltd., respectively. S.S. is grateful to the DAAD program, Germany, for the fellowship. References and Notes
Figure 4. Inhibition activity of catechin and poly(-lysine)-catechin conjugate (catechin content ) 3.1%) against xanthine oxidase, n ) 3.
These data suggest that the conjugation on a polymer more efficiently amplified the inhibitory action of catechin against hyaluronidase. The superoxide radical is a reactive oxygen species, which is formed during normal aerobic metabolism and by activated phagocytes.23 Reduction of molecular oxygen to superoxide by xanthine oxidase (XO), generating hydroxyl radicals and uric acid, is an important physiological pathway. However, an excess of superoxide radicals damages biomacromolecules both directly and indirectly by forming hydrogen peroxide or highly reactive hydroxyl radicals. Thus, XO is an important biological source of reactive oxygen species; XO is an enzyme responsible for the formation of uric acid associated with gout leading to painful inflammation in the joints.24 Figure 4 showed XO inhibitory activity assessed by evaluating uric acid formation from XO.25 The XO inhibition effect of catechin was negligible at a concentration below 300 µM, on the other hand, the conjugate exhibited an increase in XO inhibitory activity as an increasing concentration of the catechin unit. PL exhibited no inhibition activity toward XO. Poly(catechin), prepared by the enzymatic oxidative coupling, also showed high inhibition activity against XO; however, the inhibition activity of the conjugate was superior to that of poly(catechin).8 These data suggest
(1) Shima, S.; Sakai, H. Agric. Biol. Chem. 1977, 41, 1807. (2) Kunioka, M.; Choi, H. J. J. Appl. Polym. Sci. 1995, 58, 801. (3) Shima, S.; Matsuoka, H.; Iwamoto, T.; Sakai, H. J. Antibotics 1984, 37, 1449. (4) (a) Jankun, J.; Selman, S. H.; Swiercz, R.; Skrzypczak-Jankun, E. Nature 1997, 387, 561. (b) Bordoni, A.; Hrelia, S.; Angeloni, C.; Giordano, E.; Guarnieri, C.; Caldarera, C. M.; Biagi, P. L. J. Nutr. Biochem. 2002, 13, 103. (c) Nakagawa, K.; Ninomiya, M.; Okubo, T.; Aoi, N.; Juneja, L. R.; Kim, M.; Yamanaka, K.; Miyazawa, T. J. Agric. Food Chem. 1999, 47, 3967. (5) (a) Jovanovic, S. V.; Steenken, S.; Tosic, M.; Marjanovic, B.; Simic, M. G. J. Am. Chem. Soc. 1994, 116, 4846. (b) Yen, G. C.; Chen, H. Y. J. Agric. Food Chem. 1995, 43, 27. (6) (a) Lin, J.-K.; Chen, P.-C.; Ho, C.-T.; Lin-Shiau, S.-Y. J. Agric. Food Chem. 2000, 48, 2736. (b) Sartor, L.; Pezzato, E.; Dell′Aica, I.; Caniato, R.; Biggin, S.; Garbisa, S. Biochem. Pharm. 2002, 64, 229. (7) (a) Ariga, T.; Hamano, M. Agric. Biol. Chem. 1990, 54, 2499. (b) Saito, M.; Hosoyama, H.; Ariga, T.; Kataoka, S.; Yamaji, N. J. Agric. Food Chem. 1998, 46, 1460. (c) Hagerman, A. E.; Riedl, K. M.; Jones, G. A.; Sovik, K. N.; Ritchard, N. T.; Hartzfeld, P. W.; Riechel, T. L. J. Agric. Food Chem. 1998, 46, 1887. (8) (a) Kurisawa, M.; Chung, J. E.; Kim, Y. J.; Uyama, H.; Kobayashi, S. Biomacromolecules 2003, 4, 469. (b) Kurisawa, M.; Chung, J. E.; Kim, Y. J.; Uyama, H.; Kobayashi, S. Macromol. Biosci. 2003, 3, 758. (c) Kurisawa, M.; Chung, J. E.; Uyama, H.; Kobayashi, S. Chem. Commun. 2004, 294. (9) Kurisawa, M.; Chung, J. E.; Uyama, H.; Kobayashi, S. Biomacromolecules 2003, 4, 1394. (10) (a) Kim, Y. J.; Chung, J. E.; Kurisawa, M.; Uyama, H.; Kobayashi, S. Macromol. Chem. Phys. 2003, 204, 1863. (b) Chung, J. E.; Kurisawa, M.; Kim, Y. J.; Uyama, H.; Kobayashi, S. Biomacromolecules 2004, 5, 113. (c) Kim, Y. J.; Chung, J. E.; Kurisawa, M.; Uyama, H.; Kobayashi, S. Biomacromolecules 2004, 5, 474. (d) Kim, Y. J.; Chung, J. E.; Kurisawa, M.; Uyama, H.; Kobayashi, S. Biomacromolecules 2004, 5, 547. (11) (a) Kumar, G.; Smith, P. J.; Payne, G. F. Biotechnol. Bioeng. 1999, 63, 154. (b) Chen, T.; Kumar, G.; Harris, M. T.; Smith, P. J.; Payne G. F. Biotechnol. Bioeng. 2000, 70, 564. (12) Wu, L.-Q.; Embree, H. D.; Balgley, B. M.; Smith, P. J.; Payne, G. F. EnViron. Sci. Technol. 2002, 36, 3446.
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(13) Chung, J. E.; Kurisawa, M.; Tachibana, Y.; Uyama, H.; Kobayashi, S. Chem. Lett. 2003, 32, 620. (14) Ihara, N.; Tachibana, Y.; Chung, J. E.; Kurisawa, M.; Uyama, H.; Kobayashi, S. Chem. Lett. 2003, 32, 816. (15) Chung, J. E.; Kurisawa, M.; Uyama, H.; Kobayashi, S. Biotechnol. Lett. 2003, 25, 1993. (16) Whittaker, M.; Floyd, C. D.; Brown, P.; Gearing, A. J. H. Chem. ReV. 1999, 99, 2735. (17) (a) Scozzafava, A.; Supuran, C. T. J. Med. Chem. 2000, 43, 1858. (b) Gupta, S. P.; Kumaran, S. Bioorg. Med. Chem. 2003, 11, 3065. (18) Facino, R. M.; Carini, M.; Aldini, G.; Bombardelli, E.; Morazzoni, P.; Morelli, R. Arzneim.-Forsch. 1994, 44, 592. (19) A typical procedure of the conjugate production is as follows. PL (1.65 g, 10 mmol of monomer unit) was dissolved in 40 mL of water. The pH of the solution was adjusted at 7 or 8 by adding 2 N NaOH. Catechin (0.15 g, 0.50 mmol) in 4 mL of methanol was added to the solution. The reaction was started by the addition of laccase solution (4 µL, 4 units). After the reaction, the mixture was acidified by the addition of 6N HCl and subjected to purification by dialysis (cutoff
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(20) (21) (22) (23) (24) (25)
molecular weight ) 5 × 102) four times. The remaining solution was lyophilized to give the conjugate. The conjugate formation was confirmed by UV spectroscopy; a broad peak around 430 nm newly appeared by the conjugation.12 The catechin content in the conjugate was determined by elemental analysis and the enzyme inhibition was measured on the basis of the catechin unit. Yokochi, N.; Morita, T.; Yagi, T. J. Agric. Food Chem. 2003, 51, 2733. Fujitani, N.; Sakaki, S.; Yamaguchi, Y.; Takenaka, H. J. Appl. Phycol. 2001, 13, 489. Mio, K.; Stern, R. Matrix Biol. 2002, 21, 31. Fantone, J. C.; Ward, P. A. Hum. Pathol. 1985, 16, 973. Rastelli, G.; Costantino, L.; Albasini, A. J. Am. Chem. Soc. 1997, 119, 3007. Noro, T.; Oda, Y.; Miyase, T.; Ueno, A.; Fukushima, S. Chem. Pharm. Bull. 1983, 31, 3984.
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