Characterization of inhibitory effects of the potential therapeutic inhibitors, benzoic acid and pyridine derivatives, on the monophenolase and diphenolase activities of tyrosinase

Document Type: Original Article


1 Cellular and Molecular Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

2 Department of Biology, Faculty of Basic Sciences, Islamic Azad University of Science and Research, Tehran, Iran

3 Biochemistry and Biophysics Department, National Institute for Genetic Engineering and Biotechnology, Tehran, Iran

4 Department of Clinical Biochemistry and Genetics, Qazvin University of Medical Science, Qazvin, Iran


Objective(s):Involvement of tyrosinase in the synthesis of melanin and cell signaling pathway has made it an attractive target in the search for therapeutic inhibitors for treatment of different skin hyperpigmentation disorders and melanoma cancers.
Materials and Methods: In the present study, we conducted a comprehensive kinetic analysis to understand the mechanisms of inhibition imposed by 2-amino benzoic acid, 4-amino benzoic acid, nicotinic acid, and picolinic acid on the monophenolase and diphenolase activities of the mushroom tyrosinase, and then MTT assay was exploited to evaluate their toxicity on the melanoma cells.
Results: Kinetic analysis revealed that nicotinic acid and picolinic acid competitively restricted the monophenolase activity with inhibition constants (Ki) of 1.21 mM and 1.97 mM and the diphenolase activity with Kis of 2.4 mM and 2.93 mM, respectively. 2-aminobenzoic acid and 4-aminobenzoic acid inhibited the monophenolase activity in a non-competitive fashion with Kis of 5.15 µM and 3.8 µM and the diphenolase activity with Kis of 4.72 µM and 20 µM, respectively.
Conclusion: Our cell-based data revealed that only the pyridine derivatives imposed cytotoxicity in melanoma cells. Importantly, the concentrations of the inhibitors leading to 50% decrease in the cell density (IC50) werecomparable to those causing 50% drop in the enzyme activity, implying that the observed cytotoxicity is highly likely due to the tyrosinase inhibition. Moreover, our cell-based data exhibited that the pyridine derivatives acted as anti-proliferative agents, perhaps inducing cytotoxicity in the melanoma cells through inhibition of the tyrosinase activities.


1. Lerch K. Copper monoxygenases: tyrosinase and dopamine beta-monoxygenase. In: Sigel H (ed).  Metal ions in biological systems.  vol 13. New York: Marcel Dekker; 1981; pp 143–186.

2. Robb DA. Tyrosinase. In: Lontie R (ed). Copper Proteins and Copper Enzymes, vol 2. Boca Raton, FL: CRC Press; 1984, pp. 207–240.

3. Espin  JC, Varon R, Fenol LG, Gilabert MA, Garcia-Ruiz PA, Tudela J, et al. Kinetic characterization of the substrate specificity and mechanism of mushroom tyrosinase. Eur J Biochem 2000; 267:1270-1279.

4. Rodriguez-Lopez JN, Tudela J, Varon R, Garcia-Carmona F, Garcia-Caآnovas F. Analysis of a kinetic model for melanin biosynthesis pathway. J Biol Chem 1992; 267:3801-3810.

5. Sanchez-Ferrer A, Rodriguez-Lopez JN, Garcia-Canovas F, Garcia-Carmona F. Tyrosinase: a comprehensive review of its mechanism. Biochim Biophys Acta 1995; 1247:1-11.

6. Funyama M, Arakawa H, Yamamoto R, Nishino T, Shin T, Murao S. Effects of R. â arbutin on the activity of tyrosinase from mushroom and mouse melanoma. Biosci Biotechnol Biochem 1995; 59:143-144.

7. Passi S, Nazzarro-Porro M. Molecular basis of substrate and inhibitory of tyrosinase: phenolic compounds. Br J Dermatol 1981; 104:659-665.

8. Cabanes J, Chazarra S, Garcia-Carmona F. Kojic acid, A cosmetic skin whitening agent, is a slow-binding inhibitor of catecholase activity of tyrosinase. J Pharm Pharmacol 1994; 46:982-985.

9. Jiménez  M, Chazarra S, Escribano J, Cabanes J, García-Carmona F. Competitive inhibition of mushroom tyrosinase by 4-substituted benzaldehydes. J Agric Food Chem 2001; 49:4060-4063.

10. Kubo I, Kinst-Hori I. 2-Hydroxy-4-methoxy benzaldehyde: a potent tyrosinase inhibitor from African medicinal plants. Planta Med 1999; 65:19-22.

11. Wang Q, Shi Y, Song KK, Guo HY, Qiu L, Chen QX.  Inhibitory effects of 4-halobenzoic acids on the diphenolase and monophenolase activity of mushroom tyrosinase. Protein J 2004; 23:303-308

12. Qiao C, Qing-Xi C, Ling Q, Kang-Kang S, Huang H. Inhibitory effect of 4-cyanobenzaldehyde and 4-cyanobenzoic acid on mushroom (Agaricus bisporus) tyrosinase. J Protein Chem 2003; 22:607-612.

13. Bubacco L, Vijgenboom E, Gobin C, Tepper AW, Salgado J, Canters GW.  Kinetic and paramagnetic NMR investigations of the inhibition of Streptomyces antibioticus tyrosinase. J Mol Catal B–Enzym 2000; 8:27-35.

14.  Ha YM, Chung SW, Song S, Lee H, Suh H, Chung HY. 4-(6-Hydroxy-2-naphthyl)-1,3-bezendiol: A potent, new tyrosinase inhibitor. Biol Pharm  Bull 2007; 30:1711-1715.

15. Kubo I, Kinst-Hori I. Tyrosinase inhibitory activity of the olive oil flavor compounds. J Agric Food Chem1999; 47: 4574-4578.

16. Conrad JS, Dawso SR, Hubbard ER, Meyers TE, Strothkamp KG. Inhibitor binding to the binuclear active site of tyrosinase: temperature, pH and solvent deuterium isotope effects. Biochemistry 1994; 33:5739-5744.

17. Menon S, Fleck  RW, Yong G, Strothkamp  KG. Benzoic acid inhibition of the alpha-isozyme, beta-isozyme, and gammaisozyme of Agaricus bisporus tyrosinase. Arch Biochem Biophys 1990; 280:27-32.

18. Haghbeen K, Babaei KM, Saeid nematpour F, Gheibi N, Fazli, M, Alijanianzadeh M, Zolghadri jahromi S, Sariri R. surveying allosteric cooperativity and cooperative inhibition in mushroom tyrosinase. J Food Biochem 2010; 34:2, 308-328.

19. Gheibi N, Saboury AA, Haghbeen K, Rajaei F, Pahlevan AA.  Dual effects of aliphatic carboxylic acids on cresolase andcatecholase reactions of mushroom tyrosinase. J Enzyme Inhib Med Chem 2009; 24(5): 1076–1081.

20. Gheibi N, Saboury AA, Haghbeen K, Moosavi-Movahedi AA. Activity and structural changes of mushroom tyrosinase induced by n-alkyl sulfates; Coll Surf B: Biointerfaces 2005; 45:104-107.

21. Doyle A, Griffiths JB (ed). Cell and tissue culture, Laboratory procedures in biotechnology. Chichester: John Wiley & Sons; 1998.

22. Yeldjou C, Moree P, Techounwou PB. Dose and time-dependent response of human leukemia (HL-69) cells to Arsenic trioxide treatment. Int  J  Environ Res Public Health 2006;3:136-140.

23. Kermasha S, Goetghebeur M, Monfette A, Metchet M, Rovelt M. Inhibitory effects of cysteine and aromatic acids on tyrosinase activity. Phytochemistry 1993; 34:349-353.

24. Chang TS. An updated review of tyrosinase inhibitors. Int J Mol Sci 2009; 10:2440-2475.

25. Palumbo A, d’Ischia  M, Misuraca G, Prota G. Mechanism of inhibition of melanogenesis by hydroquinone. Biochim  Biophys Acta 1991; 1073:85-90.

26. Ohyama Y, Mishima Y. Melanogenesis inhibitory effects of kojic acid and its action mechanism. Fragrance J 1990; 6:53-57.

27. Chen QX, Kubo I. Kinetics of mushroom tyrosinase inhibition by quercetin. J Agric Food Chem 2002; 50: 4108-4112.

28. Gude RP, Ingle AD, Rao SGA. In vitro & in vivo studies of nicotinic acid in experimental metastasis model of B16F10 melanoma. Indian J Pharm Sci 1999; 61:287-292.

29. Ramchandran C, Peter KV, Gopalakrishnan PK. Drumstick (Moringoliefera) A multipurpose Indian vegetable. Econ Bot 1980; 34:276-283.

30. Ivanovic I, Grguric-Š S. X-Ray structure and cytotoxic activity of a picolinate ruthenium (II)–arene

complex. J Serb Chem Soc 2011; 76:53-61.

31. Xavier S, Macdonald S, Roth J, Caunt M, Akalu A, Morais D, et al. The vitamin-like dietary supplement para-aminobenzoic acid enhances the antitumor activity of ionizing radiation. Int J Radiat Oncol Biol Phys 2006; 2:517-527.

32. Bridges JW, French MR, Smith RL, Williams RT. The fate of benzoic acid in various species. Biochem J 1970; 118:47-51.