4048
U. Ghani, N. Ullah / Bioorg. Med. Chem. 18 (2010) 4042–4048
10. Asanuma, M.; Miyazaki, I.; Ogawa, N. Neurotox. Res. 2003, 5, 165.
11. Kima, Y. J.; Uyama, H. Cell. Mol. Life Sci. 2005, 62, 1707.
12. Sugumaran, M.; Nelson, E. Arch. Insect Biochem. Physiol. 1998, 38, 44.
13. Amtul, Z.; Rasheed, M.; Choudhary, M. I.; Supino, R.; Khan, K. M.; Atta-ur-
Rahman Biochem. Biophys. Res. Commun. 2004, 319, 1053.
14. Khan, K. M.; Fatima, N.; Rasheed, M.; Jalil, S.; Ambreen, N.; Perveen, S.;
Choudhary, M. I. Bioorg. Med. Chem. 2009, 17, 7816.
microplate reader (Molecular Devices, CA, USA). Inhibitory activity
was determined by the method previously reported.48 All inhibi-
tors were dissolved in DMSO and its final concentration in the
reaction mixture was 3%. Mushroom tyrosinase (28 nM; Sigma
Chemical Co., St. Louis, USA) was preincubated with the com-
pounds in 50 mM sodium phosphate buffer (pH 6.8) for 20 min
15. Chang, T. S. Int. J. Mol. Sci. 2009, 10, 2440.
at 25 °C.
L
-DOPA (final concn 0.5 mM) was added to the mixture
16. Xie, L. P.; Chen, Q. X.; Huang, H.; Wang, H. Z.; Zhang, R. Q. Biochemistry 2003, 68,
487.
17. Kim, Y. M.; Yun, J.; Lee, C. K.; Lee, H.; Min, K. R.; Kim, Y. J. Biol. Chem. 2002, 277,
and the enzyme reaction was continuously monitored by measur-
ing the change in absorbance at 475 nm. Kojic acid was used as
standard inhibitor.
16340.
18. Jones, K.; Hughes, J.; Hong, M.; Jia, Q.; Orndorff, S. Pigment Cell Res. 2002, 15,
335.
19. Sabudak, T.; Khan, M. T.; Choudhary, M. I.; Oksuz, S. Nat. Prod. Res. 2006, 20,
665.
4.3. Determination of Ki values and type of inhibition
20. Devkota, K. P.; Khan, M. T.; Ranjit, R.; Lannang, A. M.; Samreen; Choudhary, M.
I. Nat. Prod. Res. 2007, 21, 321.
21. Khan, M. T.; Choudhary, M. I.; Khan, K. M.; Rani, M.; Atta-ur-Rahman Bioorg.
Med. Chem. 2005, 13, 3385.
22. Liu, J.; Yi, W.; Wan, Y.; Ma, L.; Song, H. Bioorg. Med. Chem. 2008, 16, 1096.
23. Wei, Y.; Cao, R.; Chen, Z.; Yu, L.; Ma, L.; Song, H. Chem. Pharm. Bull. 2009, 57,
1273.
24. Yia, W.; Cao, R.; Wena, H.; Yana, Q.; Zhoua, B.; Wana, Y.; Maa, L.; Song, H.
Bioorg. Med. Chem. Lett. 2008, 18, 6490.
25. Canovas, F. G.; Tudela, J.; Madrid, C. M.; Varon, R.; Carmona, F. G.; Lozano, J. A.
Biochim. Biophys. Acta 1987, 912, 417.
26. Tudela, J.; Canovas, F. G.; Varon, R.; Jimenez, M.; Garciacarmona, F.; Lozano, J. A.
Biophys. Chem. 1988, 30, 303.
The reaction mixture contained 50 mM Na-phosphate buffer
(pH 6.8), 28 nM tyrosinase, 3% DMSO, and various concentrations
of
L-DOPA (0.2–1.0 mM). Tyrosinase was preincubated with
different concentrations of inhibitors for 20 min at 25 °C and the
reaction was initiated by adding substrate. Formation of
DOPAchrome was continuously monitored at 475 nm for 2 min in
the microplate reader.
Initial enzyme reaction velocity (lmol/min/mg) was calculated
using the linear portion of each curve expressed as
D
A475/min. The
rate of the reaction was linear for initial 2 min. Ki values and type
of inhibition were calculated by the method of Dixon28 using Grafit
software (version 4.09, Erithacus Software Ltd, Staines, UK). Mean
Ki values were expressed using SEM (standard error of mean) ob-
tained from each of four individual experiments.
27. Li, S. B.; Nie, H. L.; Zhang, H. T.; Xue, Y.; Chris, B. W.; Zhu, L. M. Acta Phys.-Chim.
Sin. 2010, 26, 215.
28. Motohashi, N.; Nishikawa, H.; Mori, I. Chem. Pharm. Bull. 1991, 39, 142.
29. Kim, S.; Jung, S. H.; Cho, C. W. Arch. Pharmacol. Res. 2008, 31, 1363.
30. Thanigaimalai, P.; Lee, K. C.; Bang, S. C.; Lee, J. H.; Yun, C. Y.; Roh, E.; Hwang, B.
Y.; Kim, Y.; Jung, S. H. Bioorg. Med. Chem. 2010, 18, 1135.
31. Thanigaimalai, P.; Lee, K. C.; Bang, S. C.; Lee, J. H.; Yun, C. Y.; Roh, E.; Hwang, B.
Y.; Kim, Y.; Jung, S. H. Bioorg. Med. Chem. 2010, 18, 1555.
32. Chen, J. S.; Wei, C.; Marshall, M. R. J. Agric. Food Chem. 1991, 39, 1897.
33. Cabanes, J.; García-Cánovas, F.; Tudela, J.; Lozano, J. A.; García-Carmona, F.
Phytochemistry 1987, 26, 917.
4.4. Dicopper center binding assay
Binding of the inhibitors to tyrosinase active site was deter-
mined by the method described elsewhere.49 Briefly, tyrosinase
was preincubated with the highest concentration of inhibitors re-
quired to achieve maximum inhibition of the enzyme. CuSO4 was
added to the pre-inhibited tyrosinase in a range of concentrations
(0.002À1.0 mM) followed by addition of substrate. The reaction
was monitored at 475 nm in the microplate reader. Any increase
in absorbance was indicative of the activity recovery of pre-inhib-
ited tyrosinase.
34. Gelder, C. W. G.; Flurkey, W. H.; Wichers, H. J. Phytochemistry 1997, 45,
1309.
35. Klabunde, T.; Eicken, C.; Sacchettini, J. C.; Krebs, B. Nat. Struct. Biol. 1998, 5,
1084 (PDB identifier: 1bt3).
36. Cuff, M. E.; Miller, K. I.; van Holde, K. E.; Hendrickson, W. A. J. Mol. Biol. 1998,
278, 855.
37. Matoba, Y.; Kumagai, T.; Yamamoto, A.; Yoshitsu, H.; Sugiyama, M. J. Biol. Chem.
2006, 281, 8981 (PDB identifiers: 1wx5, 1wxc, 1wx3, 2ahk, 2ahl, 1wx2).
38. Siegbahn, P. E. J. Biol. Inorg. Chem. 2003, 8, 567.
39. Rodriguez-Lopez, J. N.; Tudela, J.; Varon, R.; Garcia-Carmona, F.; Garcia-
Canovas, F. J. Biol. Chem. 1992, 267, 3801.
40. Dixon, M. Biochem. J. 1953, 55, 170.
41. Cabanes, J.; Chazarra, S.; García-Carmona, F. J. Pharm. Pharmacol. 1994, 46, 982.
42. Ga˛sowska, B.; Fra˛ckowiak, B.; Wojtasek, H. Biochim. Biophys. Acta 2006, 1760,
1373.
Acknowledgment
43. Miiller, G. H.; Waldmann, H. Tetrahedron Lett. 1999, 40, 3549.
44. Horning, D. E.; Muchowski, J. M. Can. J. Chem. 1972, 50, 3079.
45. Sen, S.; Srivastava, B. B. L. Indian J. Heterocycl. Chem. 2005, 14, 269.
46. Ternce, C. O. J. Heterocycl. Chem. 1984, 21, 1573.
The authors are deeply grateful to Ms. Maimoona Rasheed for
her technical support and fruitful discussions regarding synthesis
of the compounds.
47. (a) Ainsworth, C. J. Am. Chem. Soc. 1958, 80, 5201; (b) Brown, D. J.; Cowden, W.
B. Aus. J. Chem. 1983, 36, 1469; (c) Cheeseright, T. J.; Holm, M.; Lehmann, F.;
Luik, S.; Marcia Gottert, M.; Melville, J. M.; Laufer, S. J. Med. Chem. 2009, 52,
4200; (d) Chande, M. S.; Barve, P. A.; Khanwelkar, R. R.; Athalye, S. S.;
Venkataraman, D. S. Can. J. Chem. 2007, 85, 21; (e) Li, Y.; Liu, J.; Zhang, H.; Yang,
X.; Liu, Z. Bioorg. Med. Chem. Lett. 2006, 16, 2278; (f) Joshi, S.; Karnik, A. V. Synth.
Commun. 2002, 32, 111; (g) Maslat, A. O.; Abussaud, M.; Tashtoush, H.; Al-Talib,
M. Polish J. Pharmacol. 2002, 54, 55; (h) Ainsworth, C. J. Am. Chem. Soc. 1955, 78,
4475; (i) Jansen, M.; Rabe, H.; Strehless, A.; Dieler, S.; Debus, F.; Dannhardt, G.;
Akabas, M. H.; Lueddens, H. J. Med. Chem. 2008, 51, 4430; (j) Khan, K. M.;
Rasheed, M.; Ullah, Z.; Hayat, S.; Kaukab, F.; Choudhary, M. I.; Atta-ur-Rahman;
Perveen, S. Bioorg. Med. Chem. 2003, 11, 1381.
References and notes
1. Lerner, A. B.; Fitzpatrick, T. B. Physiol. Rev. 1950, 30, 91.
2. Fitzpatrick, T. B.; Miyamoto, M.; Ishikawa, K. Adv. Biol. Skin 1967, 8, 1.
3. Raper, H. S. Physiol. Rev. 1928, 8, 245.
4. Mason, H. S. Annu. Rev. Biochem. 1965, 34, 595.
5. Kubo, I.; Kinst-Hori, I.; Yokokawa, Y. J. Nat. Prod. 1994, 57, 545.
6. Oetting, W. S. Pigment Cell Res. 2000, 13, 320.
7. Hazes, B.; Magnus, K. A.; Bonaventura, C.; Bonaventura, J.; Kalk, K. H.; Hol, W. G.
Protein Sci. 1993, 2, 597.
8. Hazes, B.; Magnus, K. A.; Kalk, K. H.; Bonaventura, C.; Hol, W. G. J. Mol. Biol.
1996, 262, 532.
48. Hearing, V. J. Methods Enzymol. 1987, 142, 155.
49. Kahn, V.; Andrawis, A. Phytochemistry 1985, 24, 905.
9. Kubo, I.; Yokokawa, Y.; Kinst-Hori, I. J. Nat. Prod. 1995, 58, 739.