2-SUBSTITUTED BENZAZOLE DERIVATIVES

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48 saat. 24 saat mock. 48 saat mock. Table 1. The inhibition effect of benzothiazole and benzoxazole ring containing compounds on GST P1-1. INTRODUCTION.

2-SUBSTITUTED BENZAZOLE DERIVATIVES, SUICIDE INHIBITORS FOR GLUTATHIONE S-TRANSFERASES AND DOCKING STUDIES Yasemin Aksoy1, Deniz Ceyhan1, Yaman Muşdal1, Esin Akı2, Tuğba Ertan Bolelli2, Kayhan Bolelli2, İlkay Yıldız2, İsmail Yalçın2 1Hacettepe 2Ankara

University, Faculty of Medicine, Medical Biochemistry, Ankara

University, Faculty of Pharmacy, Pharmaceutical Chemistry, Ankara

ABSTRACT Glutathione S-transferases involved in the cancer drug resistance phenomenon, especially GSTP1-1 isoenzyme is overexpressed in many cancer cell lines and induce drug resistance. Many efforts have been made in the last years to find tight inhibitors of these enzymes to reduce their protective role in vivo. These new inhibitors are not glutathione peptidomimetic molecules and display lipophylic properties suitable for crossing the plasma membrane. We determined the GST P1-1 inhibitory activities of similar compounds, 2-substituted benzothiazol derivatives which were synthesized and the inhibitory enzyme activities tested. GST P1-1 activity was determined spectrophotometrically by measuring the absorbance change at 340 nm. The docking studies of compounds with variation in structure and activity were performed using software Discovery Studio 2.1 software based on the possible two crystal structures of the GST P1-1. The observation shows that the activity results are consistent with the docking studies based on both of the crystal structures.

INTRODUCTION Glutathione transferases (GSTs; EC 2.5.1.18) are a group of phase II detoxication enzymes, involved in the biotransformation of xenobiotic and endogenously produced compounds. They catalyze the conjugation reaction of reduced glutathione (GSH) to electrophilic compounds (1). In human cells there are seven classes of soluble cytosolic GSTs (termed alpha, mu, pi, sigma, zeta, omega and theta). GST P1-1, a member of this family, is often associated with progression of drug resistance (2). To overcome this resistance specific GST P1-1 inhibitors are needed.

Figure 3. The inhibition profile of TD11B on GST P1-1. The most potent inhibitory compound was found TD11B with the IC50 value 12 µM.

120

% Cell viability

100

Figure 1. The conjugation of glutathione with CDNB.

AIM OF THE STUDY

80

24 saat 48 saat

60

24 saat mock 48 saat mock

40 20

Design compounds specifically inhibits glutathione transferases and overcome the drug resistance in cancer cells.(Table 1)

0 0

5

10

20

40

80

160

[ TD11B], µM

METHOD

Figure 4. The cell viability effect of TD11B on MCF-7 cell lines. The most potent inhibitory compound was found TD11B with the IC50 value 12 µM.

The synthesis of the original compounds : The compounds of Benzoxazole and Benzothiazole derivatives :are s yhnthesized and controlled of purity degree s(Temiz-Arpacı, Ö., Akı-Şener, E., Yalçın, İ., Altanlar, N., “Synthesis and antimicrobial activity of some 2-[p-substituted-phenyl]benzoxazole-5-yl-arylcarboxyamides”, Arch. Der Pharm., 6, 283-288, (2002).

Discovery Studio 2.1 image

AutoDock 4.2 image

Expression and purification of recombinant hGST P1-1. The purification of human glutathione transferase P1-1 was done as the method of Kolm et al (3). Briefly; E.coli XL-1 cells containing pKXHP1 plasmid were overexpressed by using 0.2 mM IPTG at 37˚C. Soluble fractions were applied to S-hexylglutathione-Sepharose 6B affinity matrix. Eluted proteins were concentrated, dialyzed and and the puritiy of GST P1-1 was checked with SDS-PAGE. Enzymatic assay and inhibition studies. GST activity was measured spectrophotometrically by measuring the initial rate of absorbance change at 340 nm as described by Habig et al (4). Standard enzymatic assays were done in 0.1 M phosphate buffer, pH 6.5 containing 1 mM EDTA in the presence of 1 mM GSH and 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) at 30 °C. The compounds were dissolved in ethanol and the inhibition studies were done in the presence of %5 ethanol. IC50 values of the compounds were determined from the plots by using Graphpad Prism 4.0 software. Docking Analysis. The crystal structures of GST’s (2J9H and 9GSS) were obtained from Protein Data Bank. Before starting the analysis the interactions between the crystals structures and water and ions were deleted. In order to ease the tension in crystal

Figure 5. Docking analysis of TD11B with cyc48 free GST P1-1 and wild type GST P1-1 (5).

structures all missing hydrogen and side chains were added to the structures. The three dimensional structures of benzothiazol compounds were designed with Spartan software and optimized to use in docking programme. Docking studies were done with (B) (A) Discovery Studio 2.1 software.

Table 2. Docking analysis results of TD11B with different models of GST P1-1

Figure 2. The crystal structure of glutathione transferase P1-1. (A) 2JH9, crystal structure of human glutathione-stransferase p1-1 cys-free mutant in complex with S-hexylglutathione at 2.4 a resolution. (B) 9GSS, human glutathione S-transferase P1-1, complex with S-hexyl glutathione. All datas were obtained from protein data bank.

CONCLUSION

2J9H and 9GSS show %98.1 sequence similarity. Benzathiazole compounds studied were synthesized in Ankara

-Benzothiazole compound, TD 11B was selected as strong inhibitor.

University Faculty of Pharmacology in Pharmaceutical Chemistry.

-Docking analysis confirm the in vitro studies as binding energy rankings. -More specific inhibitors are needed for further studies.

RESULTS O

S NH

C

CH2

R

R: Br, F

N

REFERENCES 1)Josephy, P. D. and B. Mannervik (2006). Molecular toxicology. New York ; Oxford, Oxford University Press.

O X NO2

R TD1B TD3B TD11B TD13B

N

SO2NH

X -CH2-CH2-

R -F -Cl -Br

2)Hayes, J. D. and D. J. Pulford (1995). "The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance." Crit Rev Biochem Mol Biol 30(6): 445-600. 3)Kolm, R. H., G. Stenberg, et al. (1995). "High-level bacterial expression of human glutathione transferase P1-1 encoded by

O R1

X

NH O

N R

S1 S3 S4 S15 S16

R Br Br Br Br Br

X CH2 CH2 CH2 -

R1 H Cl Br Cl Br

semisynthetic DNA." Protein Expr Purif 6(3): 265-271. 4)Habig, W. H., M. J. Pabst, et al. (1974). "Glutathione S-transferases. The first enzymatic step in mercapturic acid formation." J Biol Chem 249(22): 7130-7139. 5) Morris GM, Huey R, Lindstrom W, Sanner MF, Belew RK, Goodsell DS, Olson AJ: AutoDock4 and AutoDockTools4: Automated docking with selective receptor flexibility. Journal of Computational Chemistry 2009, 30(16):2785-91.

Table 1. The inhibition effect of benzothiazole and benzoxazole ring containing compounds on GST P1-1.