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Table 1. The general structure of the sulfonamide bombesin analogues and the primarystructure of bombesin and its analogues, R = C 6 H 5 -SO 2 -Bombesin pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2R-[Leu 13 ]-bombesin C 6H 5-SO 2-Glu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2R-[Phe 13 ]-bombesin C 6H 5-SO 2-Glu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH 2R-[Ser 3 ,Arg 10 ,Phe 13 ] C 6H 5-SO 2-Glu-Gln-Ser-Leu-Gly-Asn-Gln-Trp-Ala-Arg-Gly-His-Phe-Met-NH 2-bombesinStepwise synthesis of the peptide analogues was achieved with diisopropylcarbodiimide/1-hydroxybenzotriazole (DIC/HOBt) in dimethylformamide (DMF) as coupling agents [7]. Inorder to achieve the prodrug formation, pGlu residue from the N-terminal of the nativebombesin sequence was replaced by Glu which has a free amino group available tocoupling with another group. Coupling of benzenesulfonyl group with the free aminoterminaland formation of the sulfonamide bond was achieved using N-methylmorpholine.After the completion of the synthesis, the resin was treated with a TFA (trifluoroaceticacid) solution to liberate the fully deprotected crude peptide conjugates. All the productswere purified by gel filtration chromatography on Sephadex G-15. Final purification wasachieved by preparative high performance liquid chromatography (Lichrosorb RP18column). ESI-MS analysis confirmed the expected peptide formulae.To analyze the sulfonamidase activity, a collection of different classes of GSTisoenzymes were investigated using sulfanilamide (4-aminobenzenesulfonamide) as amodel substrate. In particular, GSTs belonging to class alpha [human GSTA1-1, (hGSTA1-1); mouse GSTA4-4, (mGSTA1-1)], pi [human GSTP1-1, (hGSTP1-1)], sigma[Schistosoma japonicum GST, (SjGST); human spleen haematopoietic prostaglandin Dsynthase, (PDS-GST)], tau [Glycine max GST, (GmGSTU4-4)] and phi (Zea mays GST I)were examined. The higher sulfonamidase activity was observed for hGSTA1-1. Thisobservation is of particular importance since this isoenzyme is expressed in several tumoursand therefore may represent a therapeutic molecular target in cases where tumourprotectiveeffects depends upon hGSTA1-1 activity. After hGSTA1-1-mediated cleavage ofthe sulfonamide bond of the chimaeric molecule (pro-drug), the released aromaticsulfonamide moiety is conjugated with GSH, thus leading to a S-aromatic-glutathionylconjugate which is a strong inhibitor for GST. This inhibition was found to be of acompetitive-type with respect to GSH (K i = 5.1±0.8 µM) and of a non-competitive typewith respect to the aromatic substrate CDNB (K i = 8.6±0.7 µM). This effect may reduce thedrug resistance of cancer cells.AcknowledgmentsThis work was partially supported by the Hellenic General Secretariat for Research and Technology.References1. Labrou, N.E., Mello, L.V., Clonis, Y.D. Biochem. J. 358, 101-110 (2001).2. Labrou, N.E., Karavangeli, M., Tsaftaris, A., Clonis, Y.D. Planta 222, 91-97 (2005).3. Axarli, I., Labrou, N.E., Petrou, C., Rassias, N., Cordopatis, P. Clonis, Y.D. Eur. J. Med. Chem. 44,2009-2016 (2009).4. Labrou, N.E., Kotzia, G.A., Clonis, Y.D. Protein Eng. Des. Sel. 10, 741-748 (2004).5. Fields, B.G., Noble, L.R. Int. J. Pept. Prot. Res. 35 161-214 (1990).6. S. Aventis, European Patent 322348 and US Patent 5124478.7. Köning, W., Geiger, R. Chem. Ber. 103, 788-798 (1970).27