Screening for microbial inhibitors of signal transduction particularly the AKT/GSK-3B pathway

Foo , Sek Hin (2006) Screening for microbial inhibitors of signal transduction particularly the AKT/GSK-3B pathway. Masters thesis, Universiti Malaysia Sabah.

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Abstract

This study aims to screen for microbial inhibitor of GSK-3β using yeast-based screening systems. gsk-3 null mutant with four yeast GSK-3 homologs (MCK1, MDS1, MRK1 and YOL128C) disrupted , leads to (1) growth defect at 37 0C, which is rescued in transformed H 10075 (GSK-3β) and H 10079 (MCK1), alternatively by inclusion of 1.2M D-sorbitol into the growth medium. (2) Inability to utilize galactose for growth (at 25°C and 37 0C), which is suppressed in H10079 (MCK1). Growth of bul1bul2 mutant is also defective at 3rC, but distinguished in the inability to utilize glycerol. Both phenotypes of bul1bul2 mutant are suppressed in H10082 (BUL 1). Inhibitor of GSK- 3[3 was therefore expected to mimic phenotypes of gsk-3 null mutant, detected by (A) growth inhibition of H10075 (GSK-3β) and H10079 (MCK1) only at 37 0C, confirmed in inhibition of H10084 (MCK1) only at 37 0C, and non-inhibition of H10085 (∆mck1). (B) Growth inhibition of H10079 (MCK1) in galactose medium (at 25°C and 37 0C) and (C) Non-inhibition of H10082 (BUL 1) in glycerol medium (at 25°C and 37 0C). Thirty actinomycetes strains previously screened as toxic to yeast were tested on H10075 (GSK-3β). Two strains H7530 and H7667 inhibited H10075 (GSK-3[3) only at 37 0C, with minimal1.2M D-sorbitol rescue. Identical inhibition was observed when H7530 and H7667 were tested on H10079 (MCK1) and H10084 (MCK1). In galactose utilization screening , both strains inhibited H10079 (MCK1) in SG-Ura medium, 25°C with stronger inhibition at 37 0C. Another 287 actinomycetes strains, isolates from soils of Long Pasia, Lower Segama and Melalap (Crocker Range) were screened on H 1 0075 (GSK-3β) for inhibition at only 37 0C, and only two weak inhibitors were found H11329 and H11364. These weak inhibitors were not further studied. Crude extracts of H7530 and H7667 did not inhibit GSK-3[3 activity in vitro. When tested in vivo using Western blotting, H7667 inhibited p-GSK-3β (Ser-9) in MCF-7 cells. H7530 had no effect. H10082 (BUL 1) and PP1 screenings distinguished these two strains. H7530 inhibited H 1 0082 (BUL 1) in glycerol medium at 25°C and 37 0C and inhibited H10018 (GLC7) of PP1 screening at 25°C and 37 0C. H7667 was negative in the H10082 (BUL 1) screen and inhibited H 10018 (GLC7) only at 37 0C resembled inhibition seen on H 10075 (GSK-3β) at 37 0C. H7530 and H7667 were purified and bioactive peaks were identified. H7530 (F6) did not reduce p-GSK-3β (Ser-9) in MCF-7 cells. H7667 (F21) inhibited p-GSK-3β (Ser-9) but did not alter p-Akt (Thr-308) indicating H7667 (F21) could probably act directly at the Akt without affecting upstream kinases of Akt. This was substantiated by decrease in p-BAD (Ser-136). Though in vitro Akt assay did not detect any significant inhibition, H7667 (F21) may inhibit Akt in a non-A TP competitive manner or by other novel mechanisms. Despite the fact that no inhibitor of GSK-3β was found in this study, inhibitor, H7667 (F21) that affects the AktlGSK-3β pathway was identified. This justifies further investigation of H7667 (F21); to determine its specific kinase target and chemical identification of the active compound.

Item Type:Thesis (Masters)
Uncontrolled Keywords:microbial inhibitor of GSK-3β, screening system, chemical identification, kinase target
Subjects:Q Science > QK Botany
Divisions:SCHOOL > School of Science and Technology
ID Code:6499
Deposited By:IR Admin
Deposited On:08 Jul 2013 13:31
Last Modified:08 Jul 2013 13:31

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