Screening, production and characterization of inhibitors isolated from actinomycetes against protein kinases of eukaryotic signal transduction

Gan, Chai Phei (2004) Screening, production and characterization of inhibitors isolated from actinomycetes against protein kinases of eukaryotic signal transduction. Universiti Malaysia Sabah. (Unpublished)

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Abstract

Screening actinomycetes secondary metabolites is required for identification of novel bioactive molecules inhibiting on serine/threonine protein kinases, especially MKKl and GSK-3. Defects in the pathways participated by MEK and GSK-3 can lead to several diseases, such as cancer and neurodegenerative diseases. S. cerevisiae was used in work to screen for MKKI and GSK-3 inhibitors. In yeast MKKI screening system, acetone extracts and freeze-dried extracts of actinomycetes strains H7372 and H7944 showed toxicity to mutant yeast MKKlP386 . The toxicity of these strains was confirmed in yeast with human GSK-3β gene insertion screening system and Ca2+ activated yeast Mck 1 screening system. The bioactive compounds appeared to inhibit other targets that are lethal to yeast. The toxic compounds of H7372 and H7944 extracts were not temperature sensitive and dissolved totally in water. In the yeast with human GSK-3β gene insertion screening system, a total of 110 actinomycetes strains were screened. Extracts of H8934, H7530, H7667, H1110 and H8955 were identified to contain potential human GSK-3β inhibitors. The inhibited human GSK- 3f3 in yeast was unable to rescue the temperature sensitive phenotype of gsk-3 null mutant causing growth inhibition of yeast H10075 at 37oC. The GSK-3β inhibitor does not inhibit growth of yeast Hl0075 at 25°C since it was rescued by the low temperature. Human GSK-3β and yeast Mck 1 are functionally conserved because both kinases expressed under the control of glyceraldehydes-3-phosphate dehydrogenase promoter suppressed the temperature-sensitivity of gsk-3 null mutant. The five potential strains were further screened in Ca2 + -activated yeast Mck 1 screening system to investigate the potential of the GSK-3β inhibitor of inhibiting Mck 1. No inhibitor for Mck 1 can be ascertained, suggesting that the inhibitors in H8934, H7530, H7667, H8955 and H11110 were specific for GSK-3β; Mck 1 and GSK-3β may have slight difference in their functions to regulate cell growth in different pathways in yeast. In the presence of Mck 1 inhibitor, growth of mutant yeast ∆zds I will be inhibited whereas there will be no growth inhibition in mutant yeast with mck 1 deleted (∆zds 1∆mckl) on YPD. At the same time the Ca2+ sensitive phenotype ∆zdsl will be suppressed in the presence of Mck 1 inhibitor. Inhibited Mck 1 was unable to regulate Hsl 1 to cause a delay in mitosis in yeast. Toxic strains identified in the Ca2+-activated yeast Mck 1 screening are H8934 and H11110.

Item Type: Academic Exercise
Uncontrolled Keywords: protein kinase, MKKl, GSK-3, strain, screening, bioactive molecule
Subjects: Q Science > QD Chemistry
Divisions: SCHOOL > School of Science and Technology
Depositing User: MDM SITI AZIZAH IDRIS
Date Deposited: 26 Aug 2013 03:15
Last Modified: 23 Oct 2017 07:48
URI: http://eprints.ums.edu.my/id/eprint/6830

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