GSK3 beta-inhibitory activity in microfungal isolated from Sabah rainforest soils

• Main Author: Dg. Nur Azierah Fachyuni Abdul Aziz
• Format: Thesis
• Language: English; English
• Published: 2017
• Subjects: QK504-(638) Cryptogams
• Online Access: https://eprints.ums.edu.my/id/eprint/38973/1/24%20PAGES.pdf; https://eprints.ums.edu.my/id/eprint/38973/2/FULLTEXT.pdf; https://eprints.ums.edu.my/id/eprint/38973/

Glycogen synthase kinase-3β (GSK-3β) is a serine/threonine kinase that has been implicated in several diseases such as diabetes, cancer, inflammation, Alzheimer and bipolar disorder. Therefore, GSK-3β has become a prior target in drug discovery. This study was aimed to search for potential GSK-3β inhibitors in soil microfungi isolated from Sabah rainforests. In this study, a total of 122 soil samples were collected from west coast and interior division of Sabah whereby 165 microfungi strains were successfully isolated on potato dextrose agar and malt extract agar. All strain was cultured aerobically and the prepared extracts were tested using a yeast-based screening assay for their inhibitory activity on GSK-3β. The homologous genes of GSK-3 in the yeast (MCK1, MDS1, MRK1, and YOL128C) were knocked out and inserted with mammalian GSK-3β to overcome temperature-sensitive phenotype of the mutant at 37oC thus created a yeast strain that is capable to grow at both 25oC and 37oC. Positive result was scored when there is a large inhibition zone on the grown yeast at 37oC. Furthermore, targeting Cys199 residue on GSK-3β is a possible mechanism of inhibition in this assay and this residue lead to a selective inhibitor. Fourteen out of 165 strains gave detectable inhibition zones in the screening assay but only one strain namely MAN15558 isolated from Mantanani’s island gave consistent inhibitory activities at 37oC and 25oC which were 17.75 mm ± 0.35 (clear inhibition) and 11.5 mm ± 0.53 (partial inhibition), respectively when tested at 5 mg/disk of acetone crude extracts. The MAN15558 strain is classified as Aspergillus sp. based on morphology and molecular techniques using 18sRNA. This strain is also a non-virulence strain based on biochemical assay. Crude extracts of MAN15558 strain was separated into polar and non-polar layer using rapid extraction method. Non-polar layer showed potential inhibitory activity at 100 μg/disk thus further fractionation of this layer was performed using automated semi preparative HPLC. Impure fraction 2 (F2) was obtained and the inhibitory activity on GSK-3β was confirmed using screening assay (100 μg/disk) and Kinase-Glo luminescent assay (10 μg). Active F2 was analysed using LC-MS/Qtof and 639.1664 m/z detected as the potential precursor ion. Identification of compounds performed using MS/MS fragmentation data of 639.1664 m/z in MassBank programme. Five hits were obtained and two of it was predicted as the potential compound based on their chemical substructure and peak relationship. The compounds were Isoscoparin 2''-O-ferulate and Okanin 4'-(4''-acetyl-6''-p-coumarylglucoside). These compounds were predicted as anti GSK-3β agent produced by MAN15558 strain. They are flavonoid compound and contain sub-structure like ferulic acid and coumaric acid which confers anti-diabetic and anti-oxidant activity as reported extensively. Further purification and structure are required to confirm these compounds. This present study concluded that MAN15558 strain is the first soil microfungi isolated from Mantanani’s island identified as Aspergillus sp. that has produced anti GSK-3β agents targeting on Cys199 and predicted as Isoscoparin 2''-O-ferulate and Okanin 4'-(4''-acetyl-6''-p-coumarylglucoside).