Screening and characterization De inhibitors of protein Kinases and protein Phosphatases for the treatment of latent Tuberculosis

No Abstrac

Isolation of a potential anticancer agent with protein phosphatase inhibitory activity from soil-derived Penicillium sp. strain H9318

Purpose: To determine the effect of the secondary metabolites from Penicillium sp. H9318 on cytotoxicity and cell cycle progression. Methods: A yeast PP1 inhibitory screening system was carried out to confirm the presence of anti-PP1c activity in crude acetone extracts of strain H9318. The extracts were fractionated and identified as Fraction S1 and Citrinin 9318 (CTN9318). Various cancer cell lines were used to test for the toxicity of the crude acetone extracts, Fraction S1 and Citrinin 9318, using MTT viability assay. Results: It was found that a colorectal cancer cell line, HT-29, was susceptible to Fraction S1 and Citrinin 9318. A propidium iodide (PI)-incorporated DNA assay was used to show that there was G2/M arrest in HT-29 by Citrinin 9318. Conclusion: Citrinin 9318 inhibits the viability of HT-29 via mitotic block. The results suggest that Citrinin 9318 is capable of exerting cytotoxicity and mitotic arrest in a colon cancer cell line, HT29

Isolation of actinomycetes from Sabah and the screening for inhibitor against Eukaryotic signal transdustion

Actinomycetes strains isolated from 504 soil samples collected from Sabah terrestrial ecosystem were studied and screened for novel bioactive compounds inhibitory against eukaryotic signal transduction. All the soil samples were collected under trees identified to species or genus level. Isolation of Streptomyces and non-Streptomyces actinomycetes on HV (humic acid + B-vitamins) medium yielded 569 strains. Morphology characterisation of the isolated actinomycetes was carried out including aerial mycelium, substrate colour, diffusible pigment and spore morphology- on oatmeal medium while chemotaxonomic identification based on Diaminopimelic Acid. All actinomycetes strains were grown under aerobic condition in liquid culture and extracted with acetone. In this research, yeast MAPK kinase and MAP kinase phosphatase are the molecular level targeted proteins. The screening system was developed for searching MAPK kinase and MAP kinase phosphatase inhibitors. MKKIP386 and MKKI P386-MSG5 mutant yeast were used to screen for inhibitors, as these yeast kinase and phosphatase have homologous proteins in the MAP kinase signal transduction pathway in human. Strain H7553 and H7597 showed potential MAPK kinase inhibitors. The in vivo RaslRaf interaction with the yeast two hybrid screening system was used to screen against RaslRaf protein interaction inhibitor. Strain H7520 showed potential inhibitor for yeast Type 1 protein serine/threonine phosphatase (GLC7) screening system. Extract H7944 showed inhibition effect in the ERK signal transduction (the chain-reaction of phosphorylation from MEK 112 to ERK 112) when inhibited phosphorylation of MEK to ERK. Thus, strain H7944 (MBA94-2) was able to prevent activation of MEK. Strain H7944 was a potential MEK 112 inhibitor since p-galactosidase assay confirmed that H7944 do not inhibited the Ras/Raf pathway. Aktinomiset dipencilkan daripada 504 sampel tanah dikutip dari ekosistem Sabah dikaji dan penyaringan compaun bioaktif terhadap perencatan transduksi isyarat eukariot. Semua sampel tanah dikutip di bawah pokok yang dikenalpasti sehingga peringkat spesies atau genus. Pemencilan aktimomiset Streptomyces dan bukan Streptomyces menggunakan media asid humik-vitamin (HV) agar be~aya memencilkan sebanyak 569 strain aknomiset. Pencirian morfologi strain aktinomiset yang dipencilkan dilakukan melalui pencirian warna aerial miselium, warna substrak dan penyebaran warna pigment ke atas media agar oatmeal manakala pencirian kimiataksonomi dilakukan melalui teknik isomer asid diaminopimelik. Semua strain aktinomiset dikulturkan secara aerobik dalam media cecair dan diekstrak dengan menggunakan aseton. Dalam kajian ini, MAPK kinase dan MAP kinase fosfotase adalah sasaran protein molekular dalam yis. Sistem penyaringan dibangunkan untuk mencari perencat untuk MAPK kinase dan MAP kinase fosfotase. Vis mutant, MKK 1 P386 dan MKK 1 P386 -MSG5 digunakan untuk tujuan penyaringan disebabkan kinase dan fosfotase yis mempunyai persamaan dengan protein dalam transduksi isyarat MAP kinase dalam manusia. Strain H7553 dan H7597 menunjukkan potensi perencat untuk MAPK kinase. Sistem penyaringan in vivo interakasi RasIRaf dalam yis dual-hybrid digunakan untuk menyaring perencat protein interakasi RaslRaf. Ekstrak daripada aktinomiset H7520 menunjukkan potensi perencat untuk protein serine/threonine fosfotase (GLC7) dalam yis dalam penyaringan yang dijalankan. Ekstrak H7944 menunjukkan kesan perencatan dalam isyarat transduksi apabila ia merencat fosforilasi terhadap MEK daripada ERK. Ini bermakna H7944 berupaya mencegah pengaktifan MEK. Strain H7944 mempunyai potensi menjadi perencat MEK 1/2 kerana kajian p-galaktosidase yang dijalankan menunjukkan ia tidak merencat kitaran Ras/Ra

Anti-proliferative and anti-invasive properties of a purified fraction from Streptomyces sp. H7372.

Secondary metabolites from actinomycetes especially the genus Streptomyces may be one of the most important sources for novel anticancer agents. A purified fraction from a novel actinomycete strain, Streptomyces sp. H7372, was elucidated in breast cancer cells. We have isolated three purified fractions from a novel strain, Streptomyces sp. H7372. One of the fractions, designated as 31-2, exhibited the strongest growth-inhibitory effect and thereby was selected for further studies. 31-2 exerted a growth-inhibitory effect on a panel of 15 human cancer and 2 non-malignant cell lines. In MCF-7 and MDA-MB-231 breast cancer cells, 31-2 induced a cytostatic (anti-proliferative) effect without causing cytotoxicity (cell death). Our data suggest that the cytostasis resulted from cell cycle arrest at the G1 phase in MCF-7 cells and at the S phase in MDA-MB-231 cells. Western blot analysis demonstrated a modulation of phosphorylation of the Rb and CDC2 proteins and of CDK4, cyclin D1 and cyclin D3 in the 31-2-treated breast cancer cell lines. The protein levels of CDK2, CDK6, and PCNA were not affected by 31-2 treatment. 31-2 also exhibited an anti-invasive effect in MDA-MB-231 cells. However, this effect is not attributed to the modulation of proteolytic activity in MDA-MB-231 cells as the enzymatic degradation of type IV collagen was not affected by 31-2. The 31-2 is a potent cytostatic and anti-invasive agent and modulates the cell cycle pathway. Together, these results will have important implications in searching for novel approaches to treat cancer

Screening for eukaryotic signal transduction and Mycobacterium isocitrate lyase inhibitor from actinomycetes and fungi of dipterocarp rain forests at Imabak Valey, Sabah, Malaysia

A diversity of actinomycetes and fungi was isolated from various sites during the Imbak Valley Scientific Expedition 2000. A total of 144 soil samples were collected under trees that have been identified to species or genus level. Imbak Valley is a lowland dipterocarp forest, which is interestingly dominated by Dryobalanops beccarii. Isolation of Streptomyces and non-Streptomyces actinomycetes on HV medium and other specific isolation media for non-Streptomyces yielded 203 isolates from 89 soil samples. Morphological characterisation of the isolated actinomycetes was carried out based on aerial mycelium colour, substrate mycelium colour and diffusible pigment production on oatmeal medium. Nine strains of fungi were isolated from the six soil samples plated on PDA medium. All actinomycetes isolates were grown under aerobic condition in liquid culture and extracted with acetone, and used for screening against proteins involved eukaryotic signal transduction. Yeast MAPK kinase and MAP kinase phosphatase were some of the targeted proteins used in this research. MKK1P386 and MKK1P386-MSG5 mutant yeasts were used to screen for these inhibitors, as these yeast kinase and phosphatase have homologous proteins in the MAP kinase signal transduction pathway in human. No inhibitors in the extracts were found in these screenings. Type 1 protein serine/ threonine phosphatase (GLC7) in yeast was used to screen inhibitors against PP1 inhibitors and no inhibitor was found. None of the fungal extracts showed any inhibitory activities in all the screening systems. No Ras/Raf inhibitor was found in the in vivo Ras/Raf interaction with the yeast two-hybrid screening system, which used to screen for inhibitor against Ras/ Raf protein interaction inhibitor. There were 11 actinomycetes extracts that showed toxicity against yeast strain LZ (transformant of Ras/ Raf). H7667, a Streptomycete toxic to yeast is further screened for inhibitors of the GSK3-beta pathway. H7763, a Streptomyces species that showed positive in the primary screen for inhibitor of isocitrate lyase (ICL) which is not itaconic acid (known ICL inhibitor). H7240 showed the strongest susceptibility towards the resin in which the concentration of 5g/l of resin is sufficient to produce growth inhibition of the bacteria