GENOME ANALYSIS AND METABOLITE PROFILING OF SELECTED INDONESIAN Streptomyces BACTERIA WITH ANTIPLASMODIAL AND ANTICANCER ACTIVITIES

EMA DAMAYANTI, Prof. Dr. Mustofa, Apt., M.Kes.; Ir. Jaka Widada, M.P., Ph.D.; Achmad Dinoto, Ph.D.

2021 | Disertasi | DOKTOR BIOTEKNOLOGI

Malaria sebagai penyakit infeksi Plasmodium masih menghadapi permasalahan resistensi obat dan memerlukan penemuan obat baru. Bakteri Streptomyces adalah sumber utama senyawa antibiotik. Streptomyces asal Indonesia dari laut (GMY01), mangrove (SHP 22-7 dan BSE7F), dan tanah (GMR22) memiliki aktivitas antikanker, antibakteri, dan antijamur tetapi belum diketahui aktivitas antiplasmodium-nya. Penelitian ini bertujuan untuk mengidentifikasi potensi metabolit aktif sebagai antiplasmodium dan antikanker dari Streptomyces terpilih berbasis analisis whole-genome, analisis profil metabolit, dan molecular docking. Genome mining dilakukan dengan menggunakan AntiSMASH versi 5 dan BiG-FAM. Analisis profil metabolit dilakukan dengan untargeted LC-MS/MS dan targeted LC-HRMS. Bioassay-guided fractionation dilakukan dengan flash chromatography dan kolom kromatografi dengan uji in vitro antiplasmodium pada Plasmodium falciparum menggunakan metode mikroskopis dan SYBR Green I dan uji in vitro antikanker pada beberapa lini sel. Uji toksisitas dilakukan pada lini sel Vero. Klaster gen yang mengkode metabolit aktif diidentifikasi dengan RAST dan The SEED Viewer versi 2. In silico molecular docking dilakukan pada 5 protein target P. falciparum dan 4 protein target sel kanker. Analisis genome mining menunjukkan bahwa empat bakteri Streptomyces memiliki NRPS, PKS, dan terpen sebagai klaster gen dominan. Sebagian besar Streptomyces menghasilkan senyawa mengandung unsur CHON dengan BM antara 100-400 Da. Bakteri laut Streptomyces sp. GMY01 memiliki aktivitas antiplasmodium tertinggi dengan toksisitas rendah pada sel normal. Tiga metabolit aktif didapatkan dari GMY01, yaitu fraksi F2 dan F4.7 dari ekstrak etil asetat supernatan dan fraksi EMB-B dari ekstrak metanol biomassa sel. Berdasarkan indek selektivitasnya, F2, F4.7, dan EMB-B dikategorikan berturut - turut sebagai fraksi aktif, moderat, dan aktif sebagai antiplasmodium dan moderat, aktif rendah dan aktif sebagai antikanker. Senyawa dominan pada fraksi F2 adalah gabazine (C15H17N3O3), toyocamycin (C12H13N5O4), dan 4-n-pentanoylbiphenyl (C17H18O); pada fraksi F4.7 adalah isobutyranilide (C10H13NO), propoxur (C11H15NO3) dan ephedrine (C10H15NO) dan pada fraksi EMB-B adalah N-acetylneuraminyl-(2-6)-galactose (C17H29N14) dan mannotriose (C18H32O16). Analysis targeted LC-HRMS menunjukkan albaflavenone (C15H22O) terkonfirmasi sebagai senyawa aktif yang dihasilkan oleh Streptomyces sp. GMY01. Klaster gen yang mengkode albaflavenone adalah terpen dengan similaritas asam amino 100%. Analisis KEEG menunjukan GMY01 memiliki kluster gen yang mengkode biosintesis galactose dan mannose, terpen, dan alkaloid. Analisis molecular docking menunjukkan albaflavenone dan mannotriose memiliki binding affinity pada protein glutathione reductase (GR) dan glutathione-S-transferase (GST) P. falciparum dan protein autofagi (mTORC1 dan mTORC2) pada sel kanker. Hal ini mengindikasikan bahwa senyawa tersebut berpotensi sebagai kandidat obat antiplasmodium dan antikanker.

Malaria as a Plasmodium infectious disease still has drug resistance problem and a need for new drug discovery. Streptomyces bacteria are a major source of antibiotics. Indonesian Streptomyces bacteria from marine (GMY01), mangroves (SHP 22-7 and BSE7F), and forest soil (GMR22) exhibited anticancer, antibacterial, and antifungal activity but study on its antiplasmodial activity were still limited. The study aimed to identify potential active metabolites as antiplasmodial and anticancer of selected Streptomyces based on whole-genome analysis, metabolite profiling, and molecular docking. Genome mining was performed using AntiSMASH version 5 and BiG-FAM tools. Metabolite profiling was conducted using untargeted LC-MS/MS and targeted LC-HRMS. Bioassay-guided fractionation was conducted using flash and column chromatography combined with in vitro antiplasmodial on Plasmodium falciparum using microscopic and SYBR Green I method and in vitro anticancer on several cell lines. A toxicity assay was conducted on the Vero cell line. Gene clusters encoding active metabolites were identified using RAST and The SEED Viewer version 2. In silico molecular docking was performed on 5 target proteins of P. falciparum and 4 target proteins of cancer cells. Genome mining analysis revealed that four selected Indonesian Streptomyces bacteria have NRPS, PKS, and terpene as dominant biosynthesis gene clusters (BGCs). The majority of the Streptomyces produce compounds containing CHON elements with MW approximately 100-400 Da. Marine bacterium Streptomyces sp. GMY01 has the highest antiplasmodial activity with low toxicity on normal cells. Three active metabolites were obtained from GMY01, namely F2 and F4.7 from the ethyl acetate extract of supernatant and EMB-B from the methanol extract of cell biomass. Based on the selectivity index, the F2, F4.7, and EMB-B fractions were classified as active, moderate, and active as antiplasmodial, respectively. Meanwhile, F2, F4.7, and EMB-B fractions were categorized as moderate, low active, and active as anticancer, respectively. The major compounds detected in F2 fraction were gabazine (C15H17N3O3), toyocamycin (C12H13N5O4), and 4-n-pentanoylbiphenyl (C17H18O); in F4.7 fraction were isobutyranilide (C10H13NO), propoxur (C11H15NO3) and ephedrine (C10H15NO) and in EMB-B fraction was as N-acetylneuraminyl-(2-6)-galactose (C17H29N14) and mannotriose (C18H32O16). Targeted LC-HRMS analysis showed that albaflavenone (C15H22O) was confirmed as an active compound produced by Streptomyces sp. GMY01. BGCs encoding albaflavenone was terpene type with 100% deduced amino acids similarity. The KEEG map analysis showed that GMY01 has gene clusters encoding biosynthesis of galactose and mannose, terpene backbone, and alkaloids. Molecular docking analysis showed that albaflavenone and mannotriose have a binding affinity on glutathione reductase (GR) and glutathione-S-transferase (GST) of P. falciparum and on autophagy proteins (mTORC1 and mTORC2) of cancer cells. This indicated that these compounds were potential for the antiplasmodial and anticancer drug candidate.

Kata Kunci : antiplasmodial, anticancer, drug discovery, genome mining, metabolite profiling, Streptomyces