Senyawa Antimikrob Tubuh Buah Ganoderma GKR7 dan Interaksi Senyawa terhadap Escherichia coli secara In Silico

Abstract

Ganoderma telah lama dikenal dan digunakan sebagai obat tradisional di negara-negara Asia selama lebih dari 2000 tahun. Senyawa bioaktif pada Ganoderma terutama dari spesies Ganoderma lucidum telah banyak diteliti dan memiliki berbagai manfaat medis. Salah satu isolat koleksi asal Indonesia yaitu Ganoderma GKR7 juga telah diuji memiliki aktivitas antimikrob terhadap empat strain enteropatogenik Escherichia coli (EPEC) resisten antibiotik namun Ganoderma isolat GKR7 dan senyawa antimikrob tubuh buah Ganoderma GKR7 belum diidentifikasi. Selain itu, interaksi biomolekul antara senyawa antimikrob Ganoderma GKR7 dan protein reseptor E. coli belum pernah diteliti. Penelitian ini bertujuan mengidentifikasi Ganoderma isolat GKR7, senyawa antimikrob tubuh buah Ganoderma GKR7, dan interaksi biomolekul senyawa antimikrob Ganoderma GKR7 terhadap protein E. coli secara in silico. Identifikasi Ganoderma isolat GKR7 dilakukan berdasarkan pengamatan karakteristik sifat kultur, morfologi tubuh buah, dan analisis molekuler dengan ITS5/ITS4. Tubuh buah diproduksi dengan perlakuan substrat serbuk gergajian kayu (S), jerami (J), serbuk sabut kelapa (K), campuran serbuk gergajian kayu dan jerami (SJ), campuran serbuk gergajian kayu dan serbuk sabut kelapa (SK), dan campuran serbuk gergajian kayu, jerami, dan serbuk sabut kelapa (SJK). Tubuh buah dari semua perlakuan substrat tersebut selanjutnya diekstraksi dengan metode maserasi. Ekstrak kasar tubuh buah Ganoderma GKR7 diuji untuk screening senyawa antimikrob. Ekstrak terpilih selanjutnya dilakukan deteksi senyawa aktif dengan TLC-bioautografi lalu dilanjutkan dengan purifikasi parsial untuk mendapatkan fraksi aktif antimikrob. Fraksi aktif kemudian diuji terhadap enteropatogenik E. coli (EPEC) lalu dilakukan analisis senyawa dengan menggunakan LC-MS/MS. Hasil identifikasi senyawa selanjutnya dilakukan docking molekuler secara in silico untuk mengetahui senyawa antimikrob yang berinteraksi dengan protein target E. coli. Ganoderma isolat GKR7 teridentifikasi sebagai Ganoderma sichuanense. Purifikasi parsial dari ekstrak tubuh buah Ganoderma GKR7 yang diproduksi pada substrat serbuk gergajian kayu (S) menghasilkan tiga fraksi dari dua zona hambat dengan Rf 0,51, Rf antara, dan Rf 0,63. Uji aktivitas secara in vitro menunjukkan ketiga fraksi memiliki aktivitas antimikrob terhadap enteropatogenik E. coli (EPEC). Analisis senyawa melalui LC-MS/MS menghasilkan 57 senyawa yang teridentifikasi secara putatif dari ketiga fraksi. Uji secara in silico menghasilkan 25 senyawa terpilih yang berperan baik dalam penghambatan delapan protein target E. coli (GlmS, GlmU, MurA, MurB, MurD, MurG, DdlB, dan PBP3) yang terlibat pada biosintesis peptidoglikan. Senyawa 1-pentofuranosyl-2,4(1H,3H)-pyrimidine dione (CID_312160) dari fraksi zona Rf 0,51, isopropyl 1,1-dimethyl-3-{3-[(4-methylpiperazin-1-yl)methyl]-benzoyl}-1,2,3,6-tetrahydroazepino[4,5-b]indole-5-carboxylate (CID_45104531) dari fraksi Rf antara, dan 4-(1-benzofuran-3-ylmethoxy)-N-[1-[2-(3,4-dihydroxy-5-methylpiperidin-1-yl)propyl]piperidin-4-yl]-1H-indole-2-carboxamide (CID_66842043) dari fraksi zona Rf 0,63 memiliki energi afinitas yang tinggi (dengan nilai rata-rata -7,55 kkal/mol, -8,40 kkal/mol, dan -8,72 kkal/mol secara berurutan) terhadap protein target E. coli yang diujikan. Selain itu, protein GlmS, MurB, MurD, dan PBP3 memiliki interaksi terbaik dengan 25 senyawa terpilih sehingga menjadi multitarget penghambatan oleh senyawa antimikrob Ganoderma GKR7. Kajian lanjutan pada penelitian ini ialah melakukan purifikasi lebih lanjut ketiga fraksi untuk mendapatkan senyawa murni CID_312160, CID_45104531, dan CID_66842043 dan mengevaluasi kembali aktivitas senyawa tersebut.

Ganoderma has been widely used as a traditional medicinal mushroom in Asian countries for over 2000 years. The bioactive compounds of Ganoderma, foremost from Ganoderma lucidum, have been studied for various medicinal purposes. Ganoderma GKR7 is a culture collection from Indonesia that has been tested for antimicrobial activity against four enteropathogenic Escherichia coli (EPEC) strains which resistant to antibiotic. However, the Ganoderma of GKR7 isolate and its antimicrobial compounds have not been identified. Moreover, biomolecular interactions between antimicrobial compounds of Ganoderma GKR7 and proteins of E. coli have not been studied as well. This study aimed to identify the Ganoderma of GKR7 isolate, antimicrobial compounds of Ganoderma GKR7 fruiting body, and biomolecule interactions between antimicrobial compounds of Ganoderma GKR7 and proteins of E. coli through in silico study. The identification of Ganoderma of GKR7 isolate was determined by cultural features, morphological characters, and molecular analysis using ITS5/ITS4. The fruiting bodies were cultivated in several substrates of wood sawdust (S), rice straw (J), coconut coir (K), mixed of wood sawdust and rice straw (SJ), mixed of wood sawdust and coconut coir (SK), and mixed of wood sawdust, rice straw, and coconut coir (SJK). The fruiting bodies from all treatments were then extracted using the maceration method. The crude extracts of Ganoderma GKR7 fruiting bodies were tested for screening the antimicrobial compounds. The selected crude extract was then performed detection of active compounds by TLC-bioautography and partial purification was performed to obtain the antimicrobial active fractions. The active fractions were examined against enteropathogenic E. coli (EPEC) and the compounds were analyzed using LC-MS/MS. The identified compounds were performed molecular docking through in silico study to identify interactions between the antimicrobial compounds and targeted proteins of E. coli. The Ganoderma of GKR7 isolate was identified as Ganoderma sichuanense. The partial purification of Ganoderma GKR7 fruiting bodies extract that cultivated in wood sawdust (S) substrate obtained three fractions from two inhibition zones with Rf 0,51, Rf intermediate, and Rf 0,63. The in vitro assay showed that all fractions had antimicrobial activity against enteropathogenic E. coli (EPEC). Analysis of the compounds by LC-MS/MS resulted 57 putative identities from three fractions. From in silico study, 25 selected compounds were found to play a good role in the inhibition of eight E. coli proteins (GlmS, GlmU, MurA, MurB, MurD, MurG, DdlB, and PBP3) that are involved in peptidoglycan biosynthesis. Compounds of 1-pentofuranosyl-2,4(1H,3H)-pyrimidinedione (CID_312160) from fraction of Rf 0,51, isopropyl 1,1-dimethyl-3-{3-[(4-methylpiperazin-1-yl) methyl]-benzoyl}-1,2,3,6-tetrahydroazepino[4,5-b]indole-5-carboxylate (CID_45104531) from fraction of Rf intermediate, and 4-(1-benzofuran-3-ylmethoxy)-N-[1-[2-(3,4-dihydroxy-5-methylpiperidin-1yl)propyl]piperidin-4yl]-1H-indole-2-carboxamide (CID_66842043) from fraction of Rf 0,63 showed high affinity energy (average value -7,55 kcal/mol, -8,40 kcal/mol, dan -8,72 kcal/mol respectively) against targeted proteins of E. coli. Moreover, proteins of GlmS, MurB, MurD, and PBP3 had greatest interactions with 25 selected compounds thus that proteins play as multitarget of the inhibition by antimicrobial compounds of Ganoderma GKR7. Further studies from this study are to purify the three fractions to obtain pure compounds of CID_312160, CID_45104531, and CID_66842043 and evaluate the activity of these compounds.