Senyawa Aktif Penghambat Enzim Mycobacterium tubercolusis Shikimate Kinase (MtbSK) dari Mikroba Indonesia

Abstract

Kemunculan strain Mycobacterium tuberculosis (Mtb) yang resisten terhadap berbagai obat (RR-TB, MDR-TB dan XDR-TB) mendesak penemuan kerangka molekul terapeutik baru yang menargetkan jalur esensial spesifik, salah satunya adalah enzim Mycobacterium tuberculosis Shikimate Kinase (MtSK). Penelitian ini berhasil menemukan dan mengidentifikasi inhibitor baru terhadap MtSK melalui penapisan 3480 ekstrak mikroba. Pemurnian yang dipandu bioasai (bioassay-guided fractionation) terhadap kultur kapang Cosmospora sp. BioMCC-f.T-7332 menghasilkan dua senyawa golongan didepsida TPI-1 (1) dan TPI-2 (2) yang diperoleh dalam bentuk campuran tak dapat dipisahkan (inseparable mixture). Struktur molekul senyawa 1 dan 2 dielusidasi secara komprehensif menggunakan data HRMS dan NMR (1D dan 2D) serta perbandingan dengan data pustaka. Evaluasi aktivitas biologis mengungkap bahwa campuran senyawa 1 dan 2 menunjukkan aktivitas inhibisi sangat baik terhadap enzim MtSK dengan nilai IC50 = 1,49 ± 0,17 µg/mL. Namun, aktivitas ini mengalami penurunan pada tingkat seluler terhadap Mycobacterium bovis BCG (EC50 >100 µg/mL) karena kendala penetrasi senyawa dalam menembus lapisan tebal dinding sel mikobakterium. Campuran senyawa 1 dan 2 tidak memiliki efek sitotoksisitas terhadap sel MCF-7 (CC50 = 121,4 ± 8,2 µg/mL) Untuk memvalidasi mekanisme aksi terhadap enzim MtSK, penambatan molekuler (molecular docking) menunjukkan interaksi yang sangat kuat di dalam sisi aktif enzim MtSK. Senyawa campuran ini menunjukkan afinitas pengikatan tertinggi pada struktur kristal enzim MtSK (PDB: 2IYW) dengan energi bebas ikatan (?G) mencapai -8,64 ± 0,05 kcal/mol mendukung hasil eksperimen uji bioasai terhadap enzim MtSK. Sebagai kesimpulan, senyawa 1 (TPI-1) dan 2 (TPI-2) adalah senyawa aktif kelas baru terhadap enzim MtSK yang memiliki scaffold didepsida. Senyawa 1 dan 2 tidak memiliki efek samping sitotoksitas sehingga dapat dikembangkan lebih lanjut. Modifikasi struktur diharapkan dapat meningkatkan kemampuan aktivitas biologis dan kemampuan senyawa dalam menembus dinding sel sehingga menjadi kandidat obat anti-TB generasi baru.

The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb), including Rifampicin-Resistant Tuberculosis (RR-TB), Multidrug-Resistant Tuberculosis (MDR-TB), and Extensively Drug-Resistant Tuberculosis (XDR-TB), has created an urgent need for the discovery of novel therapeutic scaffolds targeting essential bacterial pathways. One such target is Mycobacterium tuberculosis shikimate kinase (MtSK). In this study, novel MtSK inhibitors were discovered and identified through the screening of 3,480 microbial extracts. Bioassay-guided fractionation of the culture of the fungus Cosmospora sp. BioMCC-f.T-7332 led to the isolation of two didepside compounds, TPI-1 (1) and TPI-2 (2), obtained as an inseparable mixture. The molecular structures of compounds 1 and 2 were comprehensively elucidated using high-resolution mass spectrometry (HRMS), one- and two-dimensional nuclear magnetic resonance (NMR) (1D and 2D NMR) spectroscopy, and comparison with published data. Biological evaluation revealed that the inseparable mixture of compounds 1 and 2 exhibited potent inhibitory activity against MtSK, with an IC50 value of 1.49 ± 0.17 µg/mL. However, its activity was markedly reduced at the cellular level against Mycobacterium bovis BCG (EC50 >100 µg/mL), most likely due to the limited penetration of the compounds through the thick mycobacterial cell wall. In addition, the mixture showed no cytotoxic effects toward MCF-7 cells, with a CC50 value of 121.4 ± 8.2 µg/mL. To validate the mechanism of action against MtSK, molecular docking analysis demonstrated strong interactions between the compound mixture and the enzyme active site. The mixture exhibited the highest binding affinity toward the MtSK crystal structure (PDB ID: 2IYW), with a binding free energy (?G) of -8.64 ± 0.05 kcal/mol, supporting the experimental findings from the enzyme bioassay. In conclusion, compounds 1 (TPI-1) and 2 (TPI-2) represent a new class of MtSK inhibitors possessing a didepside scaffold. These compounds exhibited no significant cytotoxicity and therefore represent promising lead compounds for further development. Structural optimization is expected to improve both their biological activity and their ability to penetrate the mycobacterial cell wall, thereby enhancing their potential as next-generation anti-tuberculosis drug candidates.