Nurkanto Arif, Erdian Tampubolon Joseph Calvin, Ewaldo Muhammad Farrel, Putri Ade Lia, Ratnakomala Shanti, Setiawan Ruby, Fathoni Ahmad, Palupi Kartika Dyah, Rahmawati Yulia, Waluyo Danang, Prabandari Erwahyuni Endang, Pujiyanto Sri, Sumii Yuji, Agusta Andria, Shibata Norio, Matsumoto Sohkichi, Nozaki Tomoyoshi
Research Center for Biosystematics and Evolution, Research Organization for Life Sciences and Environmental, National Research and Innovation Agency (BRIN), West Java, Indonesia.
Graduate School of Medicine, The University of Tokyo, Tokyo, Japan.
Heliyon. 2024 Aug 4;10(15):e35648. doi: 10.1016/j.heliyon.2024.e35648. eCollection 2024 Aug 15.
Tuberculosis (TB) is the foremost cause of infectious fatality globally. The primary global challenge in combatting TB lies in addressing the emergence of drug-resistant variants of the disease. However, the number of newly approved agents for treating TB has remained remarkably low over recent decades. Hence, research endeavors for discovering novel anti-TB agents are always needed. In the present study, we screened over 1,500 culture extracts from actinomycetes isolated in Indonesia for their inhibitory activity against used as a surrogate in the primary screening. The initial screening yielded approximately 6.2 % hit extracts, with a selection criterion of >80 % growth inhibition. The confirmed hit extracts were subsequently subjected to growth inhibition assay against and Approximately 20 % of the hit extracts that showed growth inhibition also exhibited efficacy against BCG and H37Rv pathogenic strain An active compound was successfully purified from a large-scale culture of the most potent representative extract by high-performance liquid chromatography and thin-layer chromatography. The structure of the active compound was elucidated by mass spectrometry and nuclear magnetic resonance. This compound displayed structural similarities to actinomycin group and exhibited robust inhibition, with IC values of 0.74, 0.02, and 0.07 μg/mL against , and , respectively. The Actinomycetes strain A612, which produced the active compound, was taxonomically classified by phylogenetic analysis of 16s rRNA gene and whole genome sequencing data as . Computational genome analysis utilizing anti-SMASH 7.0 unveiled that A612 strain harbors 40 biosynthetic gene clusters with the potential to produce 16 known (with >70 % similarity) and 24 unknown compounds. A non-ribosomal peptide synthesis (NRPS) gene cluster associated with actinomycin D biosynthesis was also identified, boasting an 85 % similarity. Molecular docking analysis of actinomycin D and 21 potential targets revealed possible interactions with multiple targets. The purified active compound inhibited recombinant shikimate kinase (SK), which validated the results obtained from the docking analysis.
结核病（TB）是全球传染性死亡的首要原因。抗击结核病的主要全球挑战在于应对该疾病耐药变体的出现。然而，近几十年来新批准的治疗结核病药物数量一直非常低。因此，始终需要开展发现新型抗结核药物的研究工作。在本研究中，我们筛选了1500多种从印度尼西亚分离的放线菌培养提取物，检测它们对用作初筛替代物的 的抑制活性。初筛产生了约6.2%的阳性提取物，选择标准为生长抑制率>80%。随后对确认的阳性提取物进行针对 和 的生长抑制试验。约20%显示生长抑制的阳性提取物对卡介苗（BCG）和H37Rv致病菌株也具有疗效。通过高效液相色谱和薄层色谱从最有效的代表性提取物的大规模培养物中成功纯化出一种活性化合物。通过质谱和核磁共振确定了该活性化合物的结构。该化合物与放线菌素组具有结构相似性，并表现出强大的抑制作用，对 、 和 的IC值分别为0.74、0.02和0.07μg/mL。通过对16s rRNA基因和全基因组测序数据进行系统发育分析，对产生该活性化合物的放线菌菌株A612进行了分类学分类，结果为 。利用anti-SMASH 7.0进行的计算基因组分析表明，A612菌株含有40个生物合成基因簇，有可能产生16种已知（相似度>70%）和24种未知化合物。还鉴定了一个与放线菌素D生物合成相关的非核糖体肽合成（NRPS）基因簇，相似度为85%。对放线菌素D和21个潜在 靶点的分子对接分析揭示了与多个靶点的可能相互作用。纯化的活性化合物抑制重组莽草酸激酶（SK），这验证了对接分析获得的结果。
