School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, 4072, Australia.
Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Ein Helwan, Helwan University, Helwan, Egypt.
Chemistry. 2020 Jul 22;26(41):8958-8968. doi: 10.1002/chem.202000899. Epub 2020 Jul 1.
Ketol-acid reductoisomerase (KARI), the second enzyme in the branched-chain amino acid biosynthesis pathway, is a potential drug target for bacterial infections including Mycobacterium tuberculosis. Here, we have screened the Medicines for Malaria Venture Pathogen Box against purified M. tuberculosis (Mt) KARI and identified two compounds that have K values below 200 nm. In Mt cell susceptibility assays one of these compounds exhibited an IC value of 0.8 μm. Co-crystallization of this compound, 3-((methylsulfonyl)methyl)-2H-benzo[b][1,4]oxazin-2-one (MMV553002), in complex with Staphylococcus aureus KARI, which has 56 % identity with Mt KARI, NADPH and Mg yielded a structure to 1.72 Å resolution. However, only a hydrolyzed product of the inhibitor (i.e. 3-(methylsulfonyl)-2-oxopropanic acid, missing the 2-aminophenol attachment) is observed in the active site. Surprisingly, Mt cell susceptibility assays showed that the 2-aminophenol product is largely responsible for the anti-TB activity of the parent compound. Thus, 3-(methylsulfonyl)-2-oxopropanic acid was identified as a potent KARI inhibitor that could be further explored as a potential biocidal agent and we have shown 2-aminophenol, as an anti-TB drug lead, especially given it has low toxicity against human cells. The study highlights that careful analysis of broad screening assays is required to correctly interpret cell-based activity data.
酮酸还原异构酶（KARI）是支链氨基酸生物合成途径中的第二酶，是包括结核分枝杆菌在内的细菌感染的潜在药物靶点。在这里，我们针对纯化的结核分枝杆菌（Mt）KARI 筛选了抗疟疾药物风险基金病原体库，并鉴定出两种 K 值低于 200nm 的化合物。在 Mt 细胞敏感性测定中，其中一种化合物的 IC 值为 0.8μm。该化合物 3-((甲基磺酰基)甲基)-2H-苯并[b][1,4]恶嗪-2-酮（MMV553002）与金黄色葡萄球菌 KARI 共结晶，该 KARI 与 Mt KARI 的同源性为 56%，与 NADPH 和 Mg 共结晶，得到了分辨率为 1.72Å 的结构。然而，在活性部位仅观察到抑制剂的水解产物（即 3-(甲基磺酰基)-2-氧代丙酸，缺少 2-氨基酚连接物）。令人惊讶的是，Mt 细胞敏感性测定表明，2-氨基酚产物是母体化合物抗结核活性的主要原因。因此，3-(甲基磺酰基)-2-氧代丙酸被鉴定为一种有效的 KARI 抑制剂，可进一步作为潜在的杀菌剂进行探索，我们已经证明 2-氨基酚作为一种抗结核药物先导化合物，特别是因为它对人细胞的毒性低。该研究强调，需要仔细分析广泛的筛选测定，以正确解释基于细胞的活性数据。
