Human carbonic anhydrase isozyme II has been used as protein target for disorder treatment including glaucoma. Current clinically used sulfonamide-based CA inhibitors can induce side effects, and so alternatives are required. This study aimed to investigate a natural CA inhibitor from Murraya paniculata. The previously developed yeast-based assay was used to screen 14 compounds isolated from M. paniculata and identified by NMR analysis for anti-human CA isozyme II (hCAII) activity. Cytotoxicity of the compounds was also tested using the same yeast-based assay but in a different cultivation condition. Two flavonoid candidate compounds, 5, 6, 7, 8, 3', 4', 5'-heptamethoxyflavone (4) and 3 ,5, 7, 8, 3', 4', 5'-heptamethoxyflavone (9), showed potent inhibitory activity against hCAII with a minimal effective concentration of 10.8 and 21.5 μM, respectively, while they both exhibited no cytotoxic effect even at the highest concentration tested (170 μM). The results from an in vitro esterase assay of the two candidates confirmed their hCAII inhibitory activity with IC₅₀ values of 24.0 and 34.3 μM, respectively. To investigate the potential inhibition mechanism of compound 4, in silico molecular docking was performed using the FlexX and Swissdock software. This revealed that compound 4 coordinated with the Zn²⁺ ion in the hCAII active site through its methoxy oxygen at a distance of 1.60 Å (FlexX) or 2.29 Å (Swissdock). The interaction energy of compound 4 with hCAII was -13.36 kcal/mol. Thus, compound 4 is a potent novel flavonoid-based hCAII inhibitor and may be useful for further anti-CAII design and development.

中文翻译：

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来自九里香的两种基于类黄酮的化合物作为新型人碳酸酐酶同工酶 II 抑制剂通过基于刃天青酵母的测定法检测。

人碳酸酐酶同工酶 II 已被用作包括青光眼在内的疾病治疗的蛋白质靶点。目前临床上使用的基于磺胺类的 CA 抑制剂会引起副作用，因此需要替代品。本研究旨在研究一种来自九里香的天然 CA抑制剂。先前开发的基于酵母的测定用于筛选从M. paniculata分离的 14 种化合物并通过 NMR 分析鉴定抗人 CA 同工酶 II (hCAII) 活性。还使用相同的基于酵母的测定法但在不同的培养条件下测试了化合物的细胞毒性。两种类黄酮候选化合物，5, 6, 7, 8, 3', 4', 5'-七甲氧基黄酮 (4) 和 3,5, 7, 8, 3', 4', 5'-七甲氧基黄酮 (9)，表明对 hCAII 的有效抑制活性分别为 10.8 和 21.5 μM，而即使在测试的最高浓度（170 μM）下，它们都没有表现出细胞毒性作用。两种候选物的体外酯酶测定结果证实了它们具有 IC _{50的 hCAII 抑制活性}值分别为 24.0 和 34.3 μM。为了研究化合物 4 的潜在抑制机制，使用 FlexX 和 Swissdock 软件进行了计算机分子对接。这表明化合物 4 通过其甲氧基氧与 hCAII 活性位点中的 Zn ²⁺离子配位，距离为 1.60 Å (FlexX) 或 2.29 Å (Swissdock)。化合物4与hCAII的相互作用能为-13.36 kcal/mol。因此，化合物 4 是一种有效的新型类黄酮类 hCAII 抑制剂，可用于进一步的抗 CAII 设计和开发。