Health Biotechnology Division, Pakistan Institute of Engineering and Applied Sciences, National Institute for Biotechnology and Genetic Engineering College, (NIBGE-C, PIEAS), Faisalabad, Punjab, 38000, Pakistan.
School of Biological Sciences, University of the Punjab, Lahore, 54810, Punjab, Pakistan.
BMC Complement Med Ther. 2022 Apr 2;22(1):98. doi: 10.1186/s12906-022-03578-1.
Hepatitis C virus infection is the main cause of liver ailments across the globe. Several HCV genotypes have been identified in different parts of the world. Effective drugs for combating HCV infections are available but not affordable, particularly to infected individuals from resource-limited countries. Hence, cost-effective drugs need to be developed against important HCV drug targets. As Citrus fruits naturally contain bioactive compounds with antiviral activities, the current study was designed to identify antiviral inhibitors from Citrus fruit extracts against an important drug target, NS3 protease, of HCV genotype 3a which is found predominantly in South Asian countries.
The full-length NS3 protease alone and the NS3 protease domain in fusion with the cognate NS4A cofactor were expressed in Escherichia coli, and purified by chromatographic techniques. Using the purified protein as a drug target, Citrus extracts were evaluated in a FRET assay, and active ingredients, identified using ESI-MS/MS, were docked to observe the interaction with active site residues of NS3. The best interacting compound was further confirmed through the FRET assay as the inhibitor of NS3 protease.
Fusion of the NS3 protease domain to the NS4A cofactor significantly improved the purification yield, and NS3-NS4A was functionally more active than the full-length NS3 alone. The purified protein (NS3-NS4A) was successfully employed in a validated FRET assay to evaluate 14 Citrus fruit extracts, revealing that the mesocarp extract of Citrus paradisi, and whole fruit extracts of C. sinesis, C. aurantinum, and C. reticulata significantly inhibited the protease activity of HCV NS3 protease (IC values of 5.79 ± 1.44 µg/mL, 37.19 ± 5.92 µg/mL, 42.62 ± 6.89 µg/mL, and 57.65 ± 3.81 µg/mL, respectively). Subsequent ESI-MS analysis identified a flavonoid, hesperidin, abundantly present in all the afore-mentioned Citrus extracts. Importantly, docking studies suggested that hesperidin interacts with active site residues, and acts as a potent inhibitor of NS3 protease, exhibiting an IC value of 11.34 ± 3.83 µg/mL.
A FRET assay was developed using NS3-NS4A protease, which was successfully utilized for the evaluation of Citrus fruit extracts. Hesperidin, a compound present in the Citrus extracts, was identified as the main flavonoid, which can serve as a cost-effective potent inhibitor of NS3 protease, and could be developed as a drug for antiviral therapy against HCV genotype 3a.
丙型肝炎病毒感染是全球肝脏疾病的主要原因。在世界不同地区已发现几种丙型肝炎病毒基因型。有针对丙型肝炎病毒感染的有效药物，但价格昂贵，尤其是对来自资源有限国家的感染者而言。因此，需要针对重要的丙型肝炎病毒药物靶点开发具有成本效益的药物。由于柑橘类水果天然含有具有抗病毒活性的生物活性化合物，因此本研究旨在从柑橘类水果提取物中鉴定针对丙型肝炎病毒基因型 3a 的重要药物靶点 NS3 蛋白酶的抗病毒抑制剂，丙型肝炎病毒基因型 3a 在南亚国家中发现。
全长 NS3 蛋白酶单独和与同源 NS4A 辅助因子融合的 NS3 蛋白酶结构域在大肠杆菌中表达，并通过色谱技术纯化。使用纯化的蛋白质作为药物靶点，在荧光各向异性（FRET）测定中评估柑橘提取物，并使用 ESI-MS/MS 鉴定活性成分，观察与 NS3 的活性位点残基的相互作用。使用 FRET 测定进一步确认最佳相互作用的化合物是 NS3 蛋白酶的抑制剂。
将 NS3 蛋白酶结构域与 NS4A 辅助因子融合可显著提高纯化产率，并且 NS3-NS4A 的功能比全长 NS3 更活跃。成功地将纯化的蛋白质（NS3-NS4A）用于经验证的 FRET 测定中，以评估 14 种柑橘类水果提取物，结果表明，葡萄柚的中果皮提取物以及整个甜橙、酸橙和红橘的果实提取物均能显著抑制丙型肝炎病毒 NS3 蛋白酶的活性（IC 值分别为 5.79 ± 1.44 µg/mL、37.19 ± 5.92 µg/mL、42.62 ± 6.89 µg/mL 和 57.65 ± 3.81 µg/mL）。随后的 ESI-MS 分析鉴定出一种在所有上述柑橘类提取物中含量丰富的类黄酮，柚皮苷。重要的是，对接研究表明，柚皮苷与活性位点残基相互作用，可作为 NS3 蛋白酶的有效抑制剂，其 IC 值为 11.34 ± 3.83 µg/mL。
开发了一种使用 NS3-NS4A 蛋白酶的 FRET 测定法，该测定法成功地用于评估柑橘类水果提取物。在柑橘提取物中发现的柚皮苷是一种主要的类黄酮，可作为 NS3 蛋白酶的经济有效的有效抑制剂，并可开发为针对丙型肝炎病毒基因型 3a 的抗病毒治疗药物。
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