Red Sea Research Center, Division of Biological and Environmental Science and Engineering (BESE), King Abdullah University of Science and Technology (KAUST), Thuwal 23955-6900, Saudi Arabia.
Scripps Institution of Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA.
Molecules. 2018 Jun 18;23(6):1472. doi: 10.3390/molecules23061472.
Viruses are underrepresented as targets in pharmacological screening efforts, given the difficulties of devising suitable cell-based and biochemical assays. In this study we found that a pre-fractionated organic extract of the Red Sea sponge was able to inhibit the West Nile Virus NS3 protease (WNV NS3). Using liquid chromatography⁻mass spectrometry (LC-MS) and nuclear magnetic resonance (NMR) spectroscopy, the identity of the bioactive compound was determined as a 3-alkylpyridinium with / = 190.16. Diffusion Ordered Spectroscopy (DOSY) NMR and NMR relaxation rate analysis suggest that the bioactive compound forms oligomers of up to 35 kDa. We observed that at 9.4 μg/mL there was up to 40⁻70% inhibitory activity on WNV NS3 protease in orthogonal biochemical assays for solid phase extracts (SPE) of . However, the LC-MS purified fragment was effective at inhibiting the protease up to 95% at an approximate amount of 2 µg/mL with negligible cytotoxicity to HeLa cells based on a High-Content Screening (HCS) cytological profiling strategy. To date, 3-alkylpyridinium type natural products have not been reported to show antiviral activity since the first characterization of halitoxin, or 3-alkylpyridinium, in 1978. This study provides the first account of a 3-alkylpyridinium complex that exhibits a proposed antiviral activity by inhibiting the NS3 protease. We suggest that the here-described compound can be further modified to increase its stability and tested in a cell-based assay to explore its full potential as a potential novel antiviral capable of inhibiting WNV replication.
鉴于设计合适的基于细胞和生化测定法存在困难，病毒在药理学筛选工作中的靶点代表性不足。在这项研究中，我们发现红海海绵的预分级有机提取物能够抑制西尼罗河病毒 NS3 蛋白酶（WNV NS3）。通过液相色谱-质谱（LC-MS）和核磁共振（NMR）光谱，确定生物活性化合物的身份为 3-烷基吡啶盐，I = 190.16。扩散有序光谱（DOSY）NMR 和 NMR 弛豫率分析表明，生物活性化合物形成高达 35 kDa 的寡聚物。我们观察到，在 9.4 μg/mL 时，在正交生化测定固相萃取（SPE）的提取物中，对 WNV NS3 蛋白酶的抑制活性高达 40-70%。然而，LC-MS 纯化片段在近似 2 µg/mL 时对蛋白酶的抑制作用有效，高达 95%，且基于高内涵筛选（HCS）细胞学分析策略，对 HeLa 细胞的细胞毒性可忽略不计。迄今为止，自 1978 年首次表征卤毒素或 3-烷基吡啶盐以来，尚未有报道表明 3-烷基吡啶盐型天然产物具有抗病毒活性。本研究首次描述了一种 3-烷基吡啶盐复合物，通过抑制 NS3 蛋白酶显示出拟议的抗病毒活性。我们建议进一步修饰此处描述的化合物以提高其稳定性，并在细胞测定中进行测试，以探索其作为能够抑制 WNV 复制的新型潜在抗病毒药物的全部潜力。
