Novartis Institute for Tropical Diseases, 10 Biopolis Road, Chromos Building, Singapore 138670.
J Virol. 2011 Jul;85(13):6548-56. doi: 10.1128/JVI.02510-10. Epub 2011 Apr 20.
Viral replication relies on the host to supply nucleosides. Host enzymes involved in nucleoside biosynthesis are potential targets for antiviral development. Ribavirin (a known antiviral drug) is such an inhibitor that suppresses guanine biosynthesis; depletion of the intracellular GTP pool was shown to be the major mechanism to inhibit flavivirus. Along similar lines, inhibitors of the pyrimidine biosynthesis pathway could be targeted for potential antiviral development. Here we report on a novel antiviral compound (NITD-982) that inhibits host dihydroorotate dehydrogenase (DHODH), an enzyme required for pyrimidine biosynthesis. The inhibitor was identified through screening 1.8 million compounds using a dengue virus (DENV) infection assay. The compound contains an isoxazole-pyrazole core structure, and it inhibited DENV with a 50% effective concentration (EC(50)) of 2.4 nM and a 50% cytotoxic concentration (CC(50)) of >5 μM. NITD-982 has a broad antiviral spectrum, inhibiting both flaviviruses and nonflaviviruses with nanomolar EC(90)s. We also show that (i) the compound inhibited the enzymatic activity of recombinant DHODH, (ii) an NITD-982 analogue directly bound to the DHODH protein, (iii) supplementing the culture medium with uridine reversed the compound-mediated antiviral activity, and (iv) DENV type 2 (DENV-2) variants resistant to brequinar (a known DHODH inhibitor) were cross resistant to NITD-982. Collectively, the results demonstrate that the compound inhibits DENV through depleting the intracellular pyrimidine pool. In contrast to the in vitro potency, the compound did not show any efficacy in the DENV-AG129 mouse model. The lack of in vivo efficacy is likely due to the exogenous uptake of pyrimidine from the diet or to a high plasma protein-binding activity of the current compound.
病毒复制依赖于宿主提供核苷。参与核苷生物合成的宿主酶是抗病毒药物开发的潜在靶点。利巴韦林（一种已知的抗病毒药物）就是这样一种抑制剂，它抑制鸟嘌呤的生物合成；细胞内 GTP 池的耗竭被证明是抑制黄病毒的主要机制。类似地，嘧啶生物合成途径的抑制剂可能成为潜在抗病毒药物开发的靶点。在这里，我们报告了一种新型的抗病毒化合物（NITD-982），它抑制宿主二氢乳清酸脱氢酶（DHODH），DHODH 是嘧啶生物合成所必需的酶。该抑制剂是通过使用登革热病毒（DENV）感染测定法筛选 180 万种化合物而鉴定出来的。该化合物含有异噁唑-吡唑核心结构，对 DENV 的 50%有效浓度（EC 50 ）为 2.4 nM，50%细胞毒性浓度（CC 50 ）>5 μM。NITD-982 具有广谱的抗病毒活性，对黄病毒和非黄病毒的半数有效浓度（EC 90 ）均为纳摩尔级。我们还表明：（i）该化合物抑制重组 DHODH 的酶活性；（ii）NITD-982 的类似物直接与 DHODH 蛋白结合；（iii）用尿苷补充培养基可逆转该化合物介导的抗病毒活性；（iv）对布雷奎纳（一种已知的 DHODH 抑制剂）耐药的 DENV-2 变体对 NITD-982 交叉耐药。总的来说，这些结果表明，该化合物通过耗尽细胞内嘧啶池来抑制 DENV。与体外效力相比，该化合物在 DENV-AG129 小鼠模型中没有表现出任何疗效。缺乏体内疗效可能是由于饮食中外源摄取嘧啶，或者由于目前化合物具有高血浆蛋白结合活性。
