Viruses of the Caliciviridae cause significant and sometimes lethal diseases, however despite substantial research efforts, specific antivirals are lacking. Broad-spectrum antivirals could combat multiple viral pathogens, offering a rapid solution when no therapies exist. The RNA-dependent RNA polymerase (RdRp) is an attractive antiviral target as it is essential for viral replication and lacks mammalian homologs. To focus the search for pan-Caliciviridae antivirals, the RdRp was probed with non-nucleoside inhibitors (NNIs) developed against hepatitis C virus (HCV) to reveal both allosteric ligands for structure-activity relationship enhancement, and highly-conserved RdRp pockets for antiviral targeting. The ability of HCV NNIs to inhibit calicivirus RdRp activities was assessed using in vitro enzyme and murine norovirus cell culture assays. Results revealed that three NNIs which bound the HCV RdRp Thumb I (TI) site also inhibited transcriptional activities of six RdRps spanning the Norovirus, Sapovirus and Lagovirus genera of the Caliciviridae. These NNIs included JTK-109 (RdRp inhibition range: IC₅₀ 4.3–16.6 μM), TMC-647055 (IC₅₀ range: 18.8–45.4 μM) and Beclabuvir (IC₅₀ range: 23.8–>100 μM). In silico studies and site-directed mutagenesis indicated the JTK-109 binding site was within the calicivirus RdRp thumb domain, in a pocket termed Site-B, which is highly-conserved within all calicivirus RdRps. Additionally, RdRp inhibition assays revealed that JTK-109 was antagonistic with the previously reported RdRp inhibitor pyridoxal-5′-phosphate-6-(2′-naphthylazo-6′-nitro-4′,8′-disulfonate) tetrasodium salt (PPNDS), that also binds to Site-B. Moreover, like JTK-109, PPNDS was also a potent inhibitor of polymerases from six viruses spanning the three Caliciviridae genera tested (IC₅₀ range: 0.1–2.3 μM). Together, this study demonstrates the potential for de novo development of broad-spectrum antivirals that target the highly-conserved RdRp thumb pocket, Site-B. We also revealed three broad-spectrum HCV NNIs that could be used as antiviral scaffolds for further development against caliciviruses and other viruses.

该病毒杯状病毒引起显著，有时甚至致命的疾病，然而，尽管大量的研究工作，具体的抗病毒药物缺乏。广谱抗病毒药可以对抗多种病毒病原体，在不存在任何疗法的情况下提供快速解决方案。RNA依赖性RNA聚合酶（RdRp）是有吸引力的抗病毒靶标，因为它对病毒复制至关重要，并且缺乏哺乳动物同源物。集中搜索泛杯状病毒科对于抗病毒药，用针对丙型肝炎病毒（HCV）开发的非核苷抑制剂（NNI）来探测RdRp，以揭示用于构效关系增强的变构配体，以及用于抗病毒靶向的高度保守的RdRp口袋。使用体外酶和鼠诺如病毒细胞培养测定法评估了HCV NNI抑制杯状病毒RdRp活性的能力。结果显示，其绑定的HCV病毒RdRp拇指I（TI）网站也六个RdRps跨越抑制转录活性3周的NNI诺如病毒，沙波病毒和Lagovirus的的属杯状病毒。这些NNI包括JTK-109（RdRp抑制范围：IC ₅₀ 4.3-16.6μM），TMC-647055（IC₅₀范围：18.8–45.4μM）和Beclabuvir（IC ₅₀范围：23.8–> 100μM）。在计算机研究和定点诱变中，JTK-109结合位点位于杯状病毒RdRp拇指域内，位于一个称为Site-B的口袋中，在所有杯状病毒RdRps中高度保守。此外，RdRp抑制试验表明JTK-109与先前报道的RdRp抑制剂吡ido醛-5'-磷酸-6-（2'-萘偶氮-6'-硝基-4'，8'-二磺酸盐）四钠盐（PPNDS）拮抗），这也绑定到Site-B。而且，与JTK-109一样，PPNDS也是一种有效的聚合酶抑制剂，这些酶来自六种病毒，跨越三种经过测试的杯状病毒科（IC ₅₀范围：0.1–2.3μM）。总之，这项研究表明，针对高度保守的RdRp拇指袋Site-B的广谱抗病毒药物有可能从头开发。我们还揭示了三种广谱的HCV NNI，它们可以用作抗病毒支架，以进一步对抗杯状病毒和其他病毒。