Discovery of Small-Molecule Orthopoxvirus Resolvase Inhibitors with Antiviral Activity

Poxvirus genome replication and viral maturation require
a Holliday
junction resolvase. Genetic evidence suggests that targeting the virally
encoded resolvase could offer a novel antiviral approach against orthopoxviruses.
However, orthopoxvirus resolvases have not been characterized biochemically
and pharmacologically, and inhibitors remain unknown. Herein, we have
developed and optimized the first in vitro assay
directly measuring the activity of mpox virus (MPXV) resolvase (Mpr)
and vaccinia virus (VACV) resolvase (A22). The subsequent pilot screen
of an in-house compound library using this assay identified multiple
inhibitors, each inhibiting both Mpr and A22, and conferring antiviral
activity against VACV. Computationally, these inhibitors docked well
into the active site of an AlphaFold-generated Mpr structural model.
The assay developed herein and the inhibitors identified and characterized
provide a valuable platform for developing compounds as broad-spectrum
antiviral drug leads against MPXV and other potentially emerging orthopoxviruses,
and as probes to investigate the functions of Mpr.

痘病毒（Poxvirus）的基因组复制与病毒成熟过程均依赖于霍利迪连接体解离酶（Holliday junction resolvase）。遗传学证据表明，靶向病毒编码的该解离酶，可为对抗正痘病毒（orthopoxvirus）提供全新的抗病毒策略。然而，目前尚未对正痘病毒解离酶开展生化与药理学表征，且其特异性抑制剂仍未被发现。本研究中，我们开发并优化了首个可直接检测猴痘病毒（mpox virus, MPXV）解离酶（Mpr）以及痘苗病毒（vaccinia virus, VACV）解离酶（A22）活性的体外检测体系。随后，利用该体系对自有化合物库进行初步筛选，我们获得了多款可同时抑制Mpr与A22的抑制剂，且这些抑制剂均展现出对抗痘苗病毒的抗病毒活性。通过计算建模分析发现，这些抑制剂可精准结合于AlphaFold预测构建的Mpr结构模型的活性位点内。本研究开发的体外检测体系，以及本次发现并表征的抑制剂，可为开发针对猴痘病毒及其他潜在新兴正痘病毒的广谱抗病毒先导化合物，以及作为探究Mpr功能的分子探针，提供极具价值的研究平台。