Viruses are responsible for some of the most deadly human diseases, yet available vaccines and antivirals address only a fraction of the potential viral human pathogens. Here, we provide a methodology for managing human herpesvirus (HHV) infection by covalently inactivating the HHV maturational protease via a conserved, non-catalytic cysteine (C161). Using human cytomegalovirus protease (HCMV Pr) as a model, we screened a library of disulfides to identify molecules that tether to C161 and inhibit proteolysis, then elaborated hits into irreversible HCMV Pr inhibitors that exhibit broad-spectrum inhibition of other HHV Pr homologs. We further developed an optimized tool compound targeted toward HCMV Pr and used an integrative structural biology and biochemical approach to demonstrate inhibitor stabilization of HCMV Pr homodimerization, exploiting a conformational equilibrium to block proteolysis. Irreversible HCMV Pr inhibition disrupts HCMV infectivity in cells, providing proof of principle for targeting proteolysis via a non-catalytic cysteine to manage viral infection.

病毒是一些最致命的人类疾病的罪魁祸首，但现有的疫苗和抗病毒药物只能解决一小部分潜在的人类病毒病原体。在这里，我们提供了一种通过保守的非催化半胱氨酸 (C161) 共价灭活 HHV 成熟蛋白酶来管理人类疱疹病毒 (HHV) 感染的方法。使用人巨细胞病毒蛋白酶 (HCMV Pr) 作为模型，我们筛选了二硫化物文库，以鉴定与 C161 相连并抑制蛋白水解的分子，然后将其精心设计成不可逆的 HCMV Pr 抑制剂，对其他 HHV Pr 同系物表现出广谱抑制作用。我们进一步开发了一种针对 HCMV Pr 的优化工具化合物，并使用综合结构生物学和生化方法来证明 HCMV Pr 同二聚化的抑制剂稳定性，利用构象平衡来阻止蛋白水解。不可逆的 HCMV Pr 抑制破坏细胞中的 HCMV 感染性，为通过非催化半胱氨酸靶向蛋白水解来控制病毒感染提供了原理证明。