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The approval of COVID-19 vaccines and antiviral drugs has been crucial to end the global health crisis caused by SARS-CoV-2. However, to prepare for future outbreaks from drug-resistant variants and novel zoonotic coronaviruses (CoVs), additional therapeutics with a distinct antiviral mechanism are needed. Here, we report a novel guanidine-substituted diphenylurea compound that suppresses CoV replication by interfering with the uridine-specific endoribonuclease (EndoU) activity of the viral non-structural protein-15 (nsp15). This compound, designated EPB-113, exhibits strong and selective cell culture activity against human coronavirus 229E (HCoV-229E) and also suppresses the replication of SARS-CoV-2. Viruses, selected under EPB-113 pressure, carried resistance sites at or near the catalytic His250 residue of the nsp15-EndoU domain. Although the best-known function of EndoU is to avoid induction of type I interferon (IFN-I) by lowering the levels of viral dsRNA, EPB-113 was found to mainly act via an IFN-independent mechanism, situated during viral RNA synthesis. Using a combination of biophysical and enzymatic assays with the recombinant nsp15 proteins from HCoV-229E and SARS-CoV-2, we discovered that EPB-113 enhances the EndoU cleavage activity of hexameric nsp15, while reducing its thermal stability. This mechanism explains why the virus escapes EPB-113 by acquiring catalytic site mutations which impair compound binding to nsp15 and abolish the EndoU activity. Since the EPB-113-resistant mutant viruses induce high levels of IFN-I and its effectors, they proved unable to replicate in human macrophages and were readily outcompeted by the wild-type virus upon co-infection of human fibroblast cells. Our findings suggest that antiviral targeting of nsp15 can be achieved with a molecule that induces a conformational change in this protein, resulting in higher EndoU activity and impairment of viral RNA synthesis. Based on the appealing mechanism and resistance profile of EPB-113, we conclude that nsp15 is a challenging but highly relevant drug target.新冠疫苗与抗病毒药物的获批，对于终结由严重急性呼吸综合征冠状病毒2（SARS-CoV-2）引发的全球卫生危机至关重要。然而，为应对由耐药毒株及新型人畜共患冠状病毒（CoVs）引发的未来疫情暴发，亟需开发具有独特抗病毒机制的新型治疗手段。本研究报道了一种新型胍基取代二苯基脲类化合物，其通过干扰病毒非结构蛋白15（nsp15）的尿苷特异性核糖核酸内切酶（EndoU）活性，从而抑制冠状病毒复制。该化合物被命名为EPB-113，对人冠状病毒229E（HCoV-229E）展现出强效且具有选择性的细胞培养活性，同时也能抑制SARS-CoV-2的复制。在EPB-113压力筛选下获得的耐药病毒，其nsp15-EndoU结构域的催化组氨酸250（His250）残基位点或其邻近区域存在耐药突变位点。尽管EndoU最广为人知的功能是通过降低病毒双链RNA（dsRNA）水平来避免I型干扰素（IFN-I）的诱导，但研究发现EPB-113主要通过不依赖于IFN的机制发挥作用，该作用发生于病毒RNA合成阶段。本研究结合生物物理与酶学实验，对HCoV-229E和SARS-CoV-2的重组nsp15蛋白进行检测，发现EPB-113可增强六聚体nsp15的EndoU剪切活性，同时降低其热稳定性。这一机制解释了病毒如何通过催化位点突变逃逸EPB-113的作用：这类突变会削弱化合物与nsp15的结合能力，并彻底消除EndoU的活性。由于EPB-113耐药突变病毒可诱导高水平的IFN-I及其效应分子，这类病毒无法在人巨噬细胞中复制，且在人成纤维细胞的共感染实验中，会被野生型病毒快速竞争淘汰。本研究结果表明，通过诱导nsp15发生构象变化的分子，可实现针对该靶点的抗病毒干预，这类分子能提升EndoU活性并干扰病毒RNA合成。基于EPB-113的优异作用机制与耐药特性，本研究认为nsp15是一个兼具挑战性与极高研究价值的药物靶点。