人巨细胞病毒（HCMV，人类疱疹病毒5型）是一种机会性病原体，负责导致免疫功能受损患者出现严重疾病。目前的抗病毒治疗主要依靠干扰病毒DNA复制和封装的药物。然而，现有药物的严重副作用和耐药性的出现表明需要新的抗HCMV治疗靶点。其中一个靶点是病毒碱性核酸酶（AN），这是一种在疱疹病毒科中高度保守的酶。在本研究中，我们通过证明由病毒UL98开放阅读框编码的HCMV AN是一种药物靶点，验证了该靶点。通过证明UL98缺失的HCMV突变体在细胞培养中的传播能力大大减弱。我们建立了一种适用于高通量筛选的荧光酶活测定，并将其用于小分子化合物库。最有希望的化合物是一种噻杂噻唑并[3,4-a]喹唑啉衍生物，在体内阻断了AN的活性，并在溶斑还原（PRA）和荧光还原测定（FRA）中抑制了HCMV的复制。对中间体化合物进行了测试，其中一些具有相似或更好的抑制活性。在中间体A的最有效衍生物AD-51中，抑制HCMV复制的50%有效浓度（EC50）在FRA中为0.9μM，在PRA中为1.1μM。AD-51也对甘昔洛韦、福昔洛韦和勒托莫韦耐药的HCMV具有活性。此外，它还抑制单纯疱疹病毒、Kaposi's肉瘤相关疱疹病毒和小鼠巨细胞病毒（作为HCMV模型的小鼠病毒）。这些结果表明，噻杂噻唑并[3,4-a]喹唑啉衍生物是一类针对病毒AN的新型疱疹病毒抑制剂。版权所有© 2023 Elsevier B.V. 出版。

Human cytomegalovirus (HCMV, human herpesvirus 5) is an opportunistic pathogen responsible for serious disease in immunocompromised patients. Current antiviral therapies rely predominantly on drugs interfering with viral DNA replication and packaging. However, the serious side effects of existing drugs and the emergence of drug resistance indicate the need for new targets for anti-HCMV therapy. One such target is the viral alkaline nuclease (AN), an enzyme highly conserved among the Herpesviridae. In this study, we validated the HCMV AN, encoded by the viral UL98 open reading frame, as a drug target by demonstrating that a UL98-deficient HCMV mutant is severely attenuated and shows a reduced ability to spread in cell culture. We established a fluorescence-based enzyme assay suitable for high-throughput screening and used it on a small-molecule compound library. The most promising hit, a thioxothiazolo[3,4-a]quinazoline derivative, blocked AN activity in vitro and inhibited HCMV replication in plaque reduction (PRA) and fluorescence reduction assays (FRA). Several derivatives of the hit compound were tested, some of which had similar or better inhibitory activities. The most potent derivative of hit scaffold A, compound AD-51, inhibited HCMV replication with a 50% effective concentrations (EC50) of 0.9 μM in the FRA and 1.1 μM in the PRA. AD-51 was also active against ganciclovir, foscarnet, and letermovir-resistant HCMVs. Moreover, it inhibited herpes simplex virus, Kaposi's sarcoma-associated herpesvirus, and murine CMV, a mouse virus serving as a model for HCMV. These results suggest that thioxothiazolo[3,4-a]quinazoline derivatives are a new class of herpesvirus inhibitors targeting the viral AN.Copyright © 2023. Published by Elsevier B.V.