兽研文章 | 预出版文章《科学报告》https://doi.org/10.1038/s41598-026-76801-5 《科学报告》本预出版文章提前共享，以便您更快获取新研究。该文章可引用，并具有永久DOI。最终编辑版本将自动替换本文。HSP90抑制剂对腮腺炎病毒及相关副黏病毒的抗病毒活性 Fumihiro Kato · Chaewon Bae · Aika Wakata · Masatoshi Kakizaki · Takashi Okura · Yuichiro Nakatsu · Fumio Seki · Kenji Someya · Noriyuki Otsuki · Akihide Ryo · Yusuke Matsumoto · Hideo Fukuhara · Katsumi Maenaka · Mari Ishigami-Yuasa · Minoru Kidokoro · Masaki Ochiai · Koji Ishii · Hiroshi Katoh · Makoto Takeda 收稿日期：2026年4月7日 / 接受日期：2026年10月15日 © 作者 2026 摘要 腮腺炎病毒（MuV）是副黏病毒科成员，尽管疫苗接种广泛普及，仍在全球范围内持续引起反复暴发。然而，目前尚无抗病毒药物获批用于MuV或其他副黏病毒。在本研究中，我们利用表达绿色荧光蛋白的重组MuV筛选了约1，000种小分子化合物，鉴定出六种能显著抑制病毒复制且未检测到细胞毒性的化合物。这些化合物是热休克蛋白90（HSP90）的已知抑制剂，HSP90是一种宿主伴侣蛋白，参与病毒聚合酶复合物的组装和功能。这些HSP90抑制剂表现出强效抗病毒活性，EC₅₀值处于亚微摩尔水平，且选择性指数高。此外，其抗病毒效力还扩展至其他副黏病毒，包括麻疹病毒（MeV）、犬瘟热病毒（CDV）和仙台病毒（SeV）。加药时间实验表明，这些化合物作用于病毒进入后阶段，主要在病毒RNA合成期间发挥作用。与此一致，MuV和尼帕病毒（NiV）的微型基因组实验证实了病毒RNA合成的强效抑制，且未直接抑制报告酶活性，表明其靶点特异性。总之，我们的研究结果凸显了HSP90抑制剂作为靶向宿主的抗副黏病毒药物的潜力，并支持进一步开发其用于治疗新发和再发副黏病毒感染。关键词 腮腺炎病毒 · 麻疹病毒 · 尼帕病毒 · HSP90 · 抗病毒化合物 · RNA复制 引言 MuV是副黏病毒科Orthorubulavirus属成员，在全球范围内流行。MuV感染通常引起以发热和腮腺肿胀为特征的轻至中度疾病；然而，它偶尔可导致严重并发症，包括无菌性脑膜炎和感音神经性听力损失1。尽管针对MuV的有效疫苗已经开发并广泛使用，但在多个地区尽管疫苗接种覆盖率很高，仍持续报告反复暴发2–5。除MuV外，其他
待发表文章《Scientific Reports》https://doi.org/10.1038/s41598-026-76801-5 副黏病毒作为新发或再发病原体构成重大威胁。麻疹病毒属成员，如MeV和CDV，以及亨尼帕病毒属物种如NiV，尤其值得关注6,7。MeV引起一种高度传染性疾病，特征为发热、鼻卡他、咳嗽和结膜炎，随后出现全身性斑丘疹8。CDV主要感染食肉动物并引起严重的全身性疾病；值得注意的是，最近报道了非人灵长类动物的自然感染9–12，这引发了对其作为影响人类社会的新发传染病的担忧13。NiV自2000年代初以来主要在东南亚引起了反复暴发，并与人类严重且往往致命的疾病相关。总体而言，副黏病毒的持续出现和再出现代表着日益增长的全球公共卫生关切14。尽管已有针对MuV和MeV的有效疫苗，但尚无抗病毒药物被批准用于治疗这些感染。已报道了几种候选抗病毒化合物15–19，但均未进入临床批准阶段。因此，目前MuV和MeV感染的临床管理仍以支持性治疗为主，侧重于缓解症状20,21。在CDV感染中，已报道利巴韦林和法匹拉韦等药物具有抗病毒活性；然而，支持其作为治疗性治疗的报道相对较少22,23。在NiV感染方面，目前既无许可疫苗也无抗病毒疗法可用24。副黏病毒具有约15–18 kb的单股负链RNA基因组，其复制周期在整个科中广泛保守。病毒进入后，核糖核蛋白（RNP）复合物释放到宿主细胞质中，病毒基因组在此转录为mRNA并翻译为病毒蛋白。RNP复合物由被核衣壳（N）蛋白包裹并与磷蛋白（P）和大（L）蛋白结合的病毒RNA组成，后者作为病毒RNA依赖性RNA聚合酶（RdRp）发挥作用25,26。新合成的病毒基因组RNA与N蛋白结合形成子代RNP，随后与病毒结构蛋白组装，包括基质（M）、融合（F）和受体结合糖蛋白——血凝素-神经氨酸酶（HN）、血凝素（H）或糖蛋白（G）——以产生感染性病毒粒子。重组病毒技术，特别是将报告基因整合到病毒基因组中，使得开发用于抗病毒药物发现的灵敏高通量筛选平台成为可能27–30。这些基于报告基因的系统能够有效监测病毒复制，并提供探究病毒生命周期多个阶段的优势，而非靶向单一预定步骤。我们此前开发了表达Aequorea coerulescens绿色荧光蛋白（AcGFP）的重组MuV，并成功利用该系统鉴定了新型抗MuV候选化合物30。在本研究中，我们评估了该重组MuV作为高通量筛选平台用于鉴定抗MuV候选药物的实用性。我们进一步研究了所鉴定化合物靶向的病毒生命周期阶段，并评估了它们对其他副黏病毒的抗病毒活性，包括MeV、CDV、SeV和NiV。材料与方法 细胞和病毒 A549/hSLAM和A549/hSLAM IFNAR1 KO细胞系（在我们此前的研究中建立30）培养于含10%胎牛血清（FBS）和青霉素-链霉素（Nacalai Tesque Co., Kyoto, Japan）的Dulbecco改良Eagle培养基（D-MEM）（FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan）中。VeroE6/TMPRSS2、Vero/hSLAM和Vero/dogSLAM细胞（同样在我们此前的研究中建立31–32）培养于含10% FBS、青霉素-链霉素和500 μg/mL G-418（Nacalai Tesque Co.）的D-MEM中。293T细胞培养于含10% FBS和青霉素-链霉素的D-MEM中。T7聚合酶
待刊于 Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 表达BHK-21细胞（BHK/T7-9）33购自RIKEN生物资源研究中心（BRC）细胞库（RCB4942，日本筑波），培养于含10%胎牛血清和青霉素-链霉素的Glasgow最低必需培养基（Thermo Fisher Scientific，美国马萨诸塞州沃尔瑟姆）中。原始野生型MuV Odate株和AcGFP-MuV的分子克隆已在既往研究中建立34,35。本研究所用MeV和CDV株分别源自IC-B株和Ac96I株36,37。表达RFP的SeV在既往研究中构建并惠赠38,39。化学文库筛选 为鉴定抗MuV化合物，筛选了购自Selleck Chemicals的小分子文库，该文库包含约1,000种生物活性化合物（补充表）。将A549/hSLAM IFNAR1 KO细胞以每孔3 × 10⁴个细胞接种于96孔板。24 h后，以MOI 0.01用AcGFP-MuV感染细胞，并以终浓度10 μM加入各化合物。孵育72 h后，使用荧光显微镜和BZ-X800 Analyzer软件1.4.0版测量GFP荧光强度以评估MuV复制40。为排除具有明显细胞毒性的化合物，在荧光测量后于同一筛选板中评估细胞活力。按照制造商方案向每孔加入Cell Count Reagent SF（Nacalai Tesque），并使用GloMax Discover Microplate Reader（Promega，美国威斯康星州麦迪逊）测量450 nm吸光度。已鉴定化合物的抗病毒效力评价 为确定已鉴定化合物的半数有效浓度（EC₅₀），进行了剂量-反应试验。将A549/hSLAM IFNAR1 KO细胞以每孔3 × 10⁵个细胞接种于24孔板。以MOI 0.01用AcGFP-MuV感染细胞1 h，随后将接种物替换为含指定浓度化合物的新鲜培养基。已鉴定的HSP90抑制剂在0至2 μM浓度范围内测试，而用作阳性对照的霉酚酸（MPA）在0至10 μM浓度范围内测试。孵育72 h后，使用荧光显微镜和BZ-X800 Analyzer软件1.4.0版测量GFP荧光强度以评估MuV复制40。还评估了已鉴定化合物对MeV、CDV和SeV的抗病毒活性。MeV、CDV和SeV感染分别使用A549/hSLAM细胞、Vero/dogSLAM细胞和VeroE6/TMPRSS2细胞。细胞感染各病毒后，在指定浓度化合物存在下孵育。对于MeV，收集培养上清，并通过空斑试验测定感染性病毒滴度。空斑经亚甲蓝染色显示并计数。对于CDV，在荧光显微镜下计数感染灶，病毒滴度以灶形成单位计算。对于SeV，如上文针对MuV所述，使用荧光显微镜和BZ-X800 Analyzer软件1.4.0版基于报告荧光强度评估病毒复制。EC₅₀值采用Reed-Muench法计算。已鉴定化合物的细胞毒性评估 为确定已鉴定化合物的半数细胞毒性浓度（CC₅₀），将细胞以每孔3 × 10⁴个细胞接种于96孔板，并在0至100 μM浓度范围内用各化合物系列稀释液培养3天。为评估SNX-2112在BHK/T7-9细胞中的细胞毒性，用SNX-2112系列稀释液处理细胞，浓度范围最高达100 μM，超过MuV和NiV微型基因组试验中所用的最高浓度。将细胞以每孔3 × 10⁴个细胞接种于96孔板，并在0至100浓度范围内用各化合物系列稀释液培养
预出版文章《Scientific Reports》https://doi.org/10.1038/s41598-026-76801-5 以2 μM处理2天。随后按照制造商说明书使用Cell Count Reagent SF（Nacalai Tesque）测定细胞活力。使用GloMax Discover微孔板读数仪测定450 nm处的吸光度。各浓度的吸光度值以溶剂处理对照组归一化，对照组设为100。数据以三次独立实验的平均值±标准差表示。HSP90抑制剂之间结构相似性的评估 使用在Python环境中实现的RDKit化学信息学工具包（版本2023.03.1）评估六种命中化合物（均为HSP90抑制剂）之间的化学相似性。使用Morgan指纹算法生成分子指纹，半径为2，指纹长度为1024位。基于生成的指纹计算Tanimoto系数，以定量评估每对化合物之间的结构相似性。加药时间测定实验 将A549/hSLAM IFNAR1 KO细胞以每孔3×10^5个细胞接种于12孔板中。孵育18小时后，以MOI为2的MuV感染细胞1小时。随后去除病毒接种物，用PBS洗涤细胞两次。然后，以2小时间隔向感染细胞中加入10 μM SNX-2112，直至20小时。感染20小时后收集上清液。通过空斑试验测定病毒滴度。微型基因组实验 本研究使用的MuV微型基因组基于先前报道的构建体30,35。将相应的微型基因组质粒转染BHK/T7-9细胞，并将测试化合物以不同浓度共孵育48小时。随后测定荧光素酶活性作为病毒RNA合成的指标。对于NiV微型基因组系统，编码N、P和L蛋白的质粒由Paul A. Rota博士惠赠41。本研究中构建的NiV微型基因组质粒通过将原始报告基因（氯霉素乙酰转移酶）替换为Nano荧光素酶基因而成。在微型基因组实验中，将BHK/T7-9细胞接种于24孔板，并使用TransIT-LT1转染试剂转染0.3 μg N质粒、0.2 μg P质粒、0.1 μg L质粒和0.2 μg微型基因组质粒。转染后立即加入化合物并孵育48小时，之后定量荧光素酶活性。MuV L和P/V蛋白的Western blot分析 将293T细胞接种于6孔板，并转染表达MuV N、P和L蛋白的质粒。用指定浓度的SNX-2112处理细胞36小时。随后在样品缓冲液中裂解细胞并进行SDS-PAGE。将蛋白质转移至膜上，转膜后切下约对应于100–250 kDa的膜区域，对MuV L蛋白进行免疫印迹。膜分别用抗MuV L、MuV P/V和α-微管蛋白抗体孵育。抗MuV L和P/V蛋白抗体与Katoh等人先前研究中所用抗体相同。抗α-微管蛋白抗体（克隆1E4C11）购自Proteintech公司。α-微管蛋白用作上样对照。使用ImageJ软件42定量蛋白条带强度。MuV L蛋白条带强度以α-微管蛋白归一化，并相对于溶剂处理对照组（设为100）表示。
预出版文章《Scientific Reports》https://doi.org/10.1038/s41598-026-76801-5 结果 从化合物库中筛选抗病毒化合物 我们以终浓度10 μM筛选了约1,000种小分子化合物，以鉴定MuV复制的候选抑制剂。该化合物库由具有已知药理活性的生物活性化合物组成。通过该筛选，我们鉴定出六种化合物——SNX-2112、CH5138303、PU-H71、PF-04929113、onalespib和HSP990——它们能抑制MuV复制，且均为特征明确的HSP90抑制剂（图1）。值得注意的是，PF-04929113（也称为SNX-5422）是SNX-2112的前药，与其活性形式显示出高度结构相似性（Tanimoto系数=0.777）。相比之下，其余命中化合物之间的结构相似性较低（最大Tanimoto系数≈0.13），表明这些命中化合物总体上结构多样。为定量评估抗病毒效力，我们使用基于病毒滴度的试验测定了每种HSP90抑制剂的半数最大有效浓度（EC₅₀）。所有六种化合物的EC₅₀值均低于1 μM（图2A–F）。同时，在未感染细胞中评估了细胞毒性，结果显示所有化合物的半数最大细胞毒性浓度（CC₅₀）值均超过100 μM（图2H–M）。这些结果表明，观察到的抗病毒效应并非归因于非特异性细胞毒性。图1 (A) SNX-2112、(B) CH5138303、(C) PU-H71、(D) PF-04929113、(E) HSP990和(F) Onalespib的化学结构。计算成对Tanimoto系数以可视化化合物之间的结构相似性矩阵。
预出版文章《Scientific Reports》https://doi.org/10.1038/s41598-026-76801-5 对其他副黏病毒的抗病毒活性 我们接下来使用基于报告基因的复制实验，检测所鉴定的HSP90抑制剂是否对其他副黏病毒（包括MeV、CDV和SeV）具有抗病毒活性。所有六种化合物均强效
预出版文章《Scientific Reports》https://doi.org/10.1038/s41598-026-76801-5 抑制了MeV和CDV的复制，其EC₅₀值仍处于亚微摩尔范围（表1）。在某些情况下，病毒滴度下降超过四个数量级。相比之下，虽然也观察到对SeV的抗病毒活性，但EC₅₀值较高（约1–2 μM），且总体抑制程度较弱，导致病毒复制大约下降十倍。通过加药时间实验估计复制的抑制步骤 为确定SNX-2112所靶向的病毒生命周期阶段——该化合物对MuV、MeV尤其是CDV表现出强抗病毒活性——我们进行了加药时间实验，即在感染后不同时间点加入化合物，随后在感染后20小时定量病毒滴度。SNX-2112在感染后8小时内加入时仍保持强抗病毒活性，而当加入时间进一步延迟时，其抑制作用逐渐下降（图3A）。这些结果表明，SNX-2112作用于MuV生命周期的进入后阶段，最可能是在RNA合成期间。在微型基因组实验中抑制病毒基因组复制 为直接评估SNX-2112是否抑制病毒RNA合成，我们采用了MuV微型基因组实验，其中荧光素酶活性作为病毒RNA合成的读数。SNX-2112处理导致荧光素酶活性呈剂量依赖性抑制，EC₅₀为101 nM（图3B），与使用感染性病毒获得的结果一致。为排除SNX-2112直接干扰荧光素酶酶活性的可能性，将细胞单独转染荧光素酶表达质粒并用SNX-2112处理。在这些条件下，荧光素酶活性未降低（图3C），证实该化合物抑制的是病毒RNA合成而非报告酶活性。即使浓度高达100 μM，SNX-2112也未明显影响细胞活力（图3E），表明微型基因组来源的荧光素酶活性降低不能归因于细胞毒性。评估SNX-2112对其他病毒种类的抗病毒活性 由于我们的设施不具备处理感染性NiV的条件，对NiV的抗病毒活性使用微型基因组复制/转录实验进行评估。SNX-2112以剂量依赖性方式有效抑制NiV微型基因组活性（图3D）。在NiV微型基因组系统中获得的EC₅₀值为14.6 nM，表明SNX-2112对不同副黏病毒属的RNA合成发挥保守的抑制作用。图2 (A) SNX-2112、(B) CH5138303、(C) PU-H71、(D) PF-04929113、(E) HSP990、(F) Onalespib和(G) 霉酚酸对MuV的抗病毒活性。每种化合物经系列稀释后与病毒共孵育。孵育后，通过定量GFP荧光强度评估抗病毒活性。各浓度下的GFP荧光强度以病毒感染、溶剂处理的对照归一化，对照设为100。所鉴定的HSP90抑制剂测试浓度为0至2 μM，而作为阳性对照的MPA测试浓度为0至10 μM。H) SNX-2112、I) CH5138303、(J) PU-H71、(K) PF-04929113、(L) HSP990、(M) Onalespib和(N) 霉酚酸的细胞毒性。每种化合物经系列稀释后与A549/hSLAM IFNAR1 KO细胞孵育。各浓度下的吸光度值以模拟处理对照归一化，对照设为100。抗病毒活性和细胞活力显示在单独的面板中，因为用于EC₅₀和CC₅₀测定的浓度范围差异很大。数据以三次独立实验的平均值 ± 标准差表示。
预出版文章《Scientific Reports》https://doi.org/10.1038/s41598-026-76801-5 SNX-2112降低MuV L蛋白表达 为探究本研究中鉴定出的HSP90抑制剂是否通过类似于既往报道的机制影响MuV L蛋白稳定性，我们分析了SNX-2112处理后MuV L蛋白的胞内水平。将表达MuV N、P和L蛋白的质粒转染293T细胞，并以指定浓度的SNX-2112处理。随后通过Western blotting检测MuV L和P/V蛋白水平。结果显示，作为内参加载对照的α-微管蛋白及MuV P/V蛋白的表达水平在任何SNX-2112浓度下均未见明显降低。相反，MuV L蛋白水平在较高SNX-2112浓度下显著下降（图3F和G）。这些发现与既往报道一致，即HSP90有助于MuV L蛋白的稳定性，并进一步支持SNX-2112至少部分通过降低L蛋白稳定性来抑制MuV RNA合成的观点。讨论 在本研究中，我们通过使用重组报告病毒的高通量筛选策略，鉴定出多种对MuV具有强效抗病毒活性的小分子化合物。在命中化合物中，有六种是已知的热休克蛋白90（HSP90）抑制剂，所有这些化合物均能强烈抑制MuV复制，且未检测到细胞毒性。这些发现凸显了靶向宿主伴侣蛋白通路作为抗副黏病毒策略的可行性。HSP90在众多客户蛋白的折叠、稳定和功能性组装中发挥关键作用，包括病毒聚合酶组分。既往研究表明，HSP90是MuV高效RNA合成所必需的35，且具有HSP90抑制活性的化合物（如17-AAG）不仅对MuV，还对MeV和NiV表现出抗病毒作用43。尽管17-AAG显示出强效抗病毒活性，但也有报道其具有高细胞毒性。相比之下，本研究中鉴定出的全部六种化合物的CC₅₀值均大于100 μM，因而具有高选择性指数。我们的筛选命中物中HSP90抑制剂的富集与这些早期观察结果一致，并进一步支持MuV复制高度依赖HSP90介导的伴侣蛋白活性的观点。尽管尚未进行体内毒性研究，但这些化合物代表了抗病毒药物开发的有前景的候选物。本研究中鉴定出的全部六种HSP90抑制剂均表现出亚微摩尔EC₅₀值和超过100 μM的CC₅₀值，因而具有高选择性指数。这些结果表明，观察到的抗病毒效应不太可能归因于非特异性细胞毒性，而是反映了对病毒生命周期中关键宿主依赖步骤的抑制。加药时间实验显示，SNX-2112在感染后长达8小时加入时仍保持抗病毒活性，这与进入后作用模式一致。这一解释进一步得到以下支持 表1 HSP90抑制剂对其他副黏病毒的抗病毒活性及在A549/hSLAM IFNAR1 KO细胞中的细胞毒性。化合物 对MuV的EC₅₀ 对MeV的EC₅₀ 对CDV的EC₅₀ 对SeV的EC₅₀ 在A549/hSLAM IFNAR1 KO细胞中的CC₅₀ 对MuV的选择性指数（CC₅₀/EC₅₀） SNX-2112 92 nM 44 nM 67 nM 1,926 nM >100,000 nM >1087 CH5138303 141 nM 66 nM 402 nM 2,893 nM >100,000 nM >709 PU-H71 199 nM 45 nM 943 nM 3,568 nM >100,000 nM >503 PF-04929113 75 nM 86 nM 293 nM 1,936 nM >100,000 nM >1333 HSP990 166 nM 34 nM 178 nM 2,913 nM >100,000 nM >602 Onalespib 35 nM 22 nM 410 nM 3,112 nM >100,000 nM >2857
预出版文章《Scientific Reports》https://doi.org/10.1038/s41598-026-76801-5 通过微型基因组试验，证明了对病毒复制/转录活性的强效抑制，且不影响荧光素酶酶活性。重要的是，这些HSP90抑制剂的抗病毒活性并不局限于MuV。这些化合物还对其他副黏病毒表现出强效抑制作用，包括MeV、CDV和NiV，提示其在副黏病毒中具有广谱抗病毒潜力。相比之下，对SeV的抗病毒效果不太明显。这一差异可能反映了不同副黏病毒属对HSP90依赖程度的差异。例如，正风疹病毒属和麻疹病毒属的RNA依赖性RNA聚合酶复合物可能比呼吸道病毒属更依赖HSP90的分子伴侣功能。或者，病毒聚合酶或辅因子蛋白的结构或序列差异可能影响对HSP90抑制的敏感性44–46。鉴于NiV的高致病性及缺乏获批的抗病毒疗法，SNX-2112在微型基因组系统中抑制NiV复制/转录活性的能力尤其值得注意。宿主导向的抗病毒策略，如HSP90抑制，可能通过限制耐药性的出现并使对新发或再发病毒威胁的快速响应成为可能而具有优势。尽管这些化合物在体外表现出极低的细胞毒性，但仍需进一步研究以评估其体内安全性特征。特别是，药代动力学、组织分布和潜在脱靶效应的评估对于治疗开发至关重要。此外，详细的生化和蛋白质组学分析对于阐明HSP90与副黏病毒聚合酶复合物之间的分子相互作用将是必要的。总之，我们的研究表明，包括SNX-2112在内的HSP90抑制剂有效抑制MuV和其他副黏病毒的复制，可能通过靶向病毒RNA合成的进入后步骤发挥作用。这些化合物的广谱抗病毒活性和高选择性凸显了其作为宿主导向抗病毒药物的潜力。这些发现为进一步开发针对HSP90的策略以对抗副黏病毒感染提供了基础。
预出版文章《Scientific Reports》https://doi.org/10.1038/s41598-026-76801-5
文章出版前版本《科学报告》https://doi.org/10.1038/s41598-026-76801-5 补充信息 在线版本包含补充材料，可在 https://doi.org/10.1038/s41598-026-76801-5 获取。致谢 我们感谢 Paul A. Rota 博士和 Naoto Ito 博士慷慨提供宝贵的研究试剂。我们还要感谢 Kentaro Fujita 博士、Seishiro Naito 博士和 Yuko Sasaki 博士提供材料和用品，以及在本研究全程给予的热情鼓励。我们也感谢国立感染症研究所质量管理体系中心的其他成员的热情鼓励。作者贡献 F.K.、C.B.、A.W.、M.Ka. 和 T.O. 进行了实验并分析了数据。F.K. 撰写了稿件。Y.N.、F.S.、K.S.、N.O.、Y.M.、H.F.、K.M.、M.Ki. 和 H.K. 提供了研究材料和试剂。M.I.Y. 和 M.O. 提供并管理了实验室设施。F.K.、Y.M.、A.R.、M.I.Y.、M.Ki.、M.O.、K.I.、H.K. 和 M.T. 对数据进行了解读。F.K.、K.M. 和 M.T. 为该项目获取了资助。所有作者讨论了结果、审阅了稿件并批准了最终版本。资助 本工作得到了日本学术振兴会资助，资助编号为 JP20K16020 和 JP24K10240，以及日本医疗研究开发机构（AMED）资助，资助编号为 JP22ama121037、JP223fa627005、23wm03025063j0401 和 JP223fa627001。数据可用性 支持本研究结果的所有数据均包含在本文中。其他原始数据可在合理请求下向通讯作者获取。声明 利益冲突 作者声明无利益冲突。开放获取 本文采用知识共享署名 4.0 国际许可协议授权，允许以任何媒介或格式使用、共享、改编、分发和复制，前提是您给予原作者和来源适当署名、提供知识共享许可协议的链接，并说明是否进行了修改。本文中的图片或其他第三方材料包含在文章的知识共享许可协议中，除非在材料的署名说明中另有说明。如果材料未包含在文章的知识共享许可协议中，且您的预期使用不被法定法规允许或超出许可使用范围，您需要直接向版权持有人获取许可。要查看该许可协议的副本，请访问 http://creativecommons.org/licenses/by/4.0/。图3 (A) SNX-2112 的加药时间试验。以感染复数（MOI）为 2 的病毒感染细胞，并在感染后指示时间点加入 SNX-2112。感染后 20 小时收集培养上清，并测定病毒滴度。(B) SNX-2112 处理 MuV 的微型基因组试验。用相应质粒转染细胞，随后加入系列稀释的 SNX-2112。孵育 48 小时后，测定荧光素酶活性以评估抗病毒活性。(C) SNX-2112 对荧光素酶活性的影响。用单独表达荧光素酶的质粒转染细胞，并与 10 μM SNX-2112 共孵育 48 小时。(D) SNX-2112 对 NiV 病毒 RNA 合成的抑制活性。用相应质粒转染细胞，随后加入系列稀释的 SNX-2112。孵育 48 小时后，测定荧光素酶活性以评估抗病毒活性。(E) 用指示浓度的 SNX-2112 处理细胞，浓度范围远高于 MuV 和 NiV 微型基因组试验中所用浓度。各浓度的吸光度值以溶剂处理对照归一化，对照设为 100。数据以三次独立实验的平均值 ± 标准差表示。(F) MuV L 蛋白表达的定量。L 蛋白水平以 α-微管蛋白归一化，并相对于溶剂处理对照表示，对照设为 100。数据以三次独立实验的平均值 ± 标准差表示。星号表示与溶剂处理对照相比具有统计学显著差异，采用配对双尾 Student t 检验确定（P < 0.05；P < 0.01）。(G) SNX-2112 处理后 MuV L 和 P/V 蛋白表达的 Western blot 分析。用表达 MuV N、P 和 L 蛋白的质粒转染细胞，并用指示浓度的 SNX-2112 处理，通过 Western blot 分析 L 和 P/V 蛋白水平。α-微管蛋白用作上样对照。为检测 MuV L 蛋白，在蛋白转印后物理切出约相当于 100–250 kDa 的膜区域并进行免疫印迹。
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预出版文章《科学报告》https://doi.org/10.1038/s41598-026-76801-5 1 质量管理体系中心，日本国立感染症研究所，日本健康安全研究所，东京都武藏村山市学园4-7-1，邮编208-0011，日本 2 生物信息学与整合组学系，日本国立感染症研究所，日本健康安全研究所，东京，日本 3 呼吸道病毒系，日本国立感染症研究所，日本健康安全研究所，东京，日本 4 联合兽医学院，跨境动物疫病研究中心，鹿儿岛大学，鹿儿岛，日本 5 病原体结构学分部，人兽共患病控制研究中心，北海道大学，北海道，日本 6 生物分子科学实验室，药学部，药物发现研究与教育中心，北海道大学，北海道，日本 7 微生物学系，医学部，医学研究生院，东京大学，东京，日本 8 现地址：微生物学与免疫学系，德克萨斯大学医学分部，加尔维斯顿，德克萨斯州，美国
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ARTICLE | Article in Press Scientific Reportshttps://doi.org/10.1038/s41598-026-76801-5 Scientific Reports This Article in Press is shared early to give you faster access to new research. It is citable and carries a permanent DOI. The final edited version will replace it automatically. Antiviral activity of HSP90 inhibitors against Mumps virus and related paramyxoviruses Fumihiro Kato · Chaewon Bae · Aika Wakata · Masatoshi Kakizaki · Takashi Okura · Yuichiro Nakatsu · Fumio Seki · Kenji Someya · Noriyuki Otsuki · Akihide Ryo · Yusuke Matsumoto · Hideo Fukuhara · Katsumi Maenaka · Mari Ishigami-Yuasa · Minoru Kidokoro · Masaki Ochiai · Koji Ishii · Hiroshi Katoh · Makoto Takeda Received: 7 April 2026 / Accepted: 15 October 2026 © The Author(s) 2026 Abstract Mumps virus (MuV), a member of the family Paramyxoviridae, continues to cause recurrent outbreaks worldwide despite widespread vaccination. Currently, however, no antiviral drugs have been approved for MuV or other paramyxoviruses. In this study, we screened approximately 1,000 small-molecule compounds using a recombinant MuV expressing green fluorescent protein and identified six compounds that significantly suppressed viral replication without detectable cytotoxicity. These compounds were known inhibitors of heat shock protein 90 (HSP90), a host chaperone involved in the assembly and function of viral polymerase complexes. These HSP90 inhibitors exhibited potent antiviral activity with submicromolar EC₅₀ values and high selectivity indices. Moreover, their antiviral efficacy extended to other paramyxoviruses, including measles virus (MeV), canine distemper virus (CDV), and Sendai virus (SeV). Time-of-addition assays demonstrated that these compounds act at a post-entry stage, primarily during viral RNA synthesis. Consistently, minigenome assays of MuV and Nipah virus (NiV) confirmed robust suppression of viral RNA synthesis without direct inhibition of reporter enzyme activity, indicating target specificity. Collectively, our findings highlight the potential of HSP90 inhibitors as host-directed antiviral agents against paramyxoviruses and support their further development for the treatment of emerging and re-emerging paramyxovirus infections. Keywords Mumps virus · Measles virus · Nipah virus · HSP90 · Antiviral compound · RNA replication Introduction MuV, a member of the genus Orthorubulavirus within the family Paramyxoviridae, circulates worldwide. MuV infection typically causes a mild to moderate illness characterized by fever and swelling of the parotid glands; however, it can occasionally result in severe complications, including aseptic meningitis and sensorineural hearing loss 1 . Although effective vaccines against MuV have been developed and are widely used, recurrent outbreaks continue to be reported in multiple regions despite high vaccination coverage 2–5 . In addition to MuV, other
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 paramyxoviruses pose significant threats as emerging or re-emerging pathogens. Members of the genus Morbillivirus, such as MeV and CDV, as well as Henipavirus species such as NiV, are of particular concern 6,7 . MeV causes a highly contagious disease characterized by fever, coryza, cough, and conjunctivitis, followed by the development of a generalized maculopapular rash 8 . CDV primarily infects carnivorous animals and causes severe systemic disease; notably, natural infections in non-human primates have recently been reported 9–12 , raising concerns about its potential as an emerging infectious disease affecting human society 13 . NiV has caused recurrent outbreaks since the early 2000s, mainly in Southeast Asia, and is associated with severe and often fatal disease in humans. Collectively, the continued emergence and re-emergence of paramyxoviruses represent a growing global public health concern 14 . Although effective vaccines are available for MuV and MeV, no antiviral drugs have been approved for the treatment of these infections. Several candidate antiviral compounds have been reported 15–19 , but none have progressed to clinical approval. Consequently, current clinical management of MuV and MeV infections remains largely supportive and focuses on symptom relief 20,21 . In CDV infection, antiviral activity has been reported for agents such as ribavirin and favipiravir; however, there are relatively few reports supporting their use as therapeutic treatments 22,23 . In the case of NiV infection, neither licensed vaccines nor antiviral therapies are currently available 24 . Paramyxoviruses possess a single-stranded, negative-sense RNA genome of approximately 15–18 kb, and their replication cycles are broadly conserved across the family. Viral entry is followed by the release of the ribonucleoprotein (RNP) complex into the host cell cytoplasm, where the viral genome is transcribed into mRNA and translated into viral proteins. The RNP complex consists of a viral RNA encapsidated with nucleocapsid (N) protein and associated with the phospho- (P) protein and large (L) protein that function as the viral RNA- dependent RNA polymerase (RdRp) 25,26 . Newly synthesized viral genomic RNA associates with N protein to form progeny RNPs, which are then assembled with viral structural proteins, including matrix (M), fusion (F), and receptor-binding glycoproteins—hemagglutinin-neuraminidase (HN), hemagglutinin (H), or glycoprotein (G)—to generate infectious virions. The recombinant virus technology, particularly the incorporation of reporter genes into viral genomes, has enabled the development of sensitive and high-throughput screening platforms for antiviral drug discovery 27–30 . These reporter-based systems allow efficient monitoring of viral replication and provide the advantage of interrogating multiple stages of the viral life cycle rather than targeting a single predefined step. We previously developed a recombinant MuV expressing Aequorea coerulescens green fluorescent protein (AcGFP) and successfully used this system to identify novel anti-MuV candidate compounds 30 . In the present study, we evaluated the utility of this recombinant MuV as a high-throughput screening platform for the identification of anti-MuV drug candidates. We further investigated the stage of the viral life cycle targeted by the identified compounds and assessed their antiviral activity against other paramyxoviruses, including MeV, CDV, SeV and NiV. Materials and methods Cells and viruses A549/hSLAM and A549/hSLAM IFNAR1 KO cell lines, which were established in our previous study 30 , were cultured in Dulbecco’s modified Eagle’s medium (D-MEM) (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) with 10% fetal bovine serum (FBS) and penicillin–streptomycin (Nacalai Tesque Co., Kyoto, Japan). VeroE6/TMPRSS2, Vero/hSLAM and Vero/dogSLAM cells, which were also established in our previous studies 31–32 were cultured in D-MEM with 10% FBS, penicillin–streptomycin, and 500 μg/mL G-418 (Nacalai Tesque Co.). 293T cells were cultured in D-MEM with 10% FBS and penicillin–streptomycin. T7 polymerase
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 expressing BHK-21 cells (BHK/T7-9) 33 were purchased from the RIKEN BioResource Research Center (BRC) Cell Bank (RCB4942, Tsukuba, Japan) and were cultured in Glasgow’s minimum essential medium (Thermo Fisher Scientific, Waltham, MA, USA) with 10% FBS and penicillin-streptomycin. Molecular clones of the original wild-type MuV Odate strain and AcGFP-MuV have been established in previous studies 34,35 . The MeV and CDV strains used in this study were derived from the IC-B and Ac96I strains, respectively 36,37 . An RFP expressing SeV was constructed in the previous study and kindly provided 38,39 . Chemical library screening To identify anti-MuV compounds, a small-molecule library purchased from Selleck Chemicals, consisting of approximately 1,000 biologically active compounds (Supplementary Table), was screened. A549/hSLAM IFNAR1 KO cells were seeded in 96-well plates at 3 × 10⁴ cells per well. After 24 h, the cells were infected with AcGFP-MuV at an MOI of 0.01, and each compound was added at a final concentration of 10 μM. After 72 h of incubation, MuV replication was evaluated by measuring GFP fluorescence intensity using a fluorescence microscope and BZ-X800 Analyzer software version 1.4.0. 40 To exclude compounds with apparent cytotoxicity, cell viability was evaluated in the same screening plates after fluorescence measurement. Cell Count Reagent SF (Nacalai Tesque) was added to each well according to the manufacturer’s protocol, and the absorbance at 450 nm was measured using a GloMax Discover Microplate Reader (Promega, Madison, WI, USA). Antiviral efficacy evaluation of the identified compounds To determine the half-maximal effective concentration (EC₅₀) of the identified compounds, dose-response assays were performed. A549/hSLAM IFNAR1 KO cells were seeded in 24-well plates at 3 × 10⁵ cells per well. The cells were infected with AcGFP-MuV at an MOI of 0.01 for 1 h, after which the inoculum was replaced with fresh medium containing the compounds at the indicated concentrations. The identified HSP90 inhibitors were tested at concentrations ranging from 0 to 2 μM, whereas mycophenolic acid (MPA), which was used as a positive control, was tested at concentrations ranging from 0 to 10 μM. After 72 h of incubation, MuV replication was evaluated by measuring GFP fluorescence intensity using a fluorescence microscope and BZ-X800 Analyzer software version 1.4.0. 40 The antiviral activities of the identified compounds against MeV, CDV, and SeV were also evaluated. A549/ hSLAM cells, Vero/dogSLAM cells, and VeroE6/TMPRSS2 cells were used for MeV, CDV, and SeV infection, respectively. Cells were infected with each virus and incubated in the presence of the compounds at the indicated concentrations. For MeV, culture supernatants were collected, and infectious virus titers were determined by plaque assay. Plaques were visualized by methylene blue staining and counted. For CDV, infectious foci were counted under a fluorescence microscope, and virus titers were calculated as focus-forming units. For SeV, viral replication was evaluated based on reporter fluorescence intensity using a fluorescence microscope and BZ-X800 Analyzer software version 1.4.0, as described above for MuV. The EC₅₀ values were calculated using the Reed and Muench method. Cytotoxicity assessment of the identified compounds To determine the half-maximal cytotoxic concentration (CC₅₀) of the identified compounds, cells were seeded in 96-well plates at 3 × 10⁴ cells per well and cultured with serial dilutions of each compound at concentrations ranging from 0 to 100 μM for 3 days. To assess the cytotoxicity of SNX-2112 in BHK/T7-9 cells, cells were treated with serial dilutions of SNX-2112 over a concentration range extending up to 100 μM, which exceeded the highest concentration used in the MuV and NiV minigenome assays. Cells were seeded in 96-well plates at 3 × 10⁴ cells per well and cultured with serial dilutions of each compound at concentrations ranging from 0 to 100
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 μM for 2 days. Cell viability was then measured using Cell Count Reagent SF (Nacalai Tesque) according to the manufacturer’s protocol. The absorbance at 450 nm was measured using a GloMax Discover Microplate Reader. Absorbance values at each concentration were normalized to that of the vehicle-treated control, which was set to 100. Data are presented as the mean ± SD from three independent experiments. Evaluation of structural similarity between HSP90 inhibitors The chemical similarity among the six hit compounds, which are HSP90 inhibitors, was evaluated using the RDKit cheminformatics toolkit (version 2023.03.1) implemented in a Python environment. Molecular fingerprints were generated using the Morgan fingerprinting algorithm with a radius of 2 and a fingerprint length of 1024 bits. The Tanimoto coefficient was calculated based on the generated fingerprints to quantitatively assess the structural similarity between each pair of compounds. Time-of-addition assay A549/hSLAM IFNAR1 KO cells were seeded in 12-well plates at 3 × 10 5 cells per well. After 18 h of incubation, the cells were infected with MuV at an MOI of 2 for 1 h. Next, the viral inoculum was removed, and cells were washed twice with PBS. Then, 10 μM of SNX-2112 was added to infected cells at 2 h intervals until 20 h. After 20 h of infection, the supernatant was collected. The viral titer was measured by plaque assay. Minigenome assay The MuV minigenome used in this study was based on a construct previously reported 30,35 . BHK/T7-9 cells were transfected with the corresponding minigenome plasmids, and test compounds were co-incubated at various concentrations for 48 h. Luciferase activity was then measured as an indicator of viral RNA synthesis. For the NiV minigenome system, plasmids encoding the N, P, and L proteins were generously provided by Dr. Paul A. Rota 41 . The NiV minigenome plasmid was constructed in this study by replacing the original reporter gene (chloramphenicol acetyltransferase) with a Nano luciferase gene. In the minigenome assay, BHK/T7-9 cells were seeded into 24-well plates and transfected with 0.3 μg of N plasmid, 0.2 μg of P plasmid, 0.1 μg of L plasmid, and 0.2 μg of the minigenome plasmid using the TransIT-LT1 transfection reagent. Compounds were added immediately after transfection and incubated for 48 h, after which luciferase activity was quantified. Western blot analysis of MuV L and P/V proteins 293T cells were seeded in 6-well plates and transfected with plasmids expressing MuV N, P, and L proteins. The cells were treated with SNX-2112 at the indicated concentrations for 36 h. Cells were then lysed in sample buffer and subjected to SDS-PAGE. Proteins were transferred onto a membrane, and after protein transfer, the membrane region corresponding approximately to 100–250 kDa was physically excised and subjected to immunoblotting for the MuV L protein. The membranes were probed with antibodies against MuV L, MuV P/V, and α-tubulin. The antibodies against MuV L and P/V proteins were the same as those used in a previous study by Katoh et al. The anti-α-tubulin antibody, clone 1E4C11, was purchased from Proteintech Inc. α-Tubulin was used as a loading control. Protein bands intensities were quantified using ImageJ software 42 . The intensity of the MuV L protein band was normalized to that of α-tubulin and expressed relative to the vehicle-treated control, which was set to 100.
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 Results Screening of antiviral compounds from a chemical library We screened approximately 1,000 small-molecule compounds at a final concentration of 10 μM to identify candidate inhibitors of MuV replication. The compound library consisted of biologically active compounds with known pharmacological activities. Through this screening, we identified six compounds—SNX-2112, CH5138303, PU-H71, PF-04929113, onalespib, and HSP990—that suppressed MuV replication and are well-characterized HSP90 inhibitors (Fig. 1). Notably, PF-04929113 (also known as SNX-5422), a prodrug of SNX-2112, showed high structural similarity to its active form (Tanimoto coefficient = 0.777). In contrast, structural similarity among the remaining hit compounds was low (maximum Tanimoto coefficient ≈ 0.13), indicating that the hits were structurally diverse overall. To quantitatively evaluate antiviral potency, we determined the half-maximal effective concentration (EC₅₀) of each HSP90 inhibitor using viral titer–based assays. All six compounds exhibited EC₅₀ values below 1 μM (Fig. 2A–F). In parallel, cytotoxicity was assessed in uninfected cells, revealing half-maximal cytotoxic concentration (CC₅₀) values exceeding 100 μM for all compounds (Fig. 2H–M). These results indicate that the observed antiviral effects were not attributable to nonspecific cytotoxicity. Fig. 1 Chemical structures of (A) SNX-2112, (B) CH5138303, (C) PU-H71, (D) PF-04929113, (E) HSP990, and (F) Onalespib. Pairwise Tanimoto coefficients were calculated to visualize the structural similarity matrix among the compounds.
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 Antiviral activities against the other paramyxoviruses We next examined whether the identified HSP90 inhibitors exhibited antiviral activity against other paramyxoviruses, including MeV, CDV, and SeV, using reporter-based replication assays. All six compounds potently
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 inhibited MeV and CDV replication, with EC₅₀ values remaining in the submicromolar range (Table 1). In some cases, viral titers were reduced by more than four orders of magnitude. In contrast, although antiviral activity was also observed against SeV, the EC₅₀ values were higher (approximately 1–2 μM), and the overall degree of inhibition was less pronounced, resulting in roughly tenfold reductions in viral replication. Estimating the inhibitory step of replication using time of addition assay To determine the stage of the viral life cycle targeted by SNX-2112, which showed strong antiviral activities against MuV, MeV, and especially CDV, we performed time-of-addition assays in which the compound was added at various time points post-infection, followed by quantification of viral titers at 20 h post-infection. SNX- 2112 retained robust antiviral activity when added up to 8 h post-infection, whereas its inhibitory effect gradually declined when addition was delayed further (Fig. 3A). These results suggest that SNX-2112 acts at a post-entry stage of the MuV life cycle, most likely during RNA synthesis. Inhibition of viral genome replication in a minigenome assay To directly assess whether SNX-2112 inhibits viral RNA synthesis, we employed a MuV minigenome assay in which luciferase activity serves as a readout of viral RNA synthesis. Treatment with SNX-2112 resulted in a dosedependent suppression of luciferase activity, with an EC₅₀ of 101 nM (Fig. 3B), consistent with results obtained using infectious virus. To exclude the possibility that SNX-2112 directly interferes with luciferase enzymatic activity, cells were transfected with a luciferase expression plasmid alone and treated with SNX-2112. Under these conditions, luciferase activity was not reduced (Fig. 3C), confirming that the compound inhibits viral RNA synthesis rather than reporter enzyme activity. Cell viability was not markedly affected by SNX-2112 even at concentrations up to 100 μM (Fig. 3E), indicating that the reduction in minigenome-derived luciferase activity was not attributable to cytotoxicity. Evaluation of the antiviral activity of SNX-2112 against other viral species Because our facility is not equipped to handle infectious NiV, antiviral activity against NiV was evaluated using a minigenome replication/transcription assay. SNX-2112 effectively suppressed NiV minigenome activity in a dose-dependent manner (Fig. 3D). The EC₅₀ value obtained in the NiV minigenome system was 14.6 nM, indicating that SNX-2112 exerts conserved inhibitory effects on RNA synthesis across distinct paramyxovirus genera. Fig. 2 Antiviral activity of (A) SNX-2112, (B) CH5138303, (C) PU-H71, (D) PF-04929113, (E) HSP990, (F) Onalespib and (G) Mycophenolic acid against MuV. Each compound was serially diluted and co-incubated with the virus. After incubation, antiviral activity was evaluated by quantifying GFP fluorescence intensity. GFP fluorescence intensity at each concentration was normalized to that of the virus-infected, vehicle-treated control, which was set to 100. The identified HSP90 inhibitors were tested at concentrations ranging from 0 to 2 μM, whereas MPA, used as a positive control, was tested at concentrations ranging from 0 to 10 μM. Cell cytotoxicity of H) SNX-2112, I) CH5138303, (J) PU-H71, (K) PF-04929113, (L) HSP990, (M) Onalespib and (N) Mycophenolic acid. Each compound was serially diluted and incubated with A549/hSLAM IFNAR1 KO cells. Absorbance values at each concentration were normalized to those of the mock-treated control, which was set to 100. Antiviral activity and cell viability are shown in separate panels because the concentration ranges used for EC₅₀ and CC₅₀ determination differed substantially. Data are presented as the mean ± SD from three independent experiments.
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 SNX-2112 reduces MuV L protein expression To examine whether the HSP90 inhibitors identified in this study affect MuV L protein stability through a mechanism similar to that reported previously, we analyzed the intracellular levels of MuV L protein following SNX- 2112 treatment. 293T cells were transfected with plasmids expressing MuV N, P, and L proteins and treated with SNX-2112 at the indicated concentrations. The levels of MuV L and P/V proteins were then examined by Western blotting. The results showed that the expression levels of α-tubulin, used as an internal loading control, and MuV P/V proteins were not markedly reduced at any concentration of SNX-2112. In contrast, the level of MuV L protein was significantly decreased at higher concentrations of SNX-2112 (Fig. 3F and G). These findings are consistent with the previous report that HSP90 contributes to the stability of the MuV L protein and further support the idea that SNX-2112 suppresses MuV RNA synthesis, at least in part, by reducing the stability of the L protein. Discussion In this study, we identified multiple small-molecule compounds with potent antiviral activity against MuV through a high-throughput screening strategy using a recombinant reporter virus. Among the hit compounds, six were known inhibitors of heat shock protein 90 (HSP90), all of which strongly suppressed MuV replication without detectable cytotoxicity. These findings highlight the feasibility of targeting host chaperone pathways as an antiviral strategy against paramyxoviruses. HSP90 plays a critical role in the folding, stabilization, and functional assembly of numerous client proteins, including viral polymerase components. Previous studies have suggested that HSP90 is required for efficient RNA synthesis of MuV 35 and that compounds with HSP90-inhibitory activity, such as 17-AAG, exhibit antiviral effects against not only MuV but also MeV and NiV 43 . Although 17-AAG shows potent antiviral activity, it has also been reported to be highly cytotoxic. In contrast, all six compounds identified in this study exhibited CC₅₀ values greater than 100 μM, resulting in a high selectivity index. The enrichment of HSP90 inhibitors among our screening hits is consistent with these earlier observations and further supports the notion that MuV replication is highly dependent on HSP90-mediated chaperone activity. Although in vivo toxicity studies have not yet been conducted, these compounds represent promising candidates for antiviral drug development. All six HSP90 inhibitors identified in this study exhibited submicromolar EC₅₀ values and CC₅₀ values exceeding 100 μM, resulting in high selectivity indices. These results indicate that the observed antiviral effects are unlikely to be attributable to nonspecific cytotoxicity and instead reflect inhibition of a critical host-dependent step in the viral life cycle. Time-of-addition assays showed that SNX-2112 retained antiviral activity when added up to 8 h post-infection, consistent with a post-entry mode of action. This interpretation was further supported Table 1 Antiviral activity of HSP90 inhibitors against other paramyxoviruses and cytotoxicity in A549/hSLAM IFNAR1 KO cells. CompoundEC 50 against MuV EC 50 against MeV EC 50 against CDV EC 50 against SeVCC 50 in A549/hSLAM IFNAR1 KO cells Selective Index (CC 50 /EC 50 ) Of MuV SNX-211292 nM44 nM67 nM1,926 nM>100,000 nM>1087 CH5138303141 nM66 nM402 nM2,893 nM>100,000 nM>709 PU-H71199 nM45 nM943 nM3,568 nM>100,000 nM>503 PF-0492911375 nM86 nM293 nM1,936 nM>100,000 nM>1333 HSP990166 nM34 nM178 nM2,913 nM>100,000 nM>602 Onalespib35 nM22 nM410 nM3,112 nM>100,000 nM>2857
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 by minigenome assays, which demonstrated robust inhibition of viral replication/transcription activity without affecting luciferase enzymatic activity. Importantly, the antiviral activity of these HSP90 inhibitors was not restricted to MuV. The compounds also exhibited potent inhibitory effects against other paramyxoviruses, including MeV, CDV, and NiV, suggesting broad antiviral potential within paramyxoviruses. In contrast, antiviral effects against SeV were less pronounced. This difference may reflect varying degrees of dependence on HSP90 among paramyxovirus genera. For example, the RNA-dependent RNA polymerase complexes of orthorubulaviruses and morbilliviruses may rely more heavily on HSP90 chaperone functions than those of respiroviruses. Alternatively, structural or sequence differences in viral polymerase or cofactor proteins may influence sensitivity to HSP90 inhibition 44–46 . The ability of SNX-2112 to suppress NiV replication/transcription activity in a minigenome system is particularly noteworthy, given the high pathogenicity of NiV and the lack of approved antiviral therapies. Host-directed antiviral strategies, such as HSP90 inhibition, may offer advantages by limiting the emergence of drug resistance and enabling rapid responses to emerging or re-emerging viral threats. Although the compounds exhibited minimal cytotoxicity in vitro, further studies are required to assess their safety profiles in vivo. In particular, evaluation of pharmacokinetics, tissue distribution, and potential off-target effects will be essential for therapeutic development. In addition, detailed biochemical and proteomic analyses will be necessary to elucidate the molecular interactions between HSP90 and paramyxovirus polymerase complexes. In conclusion, our study demonstrates that HSP90 inhibitors, including SNX-2112, effectively inhibit the replication of MuV and other paramyxoviruses, likely by targeting post-entry steps in viral RNA synthesis. The broad antiviral activity and high selectivity of these compounds underscore their potential as host-directed antiviral agents. These findings provide a foundation for the further development of HSP90-targeting strategies to combat paramyxovirus infections.
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5
Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 Supplementary Information The online version contains supplementary material available at https://doi.org/10.1038/ s41598-026-76801-5. Acknowledgements We thank Drs. Paul A. Rota and Naoto Ito for kindly providing invaluable research reagents. We would also like to thank Drs. Kentaro Fujita, Seishiro Naito, and Yuko Sasaki for providing materials and supplies and for their warm encouragement throughout this study. We also thank the other members of the Center for Quality Management Systems at the National Institute of Infectious Diseases for their warm encouragement. Author contributions F.K., C.B., A.W., M.Ka., and T.O. performed the experiments and analyzed the data. F.K. wrote the manuscript. Y.N., F.S., K.S., N.O., Y.M., H.F., K.M., M.Ki., and H.K. provided study materials and reagents. M.I.Y. and M.O. provided access to and managed laboratory facilities. F.K., Y.M., A.R., M.I.Y., M.Ki., M.O., K.I., H.K., and M.T. interpreted the data. F.K., K.M., and M.T. acquired funding for the project. All authors discussed the results, reviewed the manuscript, and approved the final version. Funding This work was supported by Japan Society for the Promotion of Science under Grant Number JP20K16020 and JP24K10240, and the Japan Agency for Medical Research and Development (AMED) under Grant Number JP22ama121037, JP223fa627005, 23wm03025063j0401 and JP223fa627001. Data availability All data supporting the findings of this study are included in this article. Additional raw data are available from the corresponding author upon reasonable request. Declarations Competing interests The authors declare no competing interests. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Fig. 3 (A) Time-of-addition assay of SNX-2112. Cells were infected with the virus at a multiplicity of infection (MOI) of 2, and SNX-2112 was added at the indicated time points post-infection. At 20 h post-infection, the culture supernatants were collected, and viral titers were determined. (B) Minigenome assay of MuV treated with SNX-2112. Cells were transfected with the respective plasmids, followed by the addition of serially diluted SNX-2112. After 48 h of incubation, luciferase activity was measured to evaluate antiviral activity. (C) Effect of SNX-2112 on luciferase activity. Cells were transfected with a plasmid expressing luciferase alone and co-incubated with 10 μM SNX-2112 for 48 h. (D) Inhibitory activity of SNX- 2112 on viral RNA synthesis of NiV. Cells were transfected with the respective plasmids, followed by the addition of serially diluted SNX-2112. After 48 h of incubation, luciferase activity was measured to assess antiviral activity. (E) Cells were treated with SNX-2112 at the indicated concentrations, covering a concentration range extending well above that used in the MuV and NiV minigenome assays. Absorbance values at each concentration were normalized to that of the vehicle-treated control, which was set to 100. Data are presented as the mean ± SD from three independent experiments. (F) Quantification of MuV L protein expression. L protein levels were normalized to α-tubulin and expressed relative to the vehicle-treated control, which was set to 100. Data are presented as the mean ± SD from three independent experiments. Asterisks indicate statistically significant differences compared with the vehicle-treated control, as determined by a paired two-tailed Student’s t-test (P < 0.05; P < 0.01). (G) Western blot analysis of MuV L and P/V protein expression following SNX-2112 treatment. Cells transfected with plasmids expressing MuV N, P, and L proteins were treated with SNX-2112 at the indicated concentrations, and L and P/V protein levels were analyzed by Western blotting. α-Tubulin was used as a loading control. For detection of the MuV L protein, the membrane region corresponding approximately to 100–250 kDa was physically excised after protein transfer and subjected to immunoblotting.
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Article in Press Scientific Reports https://doi.org/10.1038/s41598-026-76801-5 1 Center for Quality Management Systems, National Institute of Infectious Diseases, Japan Institute for Health Security, 4-7-1 Gakuen, Musashimurayama, Tokyo 208-0011, Japan 2 Department of Bioinformatics and Integrative Omics, National Institute of Infectious Diseases, Japan Institute for Health Security, Tokyo, Japan 3 Department of Respiratory Viruses, National Institute of Infectious Diseases, Japan Institute for Health Security, Tokyo, Japan 4 Joint Faculty of Veterinary Medicine, Transboundary Animal Diseases Research Center, Kagoshima University, Kagoshima, Japan 5 Division of Pathogen Structure, Research Center for Zoonosis Control, Hokkaido University, Hokkaido, Japan 6 Laboratory of Biomolecular Science, Faculty of Pharmaceutical Sciences, Center for Research and Education on Drug Discovery, Hokkaido University, Hokkaido, Japan 7 Department of Microbiology, Faculty of Medicine, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan 8 Present address: Departments of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX, USA