The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection.
The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection.
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DOI：
10.1371/journal.pntd.0005540
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发表时间：
2017-04
影响因子：
3.8
通讯作者：
White JM
中科院分区：
文献类型：
--
作者：
Nelson EA;Dyall J;Hoenen T;Barnes AB;Zhou H;Liang JY;Michelotti J;Dewey WH;DeWald LE;Bennett RS;Morris PJ;Guha R;Klumpp-Thomas C;McKnight C;Chen YC;Xu X;Wang A;Hughes E;Martin S;Thomas C;Jahrling PB;Hensley LE;Olinger GG Jr;White JM
磷脂酰肌醇-3-磷酸5-激酶（PIKfyve）是一种参与内体成熟的脂质激酶，其从埃博拉病毒（EBOV）感染所需的单倍体遗传筛选中出现。在这里，我们分析了阿吡莫德（一种PIKfyve抑制剂，据报道在2期临床试验中在人体中耐受良好）对EBOV和马尔堡病毒（MARV）进入和感染的影响。我们首先发现阿吡莫德阻断Huh 7、Vero E6和原代人巨噬细胞中的EBOV和MARV感染，在巨噬细胞中具有显著的效力（IC 50，10 nM）。我们接下来观察到相似剂量的阿吡莫德阻断EBOV-糖蛋白-病毒样颗粒（VLP）进入和转录-复制能力VLP感染，表明阿吡莫德的主要作用模式是作为进入抑制剂，防止病毒基因组释放到细胞质中以启动复制。在提供阿吡莫德的抗EBOV作用是通过PIKfyve的证据之后，我们表明它阻断EBOV VLP向含有尼曼-匹克C1（NPC 1）（EBOV的细胞内受体）的内溶酶体的运输。同时，阿吡莫德导致VLP在早期核内体抗原1阳性核内体中积累。我们没有检测到阿吡莫德对大量内体酸化、对组织蛋白酶B和L的活性或对胆固醇从内溶酶体输出的任何影响。因此，通过拮抗PIKfyve，阿吡莫德似乎阻断EBOV运输至其融合位点并进入细胞质。考虑到药物观察到的抗丝状病毒活性、相对未探索的进入抑制机制和报告的人体耐受性，我们建议进一步探索阿吡莫德作为治疗丝状病毒感染的治疗方案的一部分。最近在西非爆发的埃博拉病毒（EBOV）疾病突出了迫切需要开发治疗方法来帮助平息这种毁灭性的出血热病毒，特别是在地球仪周围资源有限的地区。在这里，我们表明，阿吡莫德，一种在2期临床试验中耐受性良好的研究药物，用于类风湿性关节炎，克罗恩病和银屑病，是多种细胞类型中EBOV和马尔堡病毒感染的强效抑制剂。进一步的研究表明，阿吡莫德阻断EBOV颗粒进入宿主细胞质，并且其通过阻断颗粒到达其正常进入口（在尼曼-匹克C1阳性内溶酶体中）来实现。我们的发现与磷脂酰肌醇-3-磷酸5-激酶作为阿吡莫德的分子靶点的身份一致，因为激酶及其产物磷脂酰肌醇3，5-二磷酸是晚期内吞细胞器适当成熟所需的。因此，我们建议进一步探索阿吡莫德作为治疗方案的一部分重新定位，以帮助改善丝状病毒感染的后遗症。
Phosphatidylinositol-3-phosphate 5-kinase (PIKfyve) is a lipid kinase involved in endosome maturation that emerged from a haploid genetic screen as being required for Ebola virus (EBOV) infection. Here we analyzed the effects of apilimod, a PIKfyve inhibitor that was reported to be well tolerated in humans in phase 2 clinical trials, for its effects on entry and infection of EBOV and Marburg virus (MARV). We first found that apilimod blocks infections by EBOV and MARV in Huh 7, Vero E6 and primary human macrophage cells, with notable potency in the macrophages (IC50, 10 nM). We next observed that similar doses of apilimod block EBOV-glycoprotein-virus like particle (VLP) entry and transcription-replication competent VLP infection, suggesting that the primary mode of action of apilimod is as an entry inhibitor, preventing release of the viral genome into the cytoplasm to initiate replication. After providing evidence that the anti-EBOV action of apilimod is via PIKfyve, we showed that it blocks trafficking of EBOV VLPs to endolysosomes containing Niemann-Pick C1 (NPC1), the intracellular receptor for EBOV. Concurrently apilimod caused VLPs to accumulate in early endosome antigen 1-positive endosomes. We did not detect any effects of apilimod on bulk endosome acidification, on the activity of cathepsins B and L, or on cholesterol export from endolysosomes. Hence by antagonizing PIKfyve, apilimod appears to block EBOV trafficking to its site of fusion and entry into the cytoplasm. Given the drug’s observed anti-filoviral activity, relatively unexplored mechanism of entry inhibition, and reported tolerability in humans, we propose that apilimod be further explored as part of a therapeutic regimen to treat filoviral infections. The recent outbreak of Ebola virus (EBOV) disease in Western Africa highlights the urgent need to develop therapeutics to help quell this devastating hemorrhagic fever virus, especially in resource-limited areas around the globe. Here we show that apilimod, an investigational drug that was well-tolerated in phase 2 clinical trials for rheumatoid arthritis, Crohn’s disease, and psoriasis, is a strong inhibitor of both EBOV and Marburgvirus infections in multiple cell types. Further work shows that apilimod blocks the entry of EBOV particles into the host cell cytoplasm and that it does so by blocking the particles from reaching their normal portal of entry, in Niemann-Pick C1-positive endolysosomes. Our findings are consistent with the identity of phosphatidylinositol-3-phosphate 5-kinase as the molecular target of apilimod, as the kinase and its product phosphatidylinositol 3,5-bisphosphate are required for the proper maturation of late endocytic organelles. Hence we propose that apilimod be further explored for repositioning as part of a therapeutic regimen to help ameliorate the sequelae of filoviral infections.