Nelson Elizabeth A, Dyall Julie, Hoenen Thomas, Barnes Alyson B, Zhou Huanying, Liang Janie Y, Michelotti Julia, Dewey William H, DeWald Lisa Evans, Bennett Richard S, Morris Patrick J, Guha Rajarshi, Klumpp-Thomas Carleen, McKnight Crystal, Chen Yu-Chi, Xu Xin, Wang Amy, Hughes Emma, Martin Scott, Thomas Craig, Jahrling Peter B, Hensley Lisa E, Olinger Gene G, White Judith M
Department of Cell Biology, University of Virginia, Charlottesville, Virginia, United States of America.
Integrated Research Facility, Division of Clinical Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Frederick, Maryland, United States of America.
PLoS Negl Trop Dis. 2017 Apr 12;11(4):e0005540. doi: 10.1371/journal.pntd.0005540. eCollection 2017 Apr.
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.
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，它是在一项单倍体基因筛选中被发现为埃博拉病毒（EBOV）感染所必需的。在此，我们分析了阿匹莫德（一种PIKfyve抑制剂，据报道在2期临床试验中在人体中耐受性良好）对EBOV和马尔堡病毒（MARV）进入和感染的影响。我们首先发现阿匹莫德可阻断EBOV和MARV在Huh 7、Vero E6和原代人巨噬细胞中的感染，在巨噬细胞中具有显著效力（IC50为10 nM）。接下来我们观察到，相似剂量的阿匹莫德可阻断EBOV糖蛋白病毒样颗粒（VLP）的进入以及具有转录-复制能力的VLP感染，这表明阿匹莫德的主要作用方式是作为一种进入抑制剂，阻止病毒基因组释放到细胞质中以启动复制。在提供了阿匹莫德抗EBOV作用是通过PIKfyve的证据后，我们表明它可阻断EBOV VLP转运至含有尼曼-皮克C1（NPC1，EBOV的细胞内受体）的内溶酶体。同时，阿匹莫德使VLP在早期内体抗原1阳性的内体中积累。我们未检测到阿匹莫德对整体内体酸化、组织蛋白酶B和L的活性或内溶酶体中胆固醇输出有任何影响。因此，通过拮抗PIKfyve，阿匹莫德似乎阻断了EBOV转运至其融合位点并进入细胞质。鉴于该药物观察到的抗丝状病毒活性、相对未被探索的进入抑制机制以及在人体中的耐受性报道，我们建议进一步研究阿匹莫德作为治疗丝状病毒感染治疗方案的一部分。
