The phosphatidylinositol-3-phosphate 5-kinase inhibitor apilimod blocks filoviral entry and infection

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过程的脂质激酶，经单倍体遗传筛选发现，其为埃博拉病毒（EBOV）感染所必需的宿主因子。本研究针对阿匹莫德（apilimod）——一种PIKfyve抑制剂，其在II期临床试验中被报道对人体具有良好耐受性——展开分析，探讨其对埃博拉病毒与马尔堡病毒（MARV）的入侵及感染过程的影响。我们首先证实，阿匹莫德可在Huh7、Vero E6及原代人巨噬细胞中阻断埃博拉病毒与马尔堡病毒的感染，且在巨噬细胞中展现出显著的抗病毒活性（半数抑制浓度IC50为10 nM）。后续实验观察到，同等剂量的阿匹莫德可阻断表达埃博拉病毒糖蛋白的病毒样颗粒（VLP）的入侵，以及具备转录复制能力的病毒样颗粒感染，这表明阿匹莫德的核心作用机制为入侵抑制，可阻止病毒基因组释放至细胞质以启动复制程序。在验证阿匹莫德的抗埃博拉病毒活性依赖于PIKfyve后，我们发现其可阻断埃博拉病毒样颗粒向含有埃博拉病毒细胞内受体尼曼-皮克C1蛋白（NPC1）的内溶酶体的转运过程。与此同时，阿匹莫德会导致病毒样颗粒在早期内体抗原1阳性的内体中聚集。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，或内溶酶体胆固醇外流产生任何影响。由此可见，通过拮抗PIKfyve，阿匹莫德可阻断埃博拉病毒向其融合并入侵细胞质的位点转运。鉴于该药物已被证实具有抗丝状病毒活性、其入侵抑制机制相对未被充分探索，且在人体中具有良好的耐受性，我们提议将阿匹莫德进一步作为治疗丝状病毒感染的治疗方案组成部分开展研究。