High-Throughput HIV-Cell Fusion Assay for Discovery of Virus Entry Inhibitors
High-Throughput HIV-Cell Fusion Assay for Discovery of Virus Entry Inhibitors
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DOI：
10.1089/adt.2015.639
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发表时间：
2015-04-01
影响因子：
1.8
通讯作者：
Melikyan, Gregory B.
中科院分区：
文献类型：
--
作者：
Marin, Mariana;Du, Yuhong;Melikyan, Gregory B.
HIV-1通过将其包膜膜与靶细胞膜合并来启动感染，这是一个由病毒Env糖蛋白介导的过程，随后病毒Env糖蛋白依次与CD 4和辅助受体CXCR 4或CCR 5结合。尽管HIV-1融合一直是抗病毒治疗的靶点，但该病毒已经对阻断CCR 5结合或该过程的Env重折叠步骤的药物产生了耐药性。这突出了对新型抑制剂的需求。在这里，我们调整和优化了酶促HIV-细胞融合试验，该试验报告了病毒包封的β-内酰胺酶转移到细胞质中，以384孔格式进行高通量筛选（HTS）。该测定法以HTS形式稳健地进行，并通过对小的生物活性化合物文库的中试筛选进行验证。通过筛选确定的几个命中包括嘌呤能受体拮抗剂的突出簇。功能研究表明，P2 X1受体拮抗剂选择性地抑制HIV-1融合，而不影响通过内吞途径进入细胞的不相关病毒的融合活性。P2 X1拮抗剂对HIV-细胞融合的抑制不是通过下调CD 4或辅助受体的细胞表面表达，因此暗示P2 X1受体参与HIV-1融合步骤。这些拮抗剂抑制病毒的能力，无论其辅助受体（CXCR 4或CCR 5）的偏好表明，在辅助受体结合下游的后期步骤阻断融合。未来大规模的HIV-1融合抑制剂筛选活动，使用上述功能读数，将可能揭示新型抑制剂，并提出抗病毒治疗的潜在靶点。
HIV-1 initiates infection by merging its envelope membrane with the target cell membrane, a process that is mediated by the viral Env glycoprotein following its sequential binding to CD4 and coreceptors, CXCR4 or CCR5. Although HIV-1 fusion has been a target for antiviral therapy, the virus has developed resistance to drugs blocking the CCR5 binding or Env refolding steps of this process. This highlights the need for novel inhibitors. Here, we adapted and optimized an enzymatic HIV-cell fusion assay, which reports the transfer of virus-encapsulated beta-lactamase into the cytoplasm, to high-throughput screening (HTS) with a 384-well format. The assay was robustly performed in HTS format and was validated by the pilot screen of a small library of pharmacologically active compounds. Several hits identified by screening included a prominent cluster of purinergic receptor antagonists. Functional studies demonstrated that P2X1 receptor antagonists selectively inhibited HIV-1 fusion without affecting the fusion activity of an unrelated virus that enters cells through an endocytic route. The inhibition of HIV-cell fusion by P2X1 antagonists was not through downmodulation of the cell surface expression of CD4 or coreceptors, thus implicating P2X1 receptor in the HIV-1 fusion step. The ability of these antagonists to inhibit viruses regardless of their coreceptor (CXCR4 or CCR5) preference indicates that fusion is blocked at a late step downstream of coreceptor binding. A future large-scale screening campaign for HIV-1 fusion inhibitors, using the above functional readout, will likely reveal novel classes of inhibitors and suggest potential targets for antiviral therapy.