Smith Elizabeth B, Ogert Robert A, Pechter David, Villafania Artjohn, Abbondanzo Susan J, Lin Karen, Rivera-Gines Aida, Rebsch-Mastykarz Cheryl, Monsma Frederick J
1Department of In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ, USA.
J Biomol Screen. 2014 Jan;19(1):108-18. doi: 10.1177/1087057113500074. Epub 2013 Aug 29.
The health and disease-related biology of the CXCR4 chemokine receptor presents the challenge of finding a small molecule that can bind CXCR4 and block T-cell tropic human immunodeficiency virus type 1 (HIV-1) cell entry, while preserving the ability of CXCR4 to respond to its native ligand, CXCL12. HIV entry into the host cell involves the interaction of the viral envelope glycoprotein gp120 binding to CD4, followed by a rearrangement in gp120, and subsequent interaction with the chemokine receptor CXCR4 or CCR5. These initial events can be re-created in a cell fusion assay that represents a surrogate system, mimicking the early stages of viral entry via these host cell receptors. In the current study, a T-tropic HIV cell fusion assay was established using U2OS cells expressing the envelope glycoprotein gp160 from the T-tropic HIV NL4-3 and HeLa cells expressing CD4 and CXCR4. Detection of the cell fusion event was based on a Gal4/VP16-activated β-lactamase signal and was measured by automated microscopy or laser scanning plate cytometry. Changes in morphology associated with cell fusion were combined with β-lactamase activity to generate results with robust assay statistics in both 384-well and 1536-well plates. Compounds were subsequently characterized by CXCR4 signaling assays to eliminate functional antagonists and allow the identification of a function-sparing HIV entry inhibitor.
趋化因子受体CXCR4与健康和疾病相关的生物学特性带来了一项挑战，即要找到一种能够结合CXCR4并阻断嗜T细胞的1型人类免疫缺陷病毒（HIV-1）进入细胞的小分子，同时保留CXCR4对其天然配体CXCL12作出反应的能力。HIV进入宿主细胞涉及病毒包膜糖蛋白gp120与CD4结合，随后gp120发生重排，接着与趋化因子受体CXCR4或CCR5相互作用。这些初始事件可在一种细胞融合试验中重现，该试验代表了一个替代系统，模拟了病毒通过这些宿主细胞受体进入的早期阶段。在本研究中，利用表达嗜T细胞的HIV NL4-3包膜糖蛋白gp160的U2OS细胞和表达CD4与CXCR4的HeLa细胞，建立了一种嗜T细胞的HIV细胞融合试验。细胞融合事件的检测基于Gal4/VP16激活的β-内酰胺酶信号，并通过自动显微镜或激光扫描平板细胞仪进行测量。将与细胞融合相关的形态变化与β-内酰胺酶活性相结合，在384孔板和1536孔板中生成具有稳健试验统计数据的结果。随后通过CXCR4信号试验对化合物进行表征，以排除功能拮抗剂，并鉴定出一种保留功能的HIV进入抑制剂。
