一种用于鉴定可阻断HIV-1 gp120与CCR5结合的进入抑制剂的新型检测方法。

A novel assay to identify entry inhibitors that block binding of HIV-1 gp120 to CCR5.

作者信息

Zhao Qian, He Yuxian, Alespeiti Gabriel, Debnath Asim Kumar

机构信息

Laboratory of Molecular Modeling and Drug Design, Lindsley F. Kimball Research Institute of The New York Blood Center, New York, NY 10021, USA.

出版信息

Virology. 2004 Sep 1;326(2):299-309. doi: 10.1016/j.virol.2004.06.022.

DOI:10.1016/j.virol.2004.06.022

PMID:15321703

Abstract

HIV-1 infection is initiated by the interaction of the envelope glycoprotein gp120 with the cellular receptor CD4 that triggers conformational changes in gp120 necessary for subsequent interaction with a coreceptor CCR5 (or CXCR4). The CD4-induced (CD4i) conformation of gp120 can be mimicked by a full-length single chain (FLSC) protein consisting of gp120 linked with the D1D2 domains of CD4 by a 20-amino-acid linker. We have used this protein to establish a flow cytometry-based assay and an ELISA-based assay to identify inhibitors that block the binding of gp120 to CCR5. Both assays are specific for detecting the known CCR5 antagonist TAK-779, but the ELISA-based assay was more sensitive, simple, inexpensive, and rapid; thus, it can be adapted to high throughput screening (HTS). The ELISA-based method was validated with a diverse set of known antagonists, for example, TAK-779, AOP-RANTES, PSC-RANTES, and several mAbs.

摘要

HIV-1感染是由包膜糖蛋白gp120与细胞受体CD4相互作用引发的，该相互作用会触发gp120发生构象变化，这对于其随后与共受体CCR5（或CXCR4）相互作用是必需的。gp120的CD4诱导（CD4i）构象可由一种全长单链（FLSC）蛋白模拟，该蛋白由gp120通过一个20个氨基酸的接头与CD4的D1D2结构域相连组成。我们已使用这种蛋白建立了一种基于流式细胞术的检测方法和一种基于ELISA的检测方法，以鉴定阻断gp120与CCR5结合的抑制剂。两种检测方法都对检测已知的CCR5拮抗剂TAK-779具有特异性，但基于ELISA的检测方法更灵敏、简单、廉价且快速；因此，它可适用于高通量筛选（HTS）。基于ELISA的方法已用多种已知拮抗剂进行了验证，例如TAK-779、AOP-RANTES、PSC-RANTES和几种单克隆抗体。