Daelemans Dirk, Afonina Elena, Nilsson Jakob, Werner Gudrun, Kjems Jorgen, De Clercq Erik, Pavlakis George N, Vandamme Anne-Mieke
Rega Institute for Medical Research, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium.
Proc Natl Acad Sci U S A. 2002 Oct 29;99(22):14440-5. doi: 10.1073/pnas.212285299. Epub 2002 Oct 9.
The HIV-1 Rev protein is an essential regulator of the HIV-1 mRNA expression that promotes the export of unspliced and partially spliced mRNA. The export receptor for the leucine-rich nuclear export signal (NES) of Rev has recently been recognized as CRM1. We identified a low molecular weight compound PKF050-638 as an inhibitor of HIV-1 Rev. This drug inhibits in a dose-dependent fashion Rev-dependent mRNA expression in a cellular assay for Rev function. We show that PKF050-638 is an inhibitor of the CRM1-mediated Rev nuclear export. By using a quantitative in vitro CRM1-NES cargo-binding assay, we could demonstrate that PKF050-638 disrupts CRM1-NES interaction. This mode of action is confirmed in cell culture because the drug reversibly interferes with the colocalization of CRM1 and Rev in the nucleolus of the cell. In addition, we prove that the inhibition is through direct interaction of the compound with Cys-539 of CRM1. These effects are similar to those of the known CRM1 inhibitor leptomycin B and suggest that the inhibitory effect of the compound is caused by binding to CRM1 at a similar site. The compound displayed strict structural requirements for its activity, as its enantiomer was inactive in all assays tested. These results show that we identified a drug that interferes with the CRM1-mediated nuclear export of Rev through inhibition of the CRM1-NES complex formation. The reversibility of its binding to CRM1 and its availability through chemical synthesis could make it useful for studying CRM1-mediated export pathways.
HIV-1 Rev蛋白是HIV-1 mRNA表达的关键调节因子，可促进未剪接和部分剪接的mRNA的输出。Rev富含亮氨酸的核输出信号（NES）的输出受体最近被确认为CRM1。我们鉴定出一种低分子量化合物PKF050-638作为HIV-1 Rev的抑制剂。在针对Rev功能的细胞试验中，该药物以剂量依赖的方式抑制Rev依赖的mRNA表达。我们表明PKF050-638是CRM1介导的Rev核输出的抑制剂。通过使用定量体外CRM1-NES货物结合试验，我们可以证明PKF050-638破坏了CRM1-NES相互作用。这种作用模式在细胞培养中得到证实，因为该药物可逆地干扰了CRM1和Rev在细胞核仁中的共定位。此外，我们证明抑制作用是通过该化合物与CRM1的Cys-539直接相互作用实现的。这些作用与已知的CRM1抑制剂雷帕霉素B相似，表明该化合物的抑制作用是由其在类似位点与CRM1结合引起的。该化合物对其活性表现出严格的结构要求，因为其对映体在所有测试试验中均无活性。这些结果表明，我们鉴定出一种通过抑制CRM1-NES复合物形成来干扰CRM1介导的Rev核输出的药物。其与CRM1结合的可逆性以及通过化学合成获得的可用性可能使其对研究CRM1介导的输出途径有用。
