Since nuclear export of Rev protein is essential for HIV proliferation, we have been engaged in search for inhibitors for nuclear export of Rev to disclose new anti-HIV seeds. Consequently, 1'-acetoxychavicol acetate (ACA, 1) was isolated as an active principle by using fission yeast expressing Rev-NES-contained protein and confirmed to inhibit proliferation of HIV-1 virus. Furthermore, the mechanism of action of 1 was revealed to bind to the cystein-529 in CRM1, the receptor of nuclear export signal (NES) in Rev, by the competitive experiment in the presence of the biotinylated leptomycin B probe (2). Reactants of 1 and N-acetyl-L-cystein methyl ester established 1-acetoxy-2-ene moiety as the binding site of cystein-529 in CRM1. Analysis for structure activity relationship by synthetic analogs disclosed the hydrolysis of functional group at 4-OH in 1 to be essential for bioactivity of 1. Based on the plausible mechanism of action of 1 wih Cys-529 in CRM1, activation energy to crucial transition state by molecular orbital calculation was found to be correlated with nuclear export inhibitory activity of ACA analogs. Halogenated ACA analogs designed from this calculation showed more potent activity than 1. On the other hand, homology modeling of hCRM1 by folding recognition method was performed. Interaction energies between ACA analogs and hCRM1 were shown to be correlated with bioactivity of ACA analogs. Functional groups at 1'-C positions of ACA analogs were assumed to affect interaction energy with hCRM1 by docking study. ACA analogs possessing 1'-O-acyl or 1'-O-carbamoyl functions exhibited lower interaction energy and more potent activity than 1. Furthermore, we developed assay systems to evaluate inhibitory activity for nuclear export of Rev in HeLa cells. Synthetic ACA analogs based on these rational designs showed more potent activity than 1 in HeLa cells as well as in yeast system.
