283   A Novel Inhibitor of the CRM1-Mediated Rev Export.

D. Daelemans*¹, E. Afonina², J. Nilsson³, G. Werner⁴, J. Kjems³, G. N. Pavlakis², E. De Clercq¹, and A.-M. Vandamme¹.
¹Rega Inst. for Med. Res., Leuven, Belgium;²NCI-FCRDC, Frederick, MD;³Univ. of Aarhus, Denmark; and⁴Novartis Res. Inst., Vienna, Austria.

Background: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 NES of Rev has been recognized as CRM1. We identified a low-molecular-weight compound, PKF050-638, as a specific inhibitor of HIV-1 Rev function.

Methods:The new compound was tested against Rev function in a cellular Rev assay. The effect of the compound on the nucleocytoplasmic transport of Rev was monitored using GFP fusion proteins. The RanGAP hydrolysis assay allowed us to study the effect on CRM1-NES-RanGTP complex formation in vitro .

Results:PKF050-638 caused a dose-dependent inhibition of the Rev-dependent RNA expression in a Rev-assay. Using GFP fusion proteins, we found PKF050-638 to be an inhibitor of the Rev nuclear export. Furthermore, it interfered with the nucleolar co-localization of CRM1 and Rev in human cells. Using a quantitative in vitro RanGAP hydrolysis assay, we demonstrated that PKF050-638 disrupts the CRM1-NES-RanGTP complex formation. In comparison with leptomycin B, a well-known inhibitor of the CRM1-mediated export, PKF050- 638 inhibited both wild-type S. cerevisiae and S.cerevisiae T539C mutated in its CRM1, whereas leptomycin B was only inhibitory to the mutated CRM1 yeast. Moreover, PKF050-638 displayed stereoselectivity in its activity against Rev function.

Conclusions:We identified a new drug interfering with the CRM1-mediated nuclear export through inhibition of CRM1-NES complex formation, with a site of interaction that extends beyond the site of interaction of leptomycin B.