Selective Pharmacological Targeting of a DEAD Box RNA Helicase
Figure 2
Selectivity of hippuristanol for eIF4A.
(A) Inhibition of eIF4A RNA-dependent ATPase activity by hippuristanol. ATPase assays were performed with 0.1 µg of His6-eIF4AI or His6-eIF4AII at 25°C or with 0.1 µg of His6-eIF4AIII at 37°C for 2 h with 0.1 µCi γ-32P-ATP (10 Ci/mmol). Following analysis by TLC and quantitation using a Fuji BAS 2000 phosphoimager, the percent hydrolysis was determined and set relative to the DMSO vehicle control reactions. Each value represents the average of three measurements with the error shown as the standard deviation. (B) Crosslinking of recombinant proteins to RNA in the presence of hippuristanol. 32P-labelled CAT RNA was cross-linked to 0.5–1 µg of the indicated recombinant protein in the presence or absence of hippuristanol, separated by SDS-PAGE, and visualized by autoradiography. [Note that in our hands, recombinant hDDX52 did not crosslink to RNA.] (C) Relative ATPase activity of eIF4AI, hDDX19, and hDDX52 in the presence of 50 µM hippuristanol. eIF4AI and hDDX19 where performed at 25°C for 5 minutes while hDDX52 was incubated for 60 minutes to allow for analysis to be in the linear range of ATP hydrolysis. The percent ATP hydrolysis was determined in the presence of hippuristanol and set relative to the DMSO vehicle control reactions. The results represent the average of 3 experiments with error bars signifying the standard deviation.