Fig. 1.

Design of an eIF4A inhibitor (MG-002) that potently inhibits cap-dependent mRNA translation. (A) Schematic outlining the rationale behind MG-002 development. RNA- (blue shading) and eIF4A1 (green shading)-interacting regions are highlighted on the RocA structure. Insets show close in views of a model of the MG-002 hydroxymate and nitrile groups, based on the RocA-bound crystal structure of eIF4A1 (26). (B) Assessing compound-induced clamping of eIF4A1 to FAM-labeled RNA of the indicated nucleotide composition by fluorescence polarization assay. The ΔmP (change in polarization) obtained with eIF4A1•AMPPNP •poly (NN)₈ RNA was measured for the indicated compounds at 10 µM. The ΔmP obtained relative to dimethylsulfoxide (DMSO) is shown (n = 3 ±SD). (C) Relative dissociation of pre-formed eIF4A1•ATP•Cmpd•FAM-poly (AG)₈ complexes measured as a function of time in the presence of 1,000-fold molar excess poly (AG)₈ RNA. DMSO, t_1/2 ~4.1 ± 1 min; CR-1-31B (10 µM), t_1/2 ~59 ± 6.5 min; MG-002 (10 µM), t_1/2 ~68 ± 2.8 min; eFT226 (10 µM), t_1/2 ~ 27 ± 5.8 min. Error values were calculated from the 95% CI. (D) Differential scanning fluorimetry analysis of eIF4A1 (8 µM) in the presence of the indicated compounds (15 µM), 15 µM poly (AG)₈, and 1 mM AMPPNP. The transition midpoint temperature shifts (ΔT₅₀) are: CR-1-31B, 7.3 °C; eFT226, 7 °C; MG-002, 7.2 °C; PatA, 10 °C (n = 3 ±SD). (E) Inhibition of cap-dependent (FLuc) and independent (RLuc) translation was measured in response to the indicated compounds in Krebs-2 translation extracts programmed with the noted bicistronic mRNA. IC₅₀s toward inhibition of FLuc synthesis from (CAG)-FF/HCV-IRES/Ren mRNA were: CR-1-31B, 54 ± 4 nM; eFT226, 813 ± 91 nM; MG-002, 43 ± 4 nM (n = 2 ±SD). (F) eHap1 cells were incubated in the presence of the indicated concentrations of compound for 1 h. During the last 15 min of incubation, ³⁵S-Met was added followed by TCA precipitation and quantitation of ³⁵S-Met incorporation into protein (n = 3 ±SD).