Rocaglates Induce Gain-of-Function Alterations to eIF4A and eIF4F

Abstract

Rocaglates are a diverse family of biologically active molecules that have gained tremendous interest in recent years due to their promising activities in pre-clinical cancer studies. As a result, this family of compounds has been significantly expanded through the development of efficient synthetic schemes. However, it is unknown whether all of the members of the rocaglate family act through similar mechanisms of action. Here, we present a comprehensive study comparing the biological activities of >200 rocaglates to better understand how the presence of different chemical entities influences their biological activities. Through this, we find that most rocaglates preferentially repress the translation of mRNAs containing purine-rich 5' leaders, but certain rocaglates lack this bias in translation repression. We also uncover an aspect of rocaglate mechanism of action in which the pool of translationally active eIF4F is diminished due to the sequestration of the complex onto RNA.

Keywords: eIF4A; eIF4F; interfacial inhibitor; rocaglates; translation initiation.

Figure 1.. Different Rocaglates Exhibit Distinct Biological Activities

(A) Polypurine clamping is a correlative, but not universal, predictor of cap-dependent inhibition. The ΔmP obtained with eIF4A1:poly r(AG)8 RNA was measured for each compound (10 μM) and is plotted against the fold inhibition for cap-dependent translation (2 μM) of FF-HCV-Ren mRNA in Krebs-2. Note the duplicate values for RHT (open circles) are due to two preparations of different enantiomeric purity, and the duplicate values for CR-1–31-B (dotted circles) are due to two different compound batches (see Table S1). Pearson r = −0.62; p < 0.0001. (B) Rocaglates preferentially stimulate eIF4A binding onto purine-rich RNAs. Different RNA probes were incubated in the presence of 500 nM eIF4A1 and compound for 30 min before measurement. The ΔmP in the presence of compound relative to vehicle control is presented; n = 3 ± SEM. (C) mRNA sensitivity toward CR-1–31-B is correlated to 5′ leader purine content. The inhibition of cap-dependent (FLuc) and -independent (RLuc) translation was measured in response to CR-1–31-B; n = 3 ± SEM. (D) Dose response of the indicated rocaglates in Krebs-2 extracts programmed with the indicated mRNAs; n = 3 ± SEM. See also Figures S1, S2, and S3 and Table S1.

Figure 2.. In Cellula Activity of Rocaglates

(A) Cytotoxicity of rocaglates toward NIH 3T3 (gray circle) and eIF4A1^em1JP (red triangle) cells. Cells were exposed to 40 nM compound for 4 days, and viability was measured using the sulforhodamine B (SRB) assay; n = 3 ± SEM. (B) Rocaglates show different sequence preferences for inhibiting cap-dependent translation in cellula. HEK293T cells were transfected with the indicated mRNA reporters, compounds added 1 h later, and luciferase activity measured; n = 3 ± SD. (C) Dose response of the indicated mRNAs to hippuristanol and pateamine in HEK293T cells; n = 3 ± SD. (D) Comparison of the frequencies of quadruplet motifs in 5′ leaders of eIF4A1-bound mRNAs upon CR-1–13-B and silvestrol treatments relative to DMSO. The quadruplet motifs are color-coded: blue is for W4, orange is for R4, and cyan is for NAGT and AGTN. W = A, T; R = A, G. Data are compiled in Table S2. See also Figure S4 and Table S1.

Figure 3.. Increased eIF4F Retention Time on mRNA by Rocaglates Inhibits Translation

(A) RPDs performed with m⁷GpppG-capped RNA in RRL with DMSO or 500 nM rocaglate. (B) eIF4F:RNA complexes are stabilized by rocaglates. Biotinylated m⁷GpppG-capped polypurine RNA (1 μM) was incubated in the presence of purified eIF4F (4 nM) in the presence or absence of CR-1–31-B (500 nM). A 10-fold molar excess of non-biotinylated RNA was then added to the reaction for the indicated periods of time before the streptavidin pull-down. Complexes retained on the biotinylated RNA were then assessed by immunoblotting. (C) eIF4F pre-stabilized onto m⁷GpppG(AG)₁₀-FF-HCV-Ren by CR-1–31-B represses cap-dependent translation. RNA (100 nM), eIF4F (100 nM), and CR-1–31-B (500 nM) were pre-incubated at 30°C for 10 min and then added to RRL translation extracts. (D) The presence of m⁷GpppG-capped but not ApppG-capped purine-rich RNAs sensitizes the RocA/CR-1–31-B-unresponsive m⁷GpppG(UC)₁₀-FF-HCV-Ren mRNA reporter. Translation reactions were performed in Krebs-2 extracts with 10 nM mRNA reporter and 250 nM competitor RNA; n = 3 ± SEM. (E) Addition of purified eIF4F rescues rocaglate-mediated translation inhibition. The m⁷GpppG(AG)₁₀-FF-HCV-Ren reporter was added to Krebs-2 translation extracts in the presence of eIF4F (10 nM) and 100 nM of the indicated compound; n ≥ 3 ± SEM.

Figure 4.. Rocaglates Function through a Conditional Gain-of-Function Mechanism

(A) Western blot documenting endogenous and ectopic eIF4A1 levels. (B) Ectopic expression of WT eIF4A1 sensitizes rocaglate-resistant cells to cell death. NIH 3T3 or eIF4A1^em1JP cells were infected with an empty MSCV cassette or by expressing either WT eIF4A1 or eIF4A1(F163L). Viability was assessed following a 4-day exposure to 40 nM of compound using SRB assays; n = 3 ± SEM. (C) Schematic diagram highlighting the different ways by which rocaglates target translation.