Amidino-Rocaglates: A Potent Class of eIF4A Inhibitors

Abstract

Rocaglates share a common cyclopenta[b]benzofuran core that inhibits eukaryotic translation initiation by modifying the behavior of the RNA helicase, eIF4A. Working as interfacial inhibitors, rocaglates stabilize the association between eIF4A and RNA, which can lead to the formation of steric barriers that block initiating ribosomes. There is significant interest in the development and expansion of rocaglate derivatives, as several members of this family have been shown to possess potent anti-neoplastic activity in vitro and in vivo. To further our understanding of rocaglate diversity and drug design, herein we explore the RNA clamping activity of >200 unique rocaglate derivatives. Through this, we report on the identification and characterization of a potent class of synthetic rocaglates called amidino-rocaglates. These compounds are among the most potent rocaglates documented to date and, taken together, this work offers important information that will guide the future design of rocaglates with improved biological properties.

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

Figure 1.. Rocaglates similarly enhance RNA binding of eIF4A1 and eIF4A2.

a. Schematic diagram of FP assay used to measure eIF4A:RNA association. FAM-labeled RNA probes are excited by plane-polarized light in the presence of eIF4A ± rocaglate. In the absence of eIF4A binding, the RNA probe rapidly tumbles and the emitted light becomes depolarized. Binding of eIF4A to RNA hinders probe rotation and results in polarized light emission. b. Coomassie blue staining of SDS-PAGE showing eIF4A1 and eIF4A2 preparations used herein. c. eIF4A1 and eIF4A2 possess similar RNA binding specificities. eIF4A1 or eIF4A2 (500 nM) were incubated in the presence of FAM-labelled RNA (10 nM) having the indicated sequence composition for 30 min, after which FP measurements were taken. The change in FP obtained relative to the DMSO control (which represents the eIF4A1:RNA association in the absence of compound) is presented. n = 3 ± SEM. d. Chemical structure of CR-1–31-B. e. Binding of eIF4A1 and eIF4A2 to RNA is equally responsive to CR-1–31-B. FAM labeled poly r(AG)₈ (10 nM) was mixed with the indicated concentrations of eIF4A1 or eIF4A2 either in the presence of vehicle (DMSO) or 10 µM CR-1–31-B. Reactions were equilibrated at RT for 30 min prior to measuring light polarization. n = 3 ± SEM. f. Stimulation of eIF4A1:RNA binding by CR-1–31-B shows preference for polypurine-enriched sequences. FAM-labelled RNA was incubated in the presence of 500 nM eIF4A1 and the indicated concentration of CR-1–31-B for 30 min, after which time FP measurements were obtained. The change in FP relative to vehicle controls is presented. n = 3 ± SEM. g. The extent of eIF4A1:RNA binding stimulated by CR-1–31-B scales with polypurine content. FAM-labelled RNA was incubated with 500 nM eIF4A1 and the indicated concentration of CR-1–31-B for 30 min, after which time FP measurements were obtained. The change in FP obtained relative to vehicle controls is presented. n = 3 ± SEM.

Figure 2.. Rocaglate activity profiling.

a. Chemical structure of the most commonly used rocaglates in biological studies. b. Assessing eIF4A1:poly r(AG)₈ (grey circles) or eIF4A1:poly r(UC)₈ (red circles) RNA binding by FP in the presence of 10 µM rocaglate. Values are expressed relative to the DMSO control (containing RNA and protein in the absence of compound) and data is rank ordered. n = 3 ± SEM. Expanded view to the right shows the structures of the top three rocaglate hits. The duplication of RHT and CR-1–31-B represent independent compound preparations of different enantiomeric composition (see Table S1, column I for more details). c. Change in polarization obtained with eIF4A1:poly r(AG)₈ and eIF4A2:poly r(AG)₈ RNA. Pearson r = 0.814; p <0.0001. d. Inhibition of cap-dependent and independent translation (as reflected by firefly and renilla RLU, respectively) measured in response to compound in Krebs-2 extracts programmed with mRNA. n = 3 ± SEM. e. ³²P-labeled (AG)₁₀-FF/HCV/Ren mRNA was incubated with 100 nM eIF4A1 in the presence of 500 nM compound for 10 min at RT, then added to RRL in the presence of 600 µM cycloheximide. Complexes were resolved by sedimentation through a 10%–30% glycerol gradient.

Figure 3.. ADRs represent a potent class of rocaglates.

a. The IC₅₀ for cytotoxicity against NIH/3T3 cells versus inhibition of in vitro translation is plotted for the ADR subfamily. Translation reactions were performed in RRL programmed with 10 ng/mL m⁷GpppG-(AG)₁₀-FF/HCV/Ren. n = 3. See Table S2 for SEM values. Pearson r = 0.69, p <0.001. b. Structure of CMLD012612. c. Modeling of ADR CMLD012612 into the published X-ray structure of RocA in complex with eIF4A1 and poly-(AG)₅ RNA (PDB ID: 5ZC9). Hydrogen bonds are represented by a yellow line and π-stacking interactions are shown in cyan. d. SAR analysis of ADRs.

Figure 4.. CMLD012612 inhibits tumor cell survival.

a. Inhibition of ³⁵S-methioinine incorporation in HEK293 cells following 1 h compound exposure. n = 3 ± SEM. b. Cytotoxicity of CMLD012612 towards NIH/3T3 and eIF4A1^em1JP cells following 4 day compound exposure. n = 3 ± SEM. c. CMLD012612 inhibits translation in vivo in the liver. Mice were injected with vehicle or CMLD012612 (0.5 mg/kg). Cytoplasmic extracts were prepared from livers 3 h later and resolved on 10%–50% sucrose gradients by centrifugation in an SW40 rotor at 150,000 × g for 2 h. Plotted are results of one representative experiment of two that showed similar results. The positions of 80S ribosomes and polysomes in the gradient are labeled, and the polysome/monosome (P/M) ratios indicated. d. CMLD012612 sensitizes myr-Akt/Eµ-Myc tumors to doxorubicin in vivo. Kaplan-Meier plot showing tumor-free survival of mice bearing myr-Akt/Eµ-Myc tumors following treatment with doxorubicin (Dox, red line; n = 10), CMLD012612 (solid black line; n = 10), CR-1–31-B + Dox (blue line; n = 4), or CMLD012612 + Dox (dashed black line; n = 10). p<0003 for CR-1–31-B+Dox versus Dox, and p<0.00001 for CMLD012612+Dox versus Dox.