Evidence for a Functionally Relevant Rocaglamide Binding Site on the eIF4A–RNA Complex

Translation initiation is an emerging target in oncology and neurobiology indications. Naturally derived and synthetic rocaglamide scaffolds have been used to interrogate this pathway; however, there is uncertainty regarding their precise mechanism(s) of action. We exploited the genetic tractability of yeast to define the primary effect of both a natural and a synthetic rocaglamide in a cellular context and characterized the molecular target using biochemical studies and <i>in silico</i> modeling. Chemogenomic profiling and mutagenesis in yeast identified the eIF (eukaryotic Initiation Factor) 4A helicase homologue as the primary molecular target of rocaglamides and defined a discrete set of residues near the RNA binding motif that confer resistance to both compounds. Three of the eIF4A mutations were characterized regarding their functional consequences on activity and response to rocaglamide inhibition. These data support a model whereby rocaglamides stabilize an eIF4A-RNA interaction to either alter the level and/or impair the activity of the eIF4F complex. Furthermore, <i>in silico</i> modeling supports the annotation of a binding pocket delineated by the RNA substrate and the residues identified from our mutagenesis screen. As expected from the high degree of conservation of the eukaryotic translation pathway, these observations are consistent with previous observations in mammalian model systems. Importantly, we demonstrate that the chemically distinct silvestrol and synthetic rocaglamides share a common mechanism of action, which will be critical for optimization of physiologically stable derivatives. Finally, these data confirm the value of the rocaglamide scaffold for exploring the impact of translational modulation on disease

Crystallographic data and refinement information.

a<p>Numbers in parenthesis are for the highest resolution shell (3.06-2.90).</p>b<p>R_sym = Σ|I_h−h>|/ΣI_h over all h, where I_h is the intensity of reflection h.</p>c<p>R_cryst and R_free = Σ∥F_o|−|F_c∥/Σ|F_o|, where F_o and F_c are observed and calculated amplitudes, respectively. Rfree was calculated using 5% of data excluded from the refinement.</p

Key residues involved in resistance to argyrin B are conserved in EF-G homologues.

<p>The amino acid sequence of <i>P. aeruginosa</i> EF-G1 was used to identify EF-G homologues in the indicated organisms by a BLAST search. EF-G sequences were then aligned using ClustalW, and residues conferring resistance to argyrin B are shown in bold.</p

Co-crystal structure of argyrin B bound to <i>P.aeruginosa</i> EF-G1.

<p>(<b>A</b>) The argyrin B binding pocket localizes to the flexible interface between domains III and V, distinct from the GTP/fusidic acid binding domain (**). (<b>B</b>) Inset view. (<b>C</b>) 2D protein-ligand interaction plot showing the chemical structure of the argyrin B macrocyclic polypeptide and the hydrophobic (cyan) and hydrophilic (yellow) amino-acid residues in EF-G1 which are in binding contact. (<b>D</b>) Interactions between <i>P. aeruginosa</i> EF-G (domain III in yellow and domain V in cyan) and argyrin B (gray). (<b>E</b>) Superposition of Thermus thermophilus EF-G in complex with GTP (magenta), Thermus thermophilus EF-G in complex with the ribosome (ribosome not shown) and fuscidic acid (cyan), and structure of the argyrin B-bound Pseudomonas aeroginosa EF-G (FusA1) (yellow). Superposition was done using domains I and II of each of the protein structures.</p

Susceptibility of representative bacteria and resistant mutants to argyrin B.

<p>Susceptibility determinations were conducted using the broth microdilution protocol as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042657#pone.0042657-Caughlan1" target="_blank">[26]</a>.</p>*<p>Argyrin B was not uniformly soluble and occasionally a small amount of precipitate was visible at concentrations greater than 16–32 µg/ml; therefore values here are reported as susceptibility rather than MIC. Selected on 128^a, 2^b, 4^c, or 16^d µg/ml argyrin B in solid Mueller-Hinton agar.</p

The mode of action of argyrin B is conserved in mammalian cells.

<p>(<b>A</b>) Cytotoxicity profile of argyrin B across 512 mammalian cell lines showing reduced cell viability with an IC₅₀ below 1 µM in 18 cell lines (red). (<b>B</b>) Susceptibility to argyrin B (IC₅₀ and A_max values) was compared to different cytotoxic agents across the cell line panel by calculating Pearson correlation values. (<b>C</b>) RKO and HCT116 cells were treated for 4 days with 1 µM argyrin B, and total proteins were extracted and analyzed by immunoblotting for SDHA and COX2. (<b>D</b>) Cells were transfected with non-targeting (NT) or GFM1 (encoding mEF-G1) siRNA for 7 days, and total proteins were extracted and analyzed by immunoblotting for mEF-G1 and GAPDH. (<b>E</b>) siRNA-transfected cells were treated for 7 days with increasing doses of Argyrin B or MG132, and cell viability was assessed using CellTiter Glo. A representative example of three independent experiments is shown.</p

Rescue of argyrin B-sensitivity by expression of mEF-G1 L693Q.

<p>(<b>A</b>) mEF-G1 wild-type (WT), S452L, S494F or L693Q were stably over-expressed in HCT116 and RKO, cells were treated for 7 days with increasing doses of argyrin B or MG132, and cell viability was assessed using CellTiter Glo. A representative example of two independent experiments is shown. (<b>B</b>) Increase in IC₅₀ relative to the parental cell line. Average fold increase was calculated from two independent experiments. (<b>C</b>) HCT116 and RKO stably expressing mEF-G1 WT, S452L, S494F or L693Q were lysed, and total proteins extracted and analyzed by immunoblotting for mEF-G1 and GAPDH. (<b>D</b>) Binding of argyrin B to recombinant human mEF-G1 WT, S452L, S494F or L693Q was measured by Biacore and is depicted relative to recombinant bacterial EF-G.</p