Amicoumacin A induces cancer cell death by targeting the eukaryotic ribosome

Abstract

Amicoumacin A is an antibiotic that was recently shown to target bacterial ribosomes. It affects translocation and provides an additional contact interface between the ribosomal RNA and mRNA. The binding site of amicoumacin A is formed by universally conserved nucleotides of rRNA. In this work, we showed that amicoumacin A inhibits translation in yeast and mammalian systems by affecting translation elongation. We determined the structure of the amicoumacin A complex with yeast ribosomes at a resolution of 3.1 Å. Toxicity measurement demonstrated that human cancer cell lines are more susceptible to the inhibition by this compound as compared to non-cancerous ones. This might be used as a starting point to develop amicoumacin A derivatives with clinical value.

Figure 1. Amicoumacin A inhibits mammalian mRNA translation.

(a) Chemical structure of amicoumacin A. (b) Inhibition of reporter mRNA translation by amicoumacin A in HEK293T cells. Error bars represent the standard deviations of the mean values for at least three independent experiments. (c) Inhibition of reporter mRNA translation by amicoumacin A in Krebs-2 cells S30 extract. (d) Ribosome stalling by amicoumacin A and other antibiotics in rabbit reticulocyte lysate as revealed by toe-printing assay. Cross signs denote components added to the reaction mixture. Final concentrations of the additives were as follows: 15 mM Mg(OAc)₂ (lane 1); 0.2% EtOH (lane 2); 2 mM GMPPNP and 0.2% EtOH (lane 3); 2 mM GMPPNP and 100 μM amicoumacin A (lane 4); 100 μM amicoumacin A (lane 5); 100 μM amicoumacin A and 1 mM cycloheximide (lane 6); 1 mM cycloheximide and 0.2% EtOH (lane 7); 2 mM GMPPNP, 100 μM amicoumacin A and 1 mM cycloheximide (lane 8). Note that the toe-print pattern produced by the 48S complex (lanes 3, 4 and 8) differs from that made by the elongating 80S ribosome (lanes 5–7), in accordance with the previous observation. (e) Inhibition of ribosome movement along mRNA by amicoumacin A. The antibiotic concentrations were 0, 1, 10 or 100 μM (in lanes 1–2, 3, 4 and 5–6, respectively).

Figure 2. Amicoumacin A action on yeast S. cerevisiae.

(a) Yeast culture growth in the presence of amicoumacin A. OD600 values were divided to that in a plate well without the drug and taken at the time point of 11 h (when these ratios were minimal, as shown in Supplementary Fig. 2). The maximum concentration of the drug used in this experiment (10 μM) was equal to 4.2 μg/ml amicoumacin A added into the medium. Error bars represent the standard deviations of the mean values for three replicates. (b) Inhibition of reporter mRNA in vitro translation by amicoumacin A in the yeast cell-free system. Error bars represent the standard deviations of the mean values for three independent experiments.

Figure 3. Structure of the amicoumacin A complex with yeast ribosome.

(a) Difference electron density map of amicoumacin A in complex with 80S ribosome. The map is contoured at 2σ. Amicoumacin A is shown in yellow. (b) Comparison of amicoumacin A conformation in yeast 80S and bacterial 70S ribosome. The 70S ribosome from T. thermophilus (PDB entry 4W2F) in complex with amicoumacin A (shown in blue) was aligned on the 80S ribosome based on the helix 23 in 18S or 16S rRNA (ribosomal components are omitted for clarity). (c) The binding pocket of amicoumacin A in small ribosomal subunit E-site. Two orientations of the 80S ribosome in complex with amicoumacin A are shown in the upper panel. The view from the side of the 40S head is shown on the left, and the view from the subunit interface is shown on the right (60S subunit is omitted for clarity). The 40S subunit is colored in wheat, and the 60S subunit is in light blue. The binding pocket of amicoumacin A is magnified in the lower panel. Amicoumacin A is shown in yellow, rRNA residues (in wheat, proteins uS11 and uS7) in green, and magnesium ions in brown. Interactions of amicoumacin A are depicted with the dashed lines. (d) Comparison of the amicoumacin A binding site in a yeast 80S ribosome with the one in bacterial 70S ribosome from T. thermophilus. Amicoumacin A is shown in blue, rRNA residues in grey, protein uS7 in violet, and magnesium ions in dark green (PDB entry 4W2F).