1. Volume 24, Issue 5, Pages 605-613.e5 (May 2017)
Inhibition of Eukaryotic Translation by the Antitumor Natural Product Agelastatin A 
Brandon McClary, Boris Zinshteyn, Mélanie Meyer, Morgan Jouanneau, Simone Pellegrino, Gulnara Yusupova, Anthony Schuller, Jeremy Chris P. Reyes, Junyan Lu, Zufeng Guo, Safiat Ayinde, Cheng Luo, Yongjun Dang, Daniel Romo, Marat Yusupov, Rachel Green, Jun O. Liu 
Cell Chemical Biology 
Volume 24, Issue 5, Pages e5 (May 2017)
DOI: /j.chembiol
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2. Cell Chemical Biology 2017 24, 605-613. e5DOI: (10. 1016/j. chembiol
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3. Figure 1 Chemical Structures of Agelastatin A and Congeners
Rings and key positions are labeled in the structure of agelastatin A. Structural differences between the congeners and agelastatin A are highlighted (red).
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4. Figure 2 Dose-Dependent Inhibition of Translation by AglA
(A and B) HeLa cells were incubated with varying concentrations of each compound in the presence of [3H]thymidine, [3H]uridine, or [35S]cysteine/methionine for 1 hr. Protein synthesis was measured by scintillation counting of trichloroacetic acid (TCA)-precipitated proteins on a filter. DNA synthesis and transcription were monitored by scintillation counting of nucleic acids bound to a filter.
(C) IC50 values for AglA and CHX for inhibition of protein and DNA synthesis. Mean values ± SEM (error bars) from three independent experiments are shown. IC50 values are listed ± SE.
(D) Drugs were added to a rabbit reticulocyte lysate (RRL) cocktail that included a control luciferase poly(A) mRNA (supplied by Promega), and [35S]methionine was added for 1 hr. Translated product was subjected to SDS-PAGE followed by autoradiography. AglA, (−)-agelastatin A; CHX, cycloheximide.
See also Figures S1 and S7.
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5. Figure 3 AglA Inhibits Translation Elongation
(A) Dual luciferase reporters with a conventional capped firefly luciferase followed by renilla luciferase under the translational control of HCV IRES were used in in vitro RRL translation assays in the presence of different concentrations of AglA, 4 μM PatA, and 4 μM CHX.
(B) Stress granule induction in U2OS cells stably expressing GFP-G3BP in the presence of different compounds as indicated. Images (40× objective) were captured using an Olympus B X61 fluorescence microscope.
(C) AglA inhibits dipeptide formation in vitro. Ribosome initiation complexes were pre-incubated with drug for 5 min. eEF1A, Phe-tRNAPhe, and GTP were added to complexes and dipeptide formation observed over time. PatA, pateamine A.
See also Figure S2.
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6. Figure 4 AglA Is Bound within the PTC of the Larger Ribosomal Subunit
(A and B) MA plots showing, for each rRNA nucleotide, the average DMS-dependent mutation rate on the x axis, against the fold change upon AglA treatment on the y axis. Nucleotides passing significance and fold-change cutoffs are orange, and AglA-protect nucleotides from (D) are labeled.
(C) Fold change in mutation rate, relative to a no-DMS control, for an unaffected nucleotide (A2819), and four AglA-protected nucleotides near the P site in the 25S rRNA.
(D) Molecular docking model of the interaction of AglA with the 80S ribosome of S. cerevisiae (PDB: 4U52). The numberings correspond to the yeast 25S rRNA sequence.
See also Figures S3, S4.
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7. Figure 5 Crystal Structure of AglA-80S Ribosome Complex
(A) AglA (pink) fitted in the unbiased difference map (Fo − Fc). Structure has been determined to the resolution of 3.5 Å. See Table S1 for statistics of data collection and processing.
(B) AglA (pink) forms hydrogen bonds with U2869 and U2873, a π-halogen (pyrrole bromine) interaction with U2875 of 25S rRNA (green), and an electrostatic interaction with a magnesium ion (yellow).
(C) Stacking interactions occur between A2820 and C2821 of 25S rRNA at the A site (green) and AglA (pink).
(D) Superimposition of vacant ribosome structure (orange; PDB: 4V88) and ribosome bound to AglA structure (green) displaying major movements of nucleotides A2404, C2821, and U2875 (black arrows) induced by the binding of AglA (pink) to the A site.
See also Figures S5, S6, and Table S1.
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