Article | Published:

Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin

Nature Chemical Biology volume 6, pages 209217 (2010) | Download Citation

Abstract

Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of translation elongation by both CHX and LTM.

  • Compound C15H23NO4

    Cycloheximide

  • Compound C27H39NO7

    Migrastatin

  • Compound C27H39NO7

    iso-Migrastatin

  • Compound C26H35NO6

    Lactimidomycin

  • Compound C27H41NO8

    Dorrigocin A

  • Compound C27H41NO8

    Dorrigocin B

  • Compound C26H37NO6

    2,3-Dihydro-lactimidomycin

  • Compound C27H37NO7

    16,17-Didehydro-iso-migrostatin

  • Compound C35H44BrNO8

    N-p-Bromophenacyl-iso-migrostatin

  • Compound C26H39NO6

    2,3,8,9-Tetrahydro-lactimidomycin

  • Compound C26H39NO7

    8-Desmethyl-9-deshydroxy-2,3-dihydro-17-hydroxy-iso-migrastatin

  • Compound C27H41NO7

    9-Deshydroxy-2,3-dihydro-17-hydroxy-iso-migrastatin

  • Compound C26H39NO7

    8-Desmethoxy-2,3-dihydro-17-hydroxy-iso-migrastatin

  • Compound C26H39NO7

    (S)-8-Hydroxy-2,3,8,9-tetrahydro-lactimidomycin

  • Compound C26H37NO7

    8-Desmethoxy-9-deshydroxy-8,9-didehydro-17-hydroxy-dorrigocin B

  • Compound C27H43NO6

    2,3-Dihydro-iso-migrastatin

  • Compound C26H41NO5

    8-Desmethoxy-2,3-dihydro-iso-migrastatin

  • Compound C26H41NO6

    8-Desmethyl-2,3-dihydro-iso-migrastatin

  • Compound C26H37NO6

    8-Desmethoxy-16,17-didehydro-2,3-dihydro-iso-migrastatin

  • Compound C26H37NO7

    8-Desmethyl-16,17-didehydro-2,3-dihydro-iso-migrastatin

  • Compound C27H39NO7

    16,17-Didehydro-2,3-dihydro-iso-migrastatin

  • Compound C30H46N2O9S

    3-((R)-2-Amino-2-carboxyethylthio)-iso-migrastatin

  • Compound C29H45NO8S

    3-(2-Hydroxyethylthio)-iso-migrastatin

  • Compound C26H37NO6

    8-Desmethoxy-migrastatin

  • Compound C26H37NO7

    8-Desmethyl-migrastatin

  • Compound C27H39NO8

    17-Hydroxy-migrastatin

  • Compound C26H37NO7

    8-Desmethoxy-17-hydroxy-migrastatin

  • Compound C26H37NO6

    9-Deshydroxy-8-desmethoxy-17-hydroxy-migrastatin

  • Compound C27H37NO7

    16,17-Didehydro-migrastatin

  • Compound C26H35NO6

    8-Desmethoxy-16,17-didehydro-migrastatin

  • Compound C26H39NO6

    9-Deshydroxy-8-desmethoxy-dorrigocin A

  • Compound C26H39NO8

    8-Desmethyl-dorrigocin A

  • Compound C27H41NO8

    13-epi-Dorrigocin A

  • Compound C26H39NO6

    9-Deshydroxy-8-desmethoxy-13-epi-dorrigocin A

  • Compound C26H39NO8

    8-Desmethyl-dorrigocin B

  • Compound C30H41NO8

    Ethylacetyl-lactimidomycin

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1.

    & The bacterial ribosome as a target for antibiotics. Nat. Rev. Microbiol. 3, 870–881 (2005).

  2. 2.

    , , & The mechanism by which cycloheximide and related glutarimide antibiotics inhibit peptide synthesis on reticulocyte ribosomes. J. Biol. Chem. 246, 174–181 (1971).

  3. 3.

    & Translation elongation after assembly of ribosomes on the Cricket paralysis virus internal ribosomal entry site without initiation factors or initiator tRNA. Genes Dev. 17, 181–186 (2003).

  4. 4.

    , , , & Iso-migrastatin congeners from Streptomyces platensis and generation of a glutarimide polyketide library featuring the dorrigocin, lactimidomycin, migrastatin, and NK30424 scaffolds. J. Am. Chem. Soc. 127, 11930–11931 (2005).

  5. 5.

    et al. Lactimidomycin, a new glutarimide group antibiotic. Production, isolation, structure and biological activity. J. Antibiot. (Tokyo) 45, 1433–1441 (1992).

  6. 6.

    et al. The migrastatin family: discovery of potent cell migration inhibitors by chemical synthesis. J. Am. Chem. Soc. 126, 11326–11337 (2004).

  7. 7.

    et al. Synthetic analogues of migrastatin that inhibit mammary tumor metastasis in mice. Proc. Natl. Acad. Sci. USA 102, 3772–3776 (2005).

  8. 8.

    & Dorrigocins: novel antifungal antibiotics that change the morphology of ras-transformed NIH/3T3 cells to that of normal cells. III. Biological properties and mechanism of action. J. Antibiot. (Tokyo) 47, 875–880 (1994).

  9. 9.

    et al. Dorrigocins: novel antifungal antibiotics that change the morphology of ras-transformed NIH/3T3 cells to that of normal cells. I. Taxonomy of the producing organism, fermentation and biological activity. J. Antibiot. (Tokyo) 47, 862–869 (1994).

  10. 10.

    , , & Migrastatin and dorrigocins are shunt metabolites of iso-migrastatin. J. Am. Chem. Soc. 127, 1622–1623 (2005).

  11. 11.

    et al. Engineered production of iso-migrastatin in heterologous Streptomyces hosts. Bioorg. Med. Chem. 17, 2147–2153 (2009).

  12. 12.

    et al. Migrastatin, a new inhibitor of tumor cell migration from Streptomyces sp. MK929–43F1. Taxonomy, fermentation, isolation and biological activities. J. Antibiot. (Tokyo) 53, 1130–1136 (2000a).

  13. 13.

    et al. Migrastatin, a novel 14-membered lactone from Streptomyces sp. MK929–43F1. J. Antibiot. (Tokyo) 53, 1228–1230 (2000).

  14. 14.

    & Molecular cloning and analysis of yeast gene for cycloheximide resistance and ribosomal protein L29. Nucleic Acids Res. 10, 3133–3148 (1982).

  15. 15.

    , , , & Cycloheximide resistance in yeast: the gene and its protein. Nucleic Acids Res. 11, 3123–3135 (1983).

  16. 16.

    & The list of cytoplasmic ribosomal proteins of Saccharomyces cerevisiae. Yeast 14, 471–477 (1998).

  17. 17.

    , , & Cycloheximide resistance conferred by novel mutations in ribosomal protein L41 of Chlamydomonas reinhardtii. Mol. Gen. Genet. 264, 790–795 (2001).

  18. 18.

    et al. Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts. J. Bacteriol. 174, 254–262 (1992).

  19. 19.

    et al. Altering chemosensitivity by modulating translation elongation. PLoS One 4, e5428 (2009).

  20. 20.

    , & Selective action of erythromycin on initiating ribosomes. Biochemistry 13, 4653–4659 (1974).

  21. 21.

    & Analysis of 40 S and 80 S complexes with mRNA as measured by sucrose density gradients and primer extension inhibition. J. Biol. Chem. 267, 1554–1562 (1992).

  22. 22.

    & Factorless ribosome assembly on the internal ribosome entry site of cricket paralysis virus. J. Mol. Biol. 324, 889–902 (2002).

  23. 23.

    , & Inhibitors of protein synthesis identified by a high throughput multiplexed translation screen. Nucleic Acids Res. 32, 902–915 (2004).

  24. 24.

    & Where to begin? The mechanism of translation initiation codon selection in eukaryotes. Curr. Opin. Chem. Biol. 10, 480–486 (2006).

  25. 25.

    & The molecular mechanics of eukaryotic translation. Annu. Rev. Biochem. 73, 657–704 (2004).

  26. 26.

    et al. Stimulation of mammalian translation initiation factor eIF4A activity by a small molecule inhibitor of eukaryotic translation. Proc. Natl. Acad. Sci. USA 102, 10460–10465 (2005).

  27. 27.

    et al. Mechanism of tRNA translocation on the ribosome. Mol. Biol. (Mosk.) 35, 655–665 (2001).

  28. 28.

    Mechanism and regulation of eukaryotic protein synthesis. Microbiol. Rev. 56, 291–315 (1992).

  29. 29.

    & Kinetic dissection of fundamental processes of eukaryotic translation initiation in vitro. EMBO J. 18, 6705–6717 (1999).

  30. 30.

    , , & Hypusine-containing protein eIF5A promotes translation elongation. Nature 459, 118–121 (2009).

  31. 31.

    , & Probing the structure of mouse Ehrlich ascites cell 5.8S, 18S and 28S ribosomal RNA in situ. Nucleic Acids Res. 22, 1374–1382 (1994).

  32. 32.

    et al. Structure of the mammalian 80S ribosome at 8.7 A resolution. Structure 16, 535–548 (2008).

  33. 33.

    et al. The comparative RNA web (CRW) site: an online database of comparative sequence and structure information for ribosomal, intron, and other RNAs. BMC Bioinformatics 3, 2 (2002).

  34. 34.

    & Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites. Cell 57, 585–597 (1989).

  35. 35.

    & [3′-32P]-labeling tRNA with nucleotidyltransferase for assaying aminoacylation and peptide bond formation. Methods 44, 74–80 (2008).

  36. 36.

    , & Binding of the 3′ terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation. EMBO J. 8, 3933–3938 (1989).

  37. 37.

    & Does the ribosome translate cancer? Nat. Rev. Cancer 3, 179–192 (2003).

  38. 38.

    Antitumor compounds from tunicates. Med. Res. Rev. 20, 1–27 (2000).

  39. 39.

    & Mechanism of protein synthesis inhibition by didemnin B in vitro. Biochemistry 34, 9177–9184 (1995).

  40. 40.

    , , , & Reconstitution of yeast translation initiation. Methods Enzymol. 430, 111–145 (2007).

  41. 41.

    RNA-Protein Interactions: a Practical Approach (Oxford University Press, 1998).

  42. 42.

    , & Probing the conformational changes in 5.8S, 18S and 28S rRNA upon association of derived subunits into complete 80S ribosomes. Nucleic Acids Res. 22, 2776–2783 (1994).

Download references

Acknowledgements

We are indebted to J. Boeke (Johns Hopkins University) and J. Warner (Albert Einstein College of Medicine) for the CHX-resistant strains of S. cerevisiae, J. Pelletier (McGill University) for providing us with the HCV and EMCV IRES reporter constructs and P. Sarnow (Stanford University) for providing the CrPV vector. We thank the laboratories of J. Hart, P. Englund, J. Lorsch, S. Sukumar and R. Rao for use of specialized equipment and constructive advice. This work was supported in part by grants from the US National Cancer Institute and the Flight Attendant Medical Research Institute (J.O.L.) and by US National Cancer Institute grants CA106150 and CA113297 (B.S.).

Author information

Affiliations

  1. Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Tilman Schneider-Poetsch
    • , Yongjun Dang
    • , Shridhar Bhat
    •  & Jun O Liu
  2. Division of Pharmaceutical Sciences, University of Wisconsin, Madison, Wisconsin, USA.

    • Jianhua Ju
    •  & Ben Shen
  3. Department of Molecular Biology and Genetics, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Daniel E Eyler
    •  & Rachel Green
  4. Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio, USA.

    • William C Merrick
  5. University of Wisconsin National Cooperative Drug Discovery Group, Madison, Wisconsin, USA.

    • Ben Shen
  6. Department of Chemistry, University of Wisconsin, Madison, Wisconsin, USA.

    • Ben Shen
  7. Department of Oncology, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.

    • Jun O Liu

Authors

  1. Search for Tilman Schneider-Poetsch in:

  2. Search for Jianhua Ju in:

  3. Search for Daniel E Eyler in:

  4. Search for Yongjun Dang in:

  5. Search for Shridhar Bhat in:

  6. Search for William C Merrick in:

  7. Search for Rachel Green in:

  8. Search for Ben Shen in:

  9. Search for Jun O Liu in:

Contributions

T.S.-P. and J.O.L. designed the experiments; T.S.-P., D.E.E., Y.D. and S.B. performed the experiments; J.J., W.C.M., R.G. and B.S. contributed reagents; T.S.-P., D.E.E., Y.D., R.G., B.S. and J.O.L. analyzed data and T.S.-P. and J.O.L. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Jun O Liu.

Supplementary information

PDF files

  1. 1.

    Supplementary Text and Figures

    Supplementary Methods, Supplementary Figures 1–8 and Supplementary Tables 1 and 2

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nchembio.304

Further reading