Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR

Abstract

Careful regulation of mRNA half-lives is a fundamental mechanism allowing cells to quickly respond to changing environmental conditions. The mRNA-binding Hu proteins are important for stabilization of short-lived mRNAs. Here we describe the identification and mechanistic characterization of the first low-molecular-weight inhibitors for Hu protein R (HuR) from microbial broths (Actinomyces sp.): dehydromutactin (1), MS-444 (2) and okicenone (3). These compounds interfere with HuR RNA binding, HuR trafficking, cytokine expression and T-cell activation. A mathematical and experimental analysis of the compounds' mode of action suggests that HuR homodimerizes before RNA binding and that the compounds interfere with the formation of HuR dimers. Our results demonstrate the chemical drugability of HuR; to our knowledge HuR is the first example of a drugable protein within the Hu family. MS-444, dehydromutactin and okicenone may become valuable tools for studying HuR function. An assessment of HuR inhibition as a central node in malignant processes might open up new conceptual routes toward combatting cancer.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Figure 1: Identification of HuR inhibitors by confocal high-throughput screening.
Figure 2: HuR ARE binding inhibition by 1 and 2.
Figure 3: Inhibition of HuR homodimerization by 1, 2 and 3.
Figure 4: Experimental evidence for inhibition of HuR homodimerization by 1 and 2.
Figure 5: Direct binding of 3 to HuR, monitored by microdialysis.
Figure 6: Effect of 1 and 2 on primary human T cells.
Figure 7: 2 decreases ARE cytokine expression in activated primary human monocytes.

Accession codes

Accessions

GenBank/EMBL/DDBJ

References

  1. 1

    Bakheet, T., Williams, B.R. & Khabar, K.S. ARED 3.0: the large and diverse AU-rich transcriptome. Nucleic Acids Res. 34, D111–D114 (2006).

    CAS  Article  Google Scholar 

  2. 2

    López de Silanes, I., Lal, A. & Gorospe, M. HuR - posttranscriptional paths to malignancy. RNA Biol. 2, e11–e13 (2005).

    Article  Google Scholar 

  3. 3

    López de Silanes, I. et al. Role of the RNA-binding protein HuR in colon carcinogenesis. Oncogene 22, 7146–7154 (2003).

    Article  Google Scholar 

  4. 4

    Erkinheimo, T.L. et al. Cytoplasmic HuR expression correlates with epithelial cancer cell but not with stromal cell cyclooxygenase-2 expression in mucinous ovarian carcinoma. Gynecol. Oncol. 99, 14–19 (2005).

    CAS  Article  Google Scholar 

  5. 5

    Mrena, J. et al. Prognostic significance of cyclin A in gastric cancer. Int. J. Cancer 119, 1897–1901 (2006).

    CAS  Article  Google Scholar 

  6. 6

    Erkinheimo, T.L. et al. Cytoplasmic HuR expression correlates with poor outcome and with cyclooxygenase 2 expression in serous ovarian carcinoma. Cancer Res. 63, 7591–7594 (2003).

    CAS  PubMed  Google Scholar 

  7. 7

    Denkert, C. et al. Overexpression of the embryonic-lethal abnormal vision-like protein HuR in ovarian carcinoma is a prognostic factor and is associated with increased cyclooxygenase 2 expression. Cancer Res. 64, 189–195 (2004).

    CAS  Article  Google Scholar 

  8. 8

    Denkert, C. et al. Expression of the ELAV-like protein HuR is associated with higher tumor grade and increased cyclooxygenase-2 expression in human breast carcinoma. Clin. Cancer Res. 10, 5580–5586 (2004).

    CAS  Article  Google Scholar 

  9. 9

    Dalmau, J., Furneaux, H.M., Cordon-Cardo, C. & Posner, J.B. The expression of the Hu (paraneoplastic encephalomyelitis/sensory neuronopathy) antigen in human normal and tumor tissues. Am. J. Pathol. 141, 881–886 (1992).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 10

    Dalmau, J., Furneaux, H.M., Rosenblum, M.K., Graus, F. & Posner, J.B. Detection of the anti-Hu antibody in specific regions of the nervous system and tumor from patients with paraneoplastic encephalomyelitis/sensory neuronopathy. Neurology 41, 1757–1764 (1991).

    CAS  Article  Google Scholar 

  11. 11

    Wilkinson, P.C. & Zeromski, J. Immunofluorescent detection of antibodies against neurones in sensory carcinomatous neuropathy. Brain 88, 529–583 (1965).

    CAS  Article  Google Scholar 

  12. 12

    Voltz, R. Paraneoplastic neurological syndromes: an update on diagnosis, pathogenesis, and therapy. Lancet Neurol. 1, 294–305 (2002).

    Article  Google Scholar 

  13. 13

    Liu, J. et al. Paraneoplastic encephalomyelitis antigens bind to the AU-rich elements of mRNA. Neurology 45, 544–550 (1995).

    CAS  Article  Google Scholar 

  14. 14

    Datta, K. et al. Role of elongin-binding domain of von hippel lindau gene product on HuR-mediated VPF/VEGF mRNA stability in renal cell carcinoma. Oncogene 24, 7850–7858 (2005).

    CAS  Article  Google Scholar 

  15. 15

    Heinonen, M. et al. Cytoplasmic HuR expression is a prognostic factor in invasive ductal breast carcinoma. Cancer Res. 65, 2157–2161 (2005).

    CAS  Article  Google Scholar 

  16. 16

    Carlsson, A. & Schwartz, S. Inhibitory activity of the human papillomavirus type 1 AU-rich element correlates inversely with the levels of the elav-like HuR protein in the cell cytoplasm. Arch. Virol. 145, 491–503 (2000).

    CAS  Article  Google Scholar 

  17. 17

    Denkert, C. et al. Expression of the ELAV-like protein HuR in human colon cancer: association with tumor stage and cyclooxygenase-2. Mod. Pathol. 19, 1261–1269 (2006).

    CAS  Article  Google Scholar 

  18. 18

    Zhang, J.H., Chung, T.D.Y. & Oldenburg, K.R. A simple statistical parameter for use in evaluation and validation of high throughput screening assazs. J. Biomol. Screen. 4, 67–73 (1999).

    CAS  Article  Google Scholar 

  19. 19

    Meisner, N.C. et al. mRNA openers and closers: modulating AU-rich element-controlled mRNA stability by a molecular switch in mRNA secondary structure. ChemBioChem 5, 1432–1447 (2004).

    CAS  Article  Google Scholar 

  20. 20

    Maris, C., Dominguez, C. & Allain, F.H. The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene expression. FEBS J. 272, 2118–2131 (2005).

    CAS  Article  Google Scholar 

  21. 21

    Dean, J.L.E. et al. The 3′-untranslated region of tumor necrosis factor α mRNA is a target of the mRNA-stabilizing factor HuR. Mol. Cell. Biol. 21, 721–730 (2001).

    CAS  Article  Google Scholar 

  22. 22

    Kasashima, K., Sakashita, E., Saito, K. & Sakamoto, H. Complex formation of the neuron-specific ELAV-like Hu RNA-binding proteins. Nucleic Acids Res. 30, 4519–4526 (2002).

    CAS  Article  Google Scholar 

  23. 23

    Huang, C.Y. Determination of binding stoichiometry by the continuous variation method: the Job plot. Methods Enzymol. 87, 509–525 (1982).

    CAS  Article  Google Scholar 

  24. 24

    Gallouzi, I.E. & Steitz, J.A. Delineation of mRNA export pathways by the use of cell-permeable peptides. Science 294, 1895–1901 (2001).

    CAS  Article  Google Scholar 

  25. 25

    Wang, W., Caldwell, C., Lon, S., Furneaux, H. & Gorospe, M. HuR regulates cyclin A and cyclin B1 mRNA stability during cell proliferation. EMBO J. 19, 2340–2350 (2000).

    CAS  Article  Google Scholar 

  26. 26

    Wang, W. et al. HuR regulates p21 mRNA stabilization by UV light. Mol. Cell. Biol. 20, 760–769 (2000).

    CAS  Article  Google Scholar 

  27. 27

    Lal, A., Kawai, T., Yang, X., Mazan-Mamczarz, K. & Gorospe, M. Antiapoptotic function of RNA-binding protein HuR effected through prothymosin alpha. EMBO J. 24, 1852–1862 (2005).

    CAS  Article  Google Scholar 

  28. 28

    Goldberg-Cohen, I., Furneauxb, H. & Levy, A.P. A 40-bp RNA element that mediates stabilization of vascular endothelial growth factor mRNA by HuR. J. Biol. Chem. 277, 13635–13640 (2002).

    CAS  Article  Google Scholar 

  29. 29

    Sheflin, L.G., Zou, A.P. & Spaulding, S.W. Androgens regulate the binding of endogenous HuR to the AU-rich 3′UTRs of HIF-1α and EGF mRNA. Biochem. Biophys. Res. Commun. 322, 644–651 (2004).

    CAS  Article  Google Scholar 

  30. 30

    Huwiler, A. et al. ATP potentiates interleukin-1 beta-induced MMP-9 expression in mesangial cells via recruitment of the ELAV protein HuR. J. Biol. Chem. 278, 51758–51769 (2003).

    CAS  Article  Google Scholar 

  31. 31

    Tran, H., Maurer, F. & Nagamine, Y. Stabilization of urokinase and urokinase receptor mRNAs by HuR is linked to its cytoplasmic accumulation induced by activated mitogen-activated protein kinase-activated protein kinase 2. Mol. Cell. Biol. 23, 7177–7188 (2003).

    CAS  Article  Google Scholar 

  32. 32

    Tatsuta, K., Nakanishi, S. & Takahashi, I. Preparation of MS-444 derivatives as immunosuppressive and anti-itching agents. WO patent 9832750 (1997).

  33. 33

    Snider, B.B. & Zhang, Q. Synthesis of (+-)-okicenone and (+-)-aloesaponol III. J. Org. Chem. 58, 3185–3187 (1993).

    CAS  Article  Google Scholar 

  34. 34

    Torigoe, K., Nakajima, S., Suzuki, H., Ojiri, K. & Suda, H. Antitumoric BE-34776 manufacture with Micromonospora. JP patent 06256338 (1994).

  35. 35

    Nakanishi, S., Chiba, S., Yano, H., Kawamoto, I. & Matsuda, Y. MS-444, a new inhibitor of myosin light chain kinase from Micromonospora sp. KY7123. J. Antibiot. (Tokyo) 48, 948–951 (1995).

    CAS  Article  Google Scholar 

  36. 36

    Tatsuta, K., Yoshimoto, T. & Gunji, H. Total synthesis of MS-444, a myosin light chain kinase inhibitor. J. Antibiot. (Tokyo) 50, 289–290 (1997).

    CAS  Article  Google Scholar 

  37. 37

    McDaniel, R., Ebert-Khosla, S., Fu, H., Hopwood, D.A. & Khosla, C. Engineered biosynthesis of novel polyketides: influence of a downstream enzyme on the catalytic specificity of a minimal aromatic polyketide synthase. Proc. Natl. Acad. Sci. USA 91, 11542–11546 (1994).

    CAS  Article  Google Scholar 

  38. 38

    Giannini, G. et al. Chrysanthones, a new source of fungal metabolites with potential antitumor and antiangiogenesis properties. Fitoterapia 74, 323–327 (2003).

    CAS  Article  Google Scholar 

  39. 39

    Yano, H., Nakanishi, S., Matsuda, Y., Nonomura, Y. & Sasaki, H. Anti-HIV drug. WO patent 9405283 (1993).

  40. 40

    Kask, P. et al. Two-dimensional fluorescence intensity distribution analysis: theory and applications. Biophys. J. 78, 1703–1713 (2000).

    CAS  Article  Google Scholar 

  41. 41

    Kask, P., Palo, K., Ullmann, D. & Gall, K. Fluorescence-intensity distribution analysis and its application in biomolecular detection technology. Proc. Natl. Acad. Sci. USA 96, 13756–13761 (1999).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

The authors thank R. Amstutz and J.E. DeVries for continuous support, and P. Fürst for reviewing the manuscript. This work was supported by a scholarship from the Austrian Academy of Sciences.

Author information

Affiliations

Authors

Contributions

N.-C.M. and V.U. established the screening assay. J.O. performed the high-throughput screen. L.O., C.G., S.M. and H.-U.N. isolated the compounds and elucidated the structures. N.-C.M. and M.H. performed the compound selection and mechanistic compound characterization. M.H. assessed structure-related literature information. J.-M.S. was responsible for the analytics and reagent preparation. N.-C.M. and J.-M.S. performed the microdialysis experiments. C.B. was responsible for the instrumental setup for confocal imaging experiments, which were performed by N.-C.M. and R.B. N.H. performed the cytokine expression experiments. The project was designed and coordinated by N.-C.M. and M.A. N.-C.M., M.H. and M.A. wrote the manuscript. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Nicole-Claudia Meisner or Martin Hintersteiner.

Ethics declarations

Competing interests

All authors are employed by Novartis Institutes for Biomedical Research. K.M. was employed by Novartis but is now retired.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6, Supplementary Table 1 and Supplementary Methods (PDF 2782 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Meisner, NC., Hintersteiner, M., Mueller, K. et al. Identification and mechanistic characterization of low-molecular-weight inhibitors for HuR. Nat Chem Biol 3, 508–515 (2007). https://doi.org/10.1038/nchembio.2007.14

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing