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Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib

Nature Methods volume 2, pages 357362 (2005) | Download Citation

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Abstract

Proteasome inhibitors, such as the dipeptide boronic acid bortezomib, are emerging as important tools in the treatment of the fatal hematologic malignancy multiple myeloma. Despite the recent US Food and Drug Administration approval of bortezomib (PS341, Velcade) for the treatment of refractory multiple myeloma, many of the basic pharmacologic parameters of bortezomib and its mode of action on myeloma cells remain to be determined. We describe the synthesis and use of a cell-permeant active site–directed probe, which allows profiling of proteasomal activities in living cells. When we compared proteasome activity patterns in cultured cells and crude cell extracts with this probe, we observed substantial differences, stressing the importance for bioassays compatible with live cells to ensure accuracy of such measurements. Using this probe, we investigated the in vivo subunit specificities of bortezomib and another inhibitor, MG132.

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Acknowledgements

The authors thank M.A. Leeuwenburgh for providing AdaAhx3L3VS. This work was financially supported by the National Institutes of Health (H.L.P. and P.J.G.), a grant from AstraZeneca (H.L.P. and P.J.G.), the Netherlands Organization for Scientific Research (H.O.), a National Cancer Institute SPORE grant Career Developmental Award (H.O.), the Dutch Cancer Society (H.O.), a Multiple Myeloma Research Foundation Senior Research Award (B.M.K.), the Dr. Saal van Zwanenberg Stichting (M.V.), the Austrian Academy of Sciences (E.F.), the Koninklijke Hollandsche Maatschappij der Wetenschappen (C.R.B.), and by the Stichting Fonds Doctor Catharine van Tussenbroek (C.R.B.).

Author information

Author notes

    • Martijn Verdoes

    Present address: Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, the Netherlands.

    • Celia R Berkers
    •  & Martijn Verdoes

    These authors contributed equally to this work.

Affiliations

  1. Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.

    • Celia R Berkers
    • , Martijn Verdoes
    • , Eben Lichtman
    • , Edda Fiebiger
    • , Benedikt M Kessler
    • , Hidde L Ploegh
    • , Huib Ovaa
    •  & Paul J Galardy
  2. Division of Cellular Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.

    • Celia R Berkers
    •  & Huib Ovaa
  3. Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.

    • Kenneth C Anderson
  4. Program in Hematology/Oncology, Children's Hospital Boston and Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.

    • Paul J Galardy

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Competing interests

H.P. and P.J.G. are supported in part through a grant from the Oncology Division of AstraZeneca. No reagents or potential products from AstraZeneca were used in this study. At the present time, AstraZeneca does not stand to benefit in any way from the publication of data contained herein.

Corresponding author

Correspondence to Huib Ovaa.

Supplementary information

PDF files

  1. 1.

    Supplementary Fig. 1

    Incubation of HeLa cells with inhibitor 3.

  2. 2.

    Supplementary Fig. 2

    H-H COSY NMR spectrum of inhibitor 3 in CDCl3

  3. 3.

    Supplementary Fig. 3

    1H-NMR spectrum of inhibitor 3 in CDCl3

  4. 4.

    Supplementary Fig. 4

    13C (APT) NMR spectrum of inhibitor 3 in CDCl3

  5. 5.

    Supplementary Methods

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DOI

https://doi.org/10.1038/nmeth759

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