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

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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Figure 1
Figure 2
Figure 3: Characterization of compounds 3 and 4.
Figure 4: Profiling of proteasome activity in EL4 cell extracts and cells.
Figure 5: In vivo profiling of proteasome inhibitors.

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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.).

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Correspondence to Huib Ovaa.

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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.

Supplementary information

Supplementary Fig. 1

Incubation of HeLa cells with inhibitor 3. (PDF 50 kb)

Supplementary Fig. 2

H-H COSY NMR spectrum of inhibitor 3 in CDCl3 (PDF 77 kb)

Supplementary Fig. 3

1H-NMR spectrum of inhibitor 3 in CDCl3 (PDF 24 kb)

Supplementary Fig. 4

13C (APT) NMR spectrum of inhibitor 3 in CDCl3 (PDF 16 kb)

Supplementary Methods (PDF 82 kb)

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Berkers, C., Verdoes, M., Lichtman, E. et al. Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib. Nat Methods 2, 357–362 (2005). https://doi.org/10.1038/nmeth759

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