Nature Methods2, 357 - 362 (2005)
Published online: 21 April 2005; | doi:10.1038/nmeth759
Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib
Celia R Berkers1, 2, 6, Martijn Verdoes1, 5, 6, Eben Lichtman1, Edda Fiebiger1, Benedikt M Kessler1, Kenneth C Anderson3, Hidde L Ploegh1, Huib Ovaa1, 2
& Paul J Galardy1, 4
1
Department of Pathology, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, Massachusetts 02115, USA.
2
Division of Cellular Biochemistry, Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, the Netherlands.
3
Department of Medical Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA.
4
Program in Hematology/Oncology, Children's Hospital Boston and Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 02115, USA.
5
Present address: Leiden Institute of Chemistry, Leiden University, P.O. Box 9502, 2300 RA Leiden, the Netherlands.
6
These authors contributed equally to this work.
Correspondence should be addressed to Huib Ovaa h.ovaa@nki.nl
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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