High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.
Anal Biochem
; 327(2): 176-83, 2004 Apr 15.
Article
en En
| MEDLINE
| ID: mdl-15051534
The molecular chaperone heat-shock protein 90 (HSP90) plays a key role in the cell by stabilizing a number of client proteins, many of which are oncogenic. The intrinsic ATPase activity of HSP90 is essential to this activity. HSP90 is a new cancer drug target as inhibition results in simultaneous disruption of several key signaling pathways, leading to a combinatorial approach to the treatment of malignancy. Inhibitors of HSP90 ATPase activity including the benzoquinone ansamycins, geldanamycin and 17-allylamino-17-demethoxygeldanamycin, and radicicol have been described. A high-throughput screen has been developed to identify small-molecule inhibitors that could be developed as therapeutic agents with improved pharmacological properties. A colorimetric assay for inorganic phosphate, based on the formation of a phosphomolybdate complex and subsequent reaction with malachite green, was used to measure the ATPase activity of yeast HSP90. The Km for ATP determined in the assay was 510+/-70 microM. The known HSP90 inhibitors geldanamycin and radicicol gave IC(50) values of 4.8 and 0.9 microM respectively, which compare with values found using the conventional coupled-enzyme assay. The assay was robust and reproducible (2-8% CV) and used to screen a compound collection of approximately 56,000 compounds in 384-well format with Z' factors between 0.6 and 0.8.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Ensayos de Selección de Medicamentos Antitumorales
/
Adenosina Trifosfatasas
/
Proteínas HSP90 de Choque Térmico
Tipo de estudio:
Diagnostic_studies
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Prognostic_studies
/
Screening_studies
Idioma:
En
Revista:
Anal Biochem
Año:
2004
Tipo del documento:
Article
Pais de publicación:
Estados Unidos