Sign In

A subscription to JoVE is required to view this content. Sign in or start your free trial.

In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent enzymes.

Abstract

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is dependent on its ability to hydrolyze adenosine triphosphate (ATP) to adenosine diphosphate (ADP) and free phosphate. The ATPase activity of Hsp90 is linked to its chaperoning function; ATP binds to the N-terminal domain of the Hsp90, and disrupting its binding was found to be the most successful strategy in suppressing Hsp90 function. The ATPase activity can be measured by a colorimetric malachite green assay, which determines the amount of free phosphate formed by ATP hydrolysis. Here, a procedure for determining the ATPase activity of yeast Hsp90 by using the malachite green phosphate assay kit is described. Further, detailed instructions for the discovery of Hsp90 inhibitors by taking geldanamycin as an authentic inhibitor is provided. Finally, the application of this assay protocol through the high-throughput screening (HTS) of inhibitor molecules against yeast Hsp90 is discussed.

Introduction

Heat shock protein 90 (Hsp90) is a molecular chaperone that maintains the stability of proteins responsible for the development and progression of cancer. In addition, proteins responsible for the development of resistance to antineoplastic agents are also clients of Hsp901. Hsp90 is overexpressed ubiquitously in all cancer cell types (>90% of cellular proteins), compared to normal cells where it may constitute less than 2% of total proteins. Moreover, the Hsp90 of cancer cells resides in a complex with co-chaperones, whereas in a normal cell it is present predominantly in a free, un-complexed state2,

Protocol

1. Lab-scale malachite green assay

  1. Preparation of assay buffer
    1. Prepare the assay buffer, as per the composition and preparation presented in Table 1.
  2. Preparation of phosphate standards
    1. Use 1 mM phosphate standard, provided in the malachite green assay phosphate assay kit (stored at 4 °C).
    2. Pipette 40 µL of 1 mM phosphate standard in 960 µL of ultra-pure water to obtain 40 µM phosphate solution (prem.......

Representative Results

The results of the assay are interpreted in terms of absorbance due to free phosphate ion concentration. The absorbance by free phosphate due to ATP hydrolysis by the yeast Hsp90 at 620 nm is considered as 100% ATPase activity, or zero percentage protein inhibition. The inhibition of protein leads to the cessation of ATP hydrolysis (less free phosphate). which is reflected in terms of decreased absorbance at 620 nm.

Results of lab-scale malachite green assay
The standard.......

Discussion

Hsp90 is a significant target for the discovery of novel anticancer drug molecules. Since its druggability was established in 199410, 18 molecules have reached clinical trials. At present, seven molecules are in various phases of clinical trials, either alone or in combination22. All such small molecules are N-terminal ATP binding inhibitors. The other means of inhibiting the chaperone (C-terminal inhibitors, middle domain inhibitors) have not proceeded as fast as N-termina.......

Acknowledgements

This study was supported by the Korea Research Fellowship (KRF) program, postdoctoral fellow of the National Research Foundation of Korea (NRF), funded by the ministry of science & ICT (NRF-2019H1D3A1A01102952). The authors are thankful to KIST intramural grant and Ministry of Oceans and Fisheries grant number 2MRB130 for providing financial assistance for this project.

....

Materials

NameCompanyCatalog NumberComments
1M Magnesium chloride solution in waterSigma-Aldrich63069-100ml
1M Potassium chloride solution in waterSigma-Aldrich60142-100ml
96-well plateSPL Life SciencesNot applicable
Adenosine 5′-triphosphate disodium salt hydrateSigma-AldrichA7699-5G
Biomek FX laboratory automation workstationBeckman CoulterNot applicable
Compounds 3-96Not applicableNot applicableHistidine tagged yeast Hsp90 was obtained from Dr. Chrisostomos Prodromou, School of Life Sciences, University of Sussex, United Kingdom, and protein was expressed in KIST Gangneung Institute of Natural Products. Details cannot be disclosed due to patent infringement issues.
Dimethyl sulfoxideSigma-AldrichD8418
Geldanamycin, 99% (HPLC), powderAK Scientific, Inc.V2064
Invitroge UltraPure DNase/RNase-Free Distilled WaterThermoFisher Scientific10977015
Malachite Green Phosphate Assay  Assay kitSigma-AldrichMAK307-1KT
Multi-Detection Microplate Reader Synergy HTBiotek Instruments, Inc.Not applicable
Synergy HT multi-plate readerBiotek Instruments, Inc.Not applicable
Trizma hydrochloride buffer solution, pH7.4Sigma-Aldrich93313-1L
Yeast Hsp90Not applicableNot applicableSchool of Life Sciences, University of Sussex, United Kingdom and protein was expressed in KIST Gangneung Institute of Natural Products. Primary Accession number: P02829

References

  1. Workman, P. Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone. Cancer Letters. 206 (2), 149-157 (2004).
  2. Taipale, M., Jarosz, D. F., Lindquist, S. HSP90 ....

Explore More Articles

Malachite Green AssayHsp90ATPase ActivityPhosphate DetectionATP HydrolysisInhibitorsHigh throughput ScreeningGeldanamycinSerial DilutionBlankPositive ControlAbsorbance

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved