Name: malachite green assay for the discovery of heat-shock protein 90 inhibitors
Uploaded: 2023-01-20T13:25:00
Description: Watch this Scientific Journal Video about Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors at JoVE.com

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 &#38; 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.

1. Lab-scale malachite green assay
<ol>
	<li>Preparation of assay buffer
	<ol>
		<li>Prepare the assay buffer, as per the composition and preparation presented in Table 1.</li>
	</ol>
	</li>
	<li>Preparation of phosphate standards
	<ol>
		<li>Use 1 mM phosphate standard, provided in the malachite green assay phosphate assay kit (stored at 4 &#176;C).</li>
		<li>Pipette 40 &#181;L of 1 mM phosphate standard in 960 &#181;L of ultra-pure water to obtain 40 &#181;M phosphate solution (prem...

<ol>
	<li>Workman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Combinatorial+attack+on+multistep+oncogenesis+by+inhibiting+the+Hsp90+molecular+chaperone.">Combinatorial attack on multistep oncogenesis by inhibiting the Hsp90 molecular chaperone.</a> Cancer Letters. 206 (2), 149-157 (2004).</li><li>Taipale, M., Jarosz, D. F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90+at+the+hub+of+protein+homeostasis:+emerging+mechanistic+insights.">HSP90 at the hub of protein homeostasis: emerging mechanistic insights.</a> Nature Reviews. Molecular Cell Biology. 11 (7), 515-528 (2010).</li><li>Mahalingam, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+HSP90+for+cancer+therapy.">Targeting HSP90 for cancer therapy.</a> British Journal of Cancer. 100 (10), 1523-1529 (2009).</li><li>Dutta Gupta, S., Pan, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+update+on+discovery+and+development+of+Hsp90+inhibitors+as+senolytic+agents.">Recent update on discovery and development of Hsp90 inhibitors as senolytic agents.</a> International Journal of Biological Macromolecules. 161, 1086-1098 (2020).</li><li>Fuhrmann-Stroissnigg, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+HSP90+inhibitors+as+a+novel+class+of+senolytics.">Identification of HSP90 inhibitors as a novel class of senolytics.</a> Nature Communications. 8 (1), 422 (2017).</li><li>Fuhrmann-Stroissnigg, H., Niedernhofer, L. J., Robbins, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp90+inhibitors+as+senolytic+drugs+to+extend+healthy+aging.">Hsp90 inhibitors as senolytic drugs to extend healthy aging.</a> Cell Cycle. 17 (9), 1048-1055 (2018).</li><li>Pearl, L. H., Prodromou, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+mechanism+of+the+Hsp90+molecular+chaperone+machinery.">Structure and mechanism of the Hsp90 molecular chaperone machinery.</a> Annual Review of Biochemistry. 75, 271-294 (2006).</li><li>Park, H. -. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unleashing+the+full+potential+of+Hsp90+inhibitors+as+cancer+therapeutics+through+simultaneous+inactivation+of+Hsp90,+Grp94,+and+TRAP1.">Unleashing the full potential of Hsp90 inhibitors as cancer therapeutics through simultaneous inactivation of Hsp90, Grp94, and TRAP1.</a> Experimental &amp; molecular medicine. 52 (1), 79-91 (2020).</li><li>Hoy, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pimitespib:+first+approval.">Pimitespib: first approval.</a> Drugs. 82 (13), 1413-1418 (2022).</li><li>Whitesell, L., Mimnaugh, E. G., De Costa, B., Myers, C. E., Neckers, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+heat+shock+protein+HSP90-pp60v-src+heteroprotein+complex+formation+by+benzoquinone+ansamycins:+essential+role+for+stress+proteins+in+oncogenic+transformation.">Inhibition of heat shock protein HSP90-pp60v-src heteroprotein complex formation by benzoquinone ansamycins: essential role for stress proteins in oncogenic transformation.</a> Proceedings of the National Academy of Sciences. 91 (18), 8324-8328 (1994).</li><li>Rowlands, M. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+assay+for+inhibitors+of+heat-shock+protein+90+ATPase+activity.">High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.</a> Analytical Biochemistry. 327 (2), 176-183 (2004).</li><li>Sheikha, G. A., Al-Sha'er, M. A., Taha, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Some+sulfonamide+drugs+inhibit+ATPase+activity+of+heat+shock+protein+90:+investigation+by+docking+simulation+and+experimental+validation.">Some sulfonamide drugs inhibit ATPase activity of heat shock protein 90: investigation by docking simulation and experimental validation.</a> Journal of Enzyme Inhibition and Medicinal Chemistry. 26 (5), 603-609 (2011).</li><li>Al-Sha'er, M. A., Mansi, I., Hakooz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Docking+and+pharmacophore+mapping+of+halogenated+pyridinium+derivatives+on+heat+shock+protein+90.">Docking and pharmacophore mapping of halogenated pyridinium derivatives on heat shock protein 90.</a> Journal of Chemical and Pharmaceutical Research. 7 (4), 103-112 (2015).</li><li>Al-Sha'er, M. A., Taha, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elaborate+ligand-based+modeling+reveals+new+nanomolar+heat+shock+protein+90&#945;+inhibitors.">Elaborate ligand-based modeling reveals new nanomolar heat shock protein 90&#945; inhibitors.</a> Journal of Chemical Information and Modeling. 50 (9), 1706-1723 (2010).</li><li>Al-Sha'er, M. A., Taha, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rational+exploration+of+new+pyridinium-based+HSP90&#945;+inhibitors+tailored+to+thiamine+structure.">Rational exploration of new pyridinium-based HSP90&#945; inhibitors tailored to thiamine structure.</a> Medicinal Chemistry Research. 21 (4), 487-510 (2012).</li><li>Al-Sha'er, M. A., Taha, M. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+docking-based+comparative+intermolecular+contacts+analysis+to+validate+Hsp90&#945;+docking+studies+and+subsequent+in+silico+screening+for+inhibitors.">Application of docking-based comparative intermolecular contacts analysis to validate Hsp90&#945; docking studies and subsequent in silico screening for inhibitors.</a> Journal of Molecular Modeling. 18 (11), 4843-4863 (2012).</li><li>Dutta Gupta, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2,4-dihydroxy+benzaldehyde+derived+Schiff+bases+as+small+molecule+Hsp90+inhibitors:+rational+identification+of+a+new+anticancer+lead.">2,4-dihydroxy benzaldehyde derived Schiff bases as small molecule Hsp90 inhibitors: rational identification of a new anticancer lead.</a> Bioorganic Chemistry. 59, 97-105 (2015).</li><li>Feng, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+malachite+green+assay+of+phosphate:+mechanism+and+application.">An improved malachite green assay of phosphate: mechanism and application.</a> Analytical Biochemistry. 409 (1), 144-149 (2011).</li><li>Gupta, S. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+docking+study,+synthesis+and+biological+evaluation+of+Mannich+bases+as+Hsp90+inhibitors.">Molecular docking study, synthesis and biological evaluation of Mannich bases as Hsp90 inhibitors.</a> International Journal of Biological Macromolecules. 80, 253-259 (2015).</li><li>Zhao, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+identifies+established+drugs+as+SARS-CoV-2+PLpro+inhibitors.">High-throughput screening identifies established drugs as SARS-CoV-2 PLpro inhibitors.</a> Protein &amp; Cell. 12 (11), 877-888 (2021).</li><li>Giri, A. K., Ianevski, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+for+drug+discovery+targeting+the+cancer+cell-microenvironment+interactions+in+hematological+cancers.">High-throughput screening for drug discovery targeting the cancer cell-microenvironment interactions in hematological cancers.</a> Expert Opinion on Drug Discovery. 17 (2), 181-190 (2022).</li><li>Mahapatra, D. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heat+shock+protein+90+(Hsp90)+inhibitory+potentials+of+some+chalcone+compounds+as+novel+anti-proliferative+candidates.">Heat shock protein 90 (Hsp90) inhibitory potentials of some chalcone compounds as novel anti-proliferative candidates.</a> Advanced Studies in Experimental and Clinical. , 107-122 (2021).</li><li>Jaeger, A. M., Whitesell, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HSP90:+enabler+of+cancer+adaptation.">HSP90: enabler of cancer adaptation.</a> Annual Review of Cancer Biology. 3, 275-297 (2019).</li><li>Yang, S., Xiao, H., Cao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+heat+shock+proteins+in+cancer+diagnosis,+prognosis,+metabolism+and+treatment.">Recent advances in heat shock proteins in cancer diagnosis, prognosis, metabolism and treatment.</a> Biomedicine &amp; Pharmacotherapy. 142, 112074 (2021).</li><li>Mishra, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+Hsp90&#946;-selective+inhibitors+to+overcome+detriments+associated+with+pan-Hsp90+inhibition.">The development of Hsp90&#946;-selective inhibitors to overcome detriments associated with pan-Hsp90 inhibition.</a> Journal of Medicinal Chemistry. 64 (3), 1545-1557 (2021).</li><li>Khandelwal, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure-guided+design+of+an+Hsp90beta+N-terminal+isoform-selective+inhibitor.">Structure-guided design of an Hsp90beta N-terminal isoform-selective inhibitor.</a> Nature Communications. 9 (1), 425 (2018).</li><li>Wang, Y., Koay, Y. C., McAlpine, S. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+selective+are+Hsp90+inhibitors+for+cancer+cells+over+normal+cells.">How selective are Hsp90 inhibitors for cancer cells over normal cells.</a> ChemMedChem. 12 (5), 353-357 (2017).</li><li>Panaretou, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATP+binding+and+hydrolysis+are+essential+to+the+function+of+the+Hsp90+molecular+chaperone+in+vivo.">ATP binding and hydrolysis are essential to the function of the Hsp90 molecular chaperone in vivo.</a> The EMBO Journal. 17 (16), 4829-4836 (1998).</li><li>Banerjee, M., Hatial, I., Keegan, B. M., Blagg, B. S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assay+design+and+development+strategies+for+finding+Hsp90+inhibitors+and+their+role+in+human+diseases.">Assay design and development strategies for finding Hsp90 inhibitors and their role in human diseases.</a> Pharmacology &amp; Therapeutics. 221, 107747 (2021).</li><li>Howes, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fluorescence+polarization+assay+for+inhibitors+of+Hsp90.">A fluorescence polarization assay for inhibitors of Hsp90.</a> Analytical Biochemistry. 350 (2), 202-213 (2006).</li><li>Opali&#324;ska, M., Ja&#324;ska, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAA+proteases:+guardians+of+mitochondrial+function+and+homeostasis.">AAA proteases: guardians of mitochondrial function and homeostasis.</a> Cells. 7 (10), 163 (2018).</li><li>Ambrose, A. J., Chapman, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function,+therapeutic+potential,+and+inhibition+of+Hsp70+chaperones.">Function, therapeutic potential, and inhibition of Hsp70 chaperones.</a> Journal of Medicinal Chemistry. 64 (11), 7060-7082 (2021).</li><li>Cheng, I., Mikita, N., Fishovitz, J., Frase, H., Wintrode, P., Lee, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+region+in+the+N-terminus+of+Escherichia+coli+Lon+that+affects+ATPase,+substrate+translocation+and+proteolytic+activity.">Identification of a region in the N-terminus of Escherichia coli Lon that affects ATPase, substrate translocation and proteolytic activity.</a> Journal of Molecular Biology. 418 (3-4), 208-225 (2012).</li></ol>

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...

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 (&#62;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 state2,3. In recent years, several Hsp90 inhibitors have been demonstrated to possess senolytic effects in in vitro and in vivo studies, where they have significantly improved the life span of mice4,5,6. All the aforementioned findings substantiate the fact that Hsp90 inhibitors could be effective in multiple cancer types, with fewer adverse effects and reduced chances of developing resistance. The chaperoning function of Hsp90 is accomplished by binding ATP at the N-terminal domain of Hsp90 and hydrolyzing it into ADP and free phosphate7. Small molecules that competitively bind to the ATP binding pocket of Hsp90 were found to successfully suppress the chaperoning effect of the protein. To date, this remains the best strategy for Hsp90 inhibition, which is supported by the fact that such inhibitors have reached clinical trials8. One of them, Pimitespib, was approved in Japan for the treatment of gastrointestinal stromal tumor (GIST) in June 20229. This is the first Hsp90 inhibitor approved since the druggability of the chaperone was established in 199410.
The malachite green assay is a simple, sensitive, fast, and inexpensive procedure for the detection of inorganic phosphate, suitable for automation and high-throughput screening (HTS) of compounds against its desired target11. The assay has been successfully employed for the screening of Hsp90 inhibitors in small lab-scale setups, as well as in a HTS12,13,14,15,16,17. The assay uses a colorimetric method that determines the free inorganic phosphate formed due to the ATPase activity of Hsp90. The basis of this quantification is the formation of a phosphomolybdate complex between free phosphate and molybdenum, which subsequently reacts with malachite green to generate a green color (Figure 1). This rapid color formation is measured on a spectrophotometer, or on a plate reader, between 600-660 nm18,19.
In the present protocol, the procedure for carrying out a malachite green assay with yeast Hsp90 and subsequent identification of inhibitors against the chaperone is described. The natural product molecule, geldanamycin (GA), with which the druggability of Hsp90 was first established, was taken as an authentic inhibitor10. HTS has become an integral part of the current drug discovery program, owing to the availability of a large number of molecules for testing. This technique has gained more significance in the past 2 years because of the urgent need for repurposing drugs for treating Covid-19 infection20,21. Therefore, a detailed outline for the HTS of molecules against yeast Hsp90 protein by adopting the malachite green assay method is presented.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1M Magnesium chloride solution in water</td>
			<td>Sigma-Aldrich</td>
			<td>63069-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>1M Potassium chloride solution in water</td>
			<td>Sigma-Aldrich</td>
			<td>60142-100ml</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>SPL Life Sciences</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Adenosine 5&#8242;-triphosphate disodium salt hydrate</td>
			<td>Sigma-Aldrich</td>
			<td>A7699-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Biomek FX laboratory automation workstation</td>
			<td>Beckman Coulter</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Compounds 3-96</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>Histidine 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.</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Geldanamycin, 99% (HPLC), powder</td>
			<td>AK Scientific, Inc.</td>
			<td>V2064</td>
			<td></td>
		</tr>
		<tr>
			<td>Invitroge UltraPure DNase/RNase-Free Distilled Water</td>
			<td>ThermoFisher Scientific</td>
			<td>10977015</td>
			<td></td>
		</tr>
		<tr>
			<td>Malachite Green Phosphate Assay&#160; Assay kit</td>
			<td>Sigma-Aldrich</td>
			<td>MAK307-1KT</td>
			<td></td>
		</tr>
		<tr>
			<td>Multi-Detection Microplate Reader Synergy HT</td>
			<td>Biotek Instruments, Inc.</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Synergy HT multi-plate reader</td>
			<td>Biotek Instruments, Inc.</td>
			<td>Not applicable</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizma hydrochloride buffer solution, pH7.4</td>
			<td>Sigma-Aldrich</td>
			<td>93313-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Hsp90</td>
			<td>Not applicable</td>
			<td>Not applicable</td>
			<td>School of Life Sciences, University of Sussex, United Kingdom and protein was expressed in KIST Gangneung Institute of Natural Products. Primary Accession number: P02829</td>
		</tr>
	</tbody>
</table>

malachite green assay for the discovery of heat-shock protein 90 inhibitors

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.

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...

Watch this Scientific Journal Video about Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors at JoVE.com

Malachite Green Assay for the Discovery of Heat-Shock Protein 90 Inhibitors

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.

Heat shock protein 90 (Hsp90) is a promising anticancer target because of its chaperoning effect on multiple oncogenic proteins. The activity of Hsp90 is ...

This protocol will aid in the determination of the activity for ATPase proteins by measuring the free phosphate resulting from ATP hydrolysis by the proteins. The malachite green assay is a simple, sensitive, fast, and inexpensive procedure for the detection of inorganic phosphate. It is suitable for automation and high-throughput screening of compounds against its target.This technique will play an important role in the rapid discovery of inhibitors against the Hsp90 protein, which is an important anti-cancer target. To begin, pipette 40 microliters of one-millimolar phosphate standard in 960 microliters of ultrapure water to obtain 40-micromolar phosphate solution. Add this solution as per the manufacturer's instructions to form a serial dilution of phosphate from 0-to 40-micromolar.Next, use eight microliters of yeast Hsp90 for measurable ATPase activity. Add malachite green reagent to the blank and the positive control, and check the the optical density difference between the blank and the positive control after the addition is at least 0.5 and less than 1. To prepare ATP, transfer one milligram of ATP to a vial containing 453.39 microliters of ultrapure water to obtain four-millimolar stock solution.Prepare the ATP fresh, as it can spontaneously hydrolyze over time. Add four microliters of ATP stock solution to each well to give a final well concentration of 0.2-millimolar. Add ATP as the last component to the reaction using a multi-channel pipette so that all the reactions start simultaneously.Next, prepare a 10-millimolar stock of geldanamycin by dissolving one milligram in 178.37 microliters of DMSO. Using the stock solution, prepare 4-millimolar, 0.8 millimolar, 0.16-millimolar, 0.032-millimolar, 0.0064-millimolar, and 0.00128-millimolar solution in DMSO by serial dilution. Add two microliters of these stock solutions to each well to obtain a final well concentration of 0.1-millimolar, 0.02-millimolar, 0.04-millimolar, 0.0008-millimolar, 0.00016-millimolar, and 0.000032-millimolar.Then, prepare the wells along with the wells containing the blank and negative control. The wells containing geldanamycin serve as the positive control. Prepare the 96-well plates and prepare a separate well for the compounds that show absorbance at 620 nanometers to record the absorbance at this wavelength.Do not show a separate well for geldanamycin, as geldanamycin does not show absorbance at 620 nanometers for the concentration used in this assay. Set up assay wells by adding ultrapure water to each well. Then, add the required amount of assay buffer and a compound solution such as geldanamycin solution.Add Hsp90 to the appropriate wells and shake the plates for two minutes on a plate shaker at room temperature. Add four microliters of ATP solution using a multi-channel pipette. Wrap the plate in aluminum foil and shake for two minutes on a plate shaker at room temperature at 200 rpm.Incubate the plate at 37 degrees Celsius for three hours. Stop the reaction by adding 20 microliters of malachite green reagent in the same order as ATP using a multi-channel pipette and incubate for 15 minutes at room temperature. Finally, measure the absorbance of the plate at 620 nanometers in a plate reader.In addition plate A, add 90 microliters of assay buffer in each well. In addition plate B, add 90 microliters of buffer with Hsp90 in each well. In addition plate C, add 90 microliters of ATP, and in addition plate D add 90 microliters of malachite green reagent in each well.Use an automated multi-channel dispensing system to transfer 40 microliters of solution from each well of addition plate A to main plates one and two, and plate B to main plate three and four. Transfer 20 microliters of the solution from each well of the compound plate to main plates one, main plates two, main plates three, and main plates four. Shake the plates for one minute in a shaker at 200 rpm.Transfer 20 microliters of the solution from each well of the addition plates C to main plates one, two, three, and four. Shake the plates for one minute in a shaker at 200 rpm. Incubate the plates at 37 degrees Celsius for three hours and then transfer 20 microliters of malachite green reagent from addition plate D to the wells of main plates one, two, three, and four.After a 15-minute incubation at room temperature, measure the absorbance of the plate at 620 nanometers in a plate reader. The structure of malachite green and the reactions of malachite green reagent A with free phosphate and the subsequent reaction with reagent B are shown here. A standard plot for standard phosphate concentration is represented in this figure.Here, the x-axis represents the free phosphate or pi concentration in micromolar, and the absorbance at 620 nanometers is depicted on the y-axis. The error bars represent the deviation between the two sample numbers. The percentage ATPase activity is used to measure the dose-dependent inhibition of the protein by geldanamycin.It was observed that geldanamycin inhibited the protein in a dose-dependent manner with an IC50 value of 0.85-micromolar. Here, each point is a mean of two determinations. The color development in main plate one after the addition of the malachite green agent is shown in this image.The pink color in the A11 well is due to the compound color. Similarly, the color development in main plate two is shown here. The pink color in the A11 well is due to the colored nature of the compound.The color development after the addition of the malachite green reagent in main plate three and in main plate four is shown here. The addition of ATP and malachite green reagent at the same time interval to every well is an important thing to remember when attempting this procedure.

Research

JoVE Journal

Biochemistry

Preparation and Delivery of Protein Microcrystals in Lipidic Cubic Phase for Serial Femtosecond Crystallography

Membrane proteins (MPs) are essential components of cellular membranes and primary drug targets. Rational drug design relies on precise structural ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Microcrystallography of Protein Crystals and In Cellulo Diffraction

Microcrystallography of Protein Crystals and In Cellulo Diffraction

The advent of high-quality microfocus beamlines at many synchrotron facilities has permitted the routine analysis of crystals smaller than 10 &#181;m in ...

Extracellular Protein Microarray Technology for High Throughput Detection of Low Affinity Receptor-Ligand Interactions

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A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

Assessment of Cellular Oxidation using a Subcellular Compartment-Specific Redox-Sensitive Green Fluorescent Protein

Measuring the intracellular oxidation/reduction balance provides an overview of the physiological and/or pathophysiological redox status of an organism. ...

Native Cell Membrane Nanoparticles System for Membrane Protein-Protein Interaction Analysis

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Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Pulldown Assay Coupled with Co-Expression in Bacteria Cells as a Time-Efficient Tool for Testing Challenging Protein-Protein Interactions

Pulldown is an easy and widely used protein-protein interaction assay. However, it has limitations in studying protein complexes that do not assemble ...

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...