Name: screening for thermotoga maritima membrane-bound pyrophosphatase inhibitors
Uploaded: 2019-11-23T15:00:00
Description: Watch this Scientific Journal Video about Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors at JoVE.com

This work was supported by the grants from the Jane and Aatos Erkko Foundation and the BBSRC (BB/M021610) to Adrian Goldman, the Academy of Finland (No. 308105) to Keni Vidilaseris, (No. 310297) to Henri Xhaard, and (No. 265481) to Jari Yli-Kauhaluoma, and the University of Helsinki Research Funds to Gustav Boije af Genn&#228;s. The authors thank Bernadette Gehl for her technical help during the project.

1. Protein preparation
NOTE: The expression and purification of TmPPase has been described elsewhere13.
<ol>
	<li>Prepare 10 mL of the reactivation buffer solution containing 20 mM 2-(N-morpholino)ethanesulfonic acid (MES) pH 6.5, 3.5% (v/v) glycerol, 2 mM dithiothreitol (DTT), and 0.05% dodecyl maltoside (DDM).</li>
	<li>Prepare 10 mL of the reaction mixture containing 200 mM Tris-Cl pH 8.0, 8.0 mM MgCl2, 333 mM KCl, and 67 mM NaCl. 
	NOTE: M...

<ol>
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R., Baltscheffsky, M., Baltscheffsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H+-PPases:+yesterday,+today+and+tomorrow.">H+-PPases: yesterday, today and tomorrow.</a> IUBMB Life. 59 (2), 76-83 (2007).</li><li>Liu, 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=A+vacuolar-H+-pyrophosphatase+(TgVP1)+is+required+for+microneme+secretion,+host+cell+invasion,+and+extracellular+survival+of+Toxoplasma+gondii.">A vacuolar-H+-pyrophosphatase (TgVP1) is required for microneme secretion, host cell invasion, and extracellular survival of Toxoplasma gondii.</a> Molecular Microbiology. 93 (4), 698-712 (2014).</li><li>Lemercier, 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=A+pyrophosphatase+regulating+polyphosphate+metabolism+in+acidocalcisomes+is+essential+for+Trypanosoma+brucei+virulence+in+mice.">A pyrophosphatase regulating polyphosphate metabolism in acidocalcisomes is essential for Trypanosoma brucei virulence in mice.</a> Journal of Biological Chemistry. 279 (5), 3420-3425 (2004).</li><li>Belogurov, G. 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=Membrane-bound+pyrophosphatase+of+Thermotoga+maritima+requires+sodium+for+activity.">Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity.</a> Biochemistry. 44 (6), 2088-2096 (2005).</li><li>Vidilaseris, 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=Asymmetry+in+catalysis+by+Thermotoga+maritima+membrane-bound+pyrophosphatase+demonstrated+by+a+nonphosphorus+allosteric+inhibitor.">Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a nonphosphorus allosteric inhibitor.</a> Science Advances. 5 (5), (2019).</li><li>Lin, S. M., 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=Crystal+structure+of+a+membrane-embedded+H+-translocating+pyrophosphatase.">Crystal structure of a membrane-embedded H+-translocating pyrophosphatase.</a> Nature. 484 (7394), 399-403 (2012).</li><li>Fiske, C. H., Subbarow, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+colorimetric+determination+of+phosphorus.">The colorimetric determination of phosphorus.</a> Journal of Biological Chemistry. 66 (2), 375-400 (1925).</li><li>Nagul, E. A., McKelvie, I. D., Worsfold, P., Kolev, S. 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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+molybdenum+blue+method+for+orthophosphate+determination+suitable+for+investigating+enzymatic+hydrolysis+of+organic+phosphates.">A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates.</a> Communications in Soil Science and Plant Analysis. 36 (9-10), 1373-1383 (2005).</li><li>Martin, B., Pallen, C. J., Wang, J. H., Graves, D. 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Here we report a detailed protocol for simple screening of inhibitors for membrane-bound pyrophosphatase from T. maritima in a 96 well plate format based on Vidilaseris et al.14. This protocol is inexpensive and based on 12-phosphomolybdic acid, which is formed from orthophosphate and molybdate under acidic conditions and reduced to phosphomolybdenum species with a distinct blue color12. This method is preferred over other protocols, such as the more sensitive mala...

Approximately 25% of the total cellular proteins are membrane proteins and about 60% of them are drug targets1,2. One of the potential drug targets3, membrane-bound pyrophosphatases (mPPases), are dimeric enzymes that pump proton and/or sodium ion across the membrane by hydrolysis of pyrophosphate into two orthophosphates4. mPPases can be found in various organisms5 such as bacteria, archaea, plants, and protist parasites, with the exception of humans and animals4. In protist parasites, for example Plasmodium falciparum, Toxoplasma gondii and Trypanosoma brucei, mPPases are essential for the parasite virulence6 and knockout of this expression in the parasites lead to failure in maintaining intracellular pH upon exposure to the external basic pH7. Due to their importance and lack of homologous protein present in vertebrates, mPPases can be considered as potential drug targets for protistal diseases3.
The in vitro screening of mPPase inhibitors in this work is based on a TmPPase model system. TmPPase is a sodium ion pumping and potassium ion dependent mPPase from T. maritima and has its optimum activity at 71 &#176;C8. Benefits of this enzyme are for example its ease in production and purification, good thermal stability and high specific activity. TmPPase shows both high similarity in addition to the complete conservation of the position as well as identity of all catalytic residues to the protist mPPases3,9 and to the solved structure of Vigna radiata10 mPPase. The available structures of TmPPase in different conformations are also useful for structure-based drug design experiment (as virtual screening and de novo design).
Here we report a detailed protocol for screening of TmPPase inhibitors in a 96 well plate format (Figure 1). The protocol is based on the colorimetric method of the molybdenum blue reaction, which was first developed by Fiske and Subbarow11. This method involves the formation of 12-phosphomolybdic acid from orthophosphate and molybdate under acidic conditions, which is then reduced to give characteristic blue-colored phosphomolybdenum species12.

<table>
	<tbody>
		<tr>
			<td>Adhesive sealing sheet</td>
			<td>Thermo Scientific</td>
			<td>AB0558</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium heptamolybdate tetrahydrate</td>
			<td>Merck</td>
			<td>F1412481 636</td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbic acid</td>
			<td>Sigma-Aldrich</td>
			<td>95212-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>BioLite 96Well Multidish</td>
			<td>Thermo Scientific</td>
			<td>130188</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Merck</td>
			<td>1167431000</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well PCR Tube Strips 0.2 ml without caps (120)</td>
			<td>Nippon genetics</td>
			<td>FG-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Dodecyl maltoside (DDM)</td>
			<td>Melford</td>
			<td>B2010-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck</td>
			<td>1009901001</td>
			<td></td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>Merck</td>
			<td>1000631011</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>258148-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Imidodiphosphate sodium salt</td>
			<td>Sigma-Aldrich</td>
			<td>I0631-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>L-&#945;-Phosphatidyl choline from soybean lecithin</td>
			<td>Sigma</td>
			<td>429415-100GM</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma-Aldrich</td>
			<td>8147330500</td>
			<td></td>
		</tr>
		<tr>
			<td>Multiplate 96-Well PCR Plates</td>
			<td>Bio-Rad</td>
			<td>MLL9651</td>
			<td></td>
		</tr>
		<tr>
			<td>MultiSkan Go</td>
			<td>Thermo Scientific</td>
			<td>10680879</td>
			<td></td>
		</tr>
		<tr>
			<td>Nepheloskan Ascent (Type 750)</td>
			<td>Labsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Petri dish (size 150 mm x 15 mm)</td>
			<td>Sigma-Aldrich</td>
			<td>P5981-100EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Merck</td>
			<td>104936</td>
			<td></td>
		</tr>
		<tr>
			<td>Prism 6 software</td>
			<td>GraphPad</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>QBT2 Heating block</td>
			<td>Grant Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium meta-arsenite</td>
			<td>Fisher Chemical</td>
			<td>12897692</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic (Pi)</td>
			<td>Sigma</td>
			<td>S0876-1KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyrophosphate dibasic</td>
			<td>Fluka</td>
			<td>71501-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trisodium citrate dihydrate</td>
			<td>Fluka</td>
			<td>71404-1KG</td>
			<td></td>
		</tr>
	</tbody>
</table>

screening for thermotoga maritima membrane-bound pyrophosphatase inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

In this protocol, eight compounds (1&#8722;8) were tested (Figure 2A) together with IDP, a common inhibitor of pyrophosphatases, as a positive control. Each compound was tested at three different concentrations (1 &#181;M, 5 &#181;M and 20 &#181;M) in triplicate. The workflow of the screening is depicted in Figure 1, starting from sample and reagent preparation until the absorbance measurement at 860 nm.
At the end of this protocol, a...

Watch this Scientific Journal Video about Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors at JoVE.com

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Here we present a screening method for membrane-bound pyrophosphatase (from Thermotoga maritima) inhibitors based on the molybdenum blue reaction in a 96 well plate format.

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave ...

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