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>
	<li>Terstappen, G. C., Reggiani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+research+in+drug+discovery.">In silico research in drug discovery.</a> Trends in Pharmacological Sciences. 22 (1), 23-26 (2001).</li><li>Rask-Andersen, M., Almen, M. S., Schioth, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+the+exploitation+of+novel+drug+targets.">Trends in the exploitation of novel drug targets.</a> Nature Reviews Drug Discovery. 10 (8), 579-590 (2011).</li><li>Shah, N. R., Vidilaseris, K., Xhaard, H., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integral+membrane+pyrophosphatases:+a+novel+drug+target+for+human+pathogens.">Integral membrane pyrophosphatases: a novel drug target for human pathogens.</a> AIMS Biophysics. 3 (1), 171-194 (2016).</li><li>Baykov, A. A., Malinen, A. M., Luoto, H. H., Lahti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrophosphate-Fueled+Na++and+H++Transport+in+Prokaryotes.">Pyrophosphate-Fueled Na+ and H+ Transport in Prokaryotes.</a> Microbiology and Molecular Biology Reviews. 77 (2), 267-276 (2013).</li><li>Serrano, A., Perez-Castineira, J. 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. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molybdenum+blue+reaction+for+the+determination+of+orthophosphate+revisited:+Opening+the+black+box.">The molybdenum blue reaction for the determination of orthophosphate revisited: Opening the black box.</a> Analytica Chimica Acta. 890, 60-82 (2015).</li><li>Kellosalo, J., Kajander, T., Palmgren, M. G., Lopez-Marques, R. L., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+expression+and+purification+of+membrane-bound+pyrophosphatases.">Heterologous expression and purification of membrane-bound pyrophosphatases.</a> Protein Expression and Purification. 79 (1), 25-34 (2011).</li><li>Vidilaseris, K., Kellosalo, J., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+orthophosphate+determination+of+thermostable+membrane-bound+pyrophosphatase+activity.">A high-throughput method for orthophosphate determination of thermostable membrane-bound pyrophosphatase activity.</a> Analytical Methods. 10 (6), 646-651 (2018).</li><li>He, Z. Q., Honeycutt, C. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorinated+tyrosine+phosphates+to+probe+the+substrate+specificity+of+the+low+molecular+weight+phosphatase+activity+of+calcineurin.">Use of fluorinated tyrosine phosphates to probe the substrate specificity of the low molecular weight phosphatase activity of calcineurin.</a> Journal of Biological Chemistry. 260 (28), 14932-14937 (1985).</li><li>Strauss, J., Wilkinson, C., Vidilaseris, K., Harborne, S. P. D., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+strategy+to+determine+the+dependence+of+membrane-bound+pyrophosphatases+on+K++as+a+cofactor.">A simple strategy to determine the dependence of membrane-bound pyrophosphatases on K+ as a cofactor.</a> Methods in Enzymology. 607, 131-156 (2018).</li></ol>

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 ...

Our protocol works as a tool to find compounds that inhibits de-activity of membrane bound pyrophosphatase. And this inhibitors may be used in the treatment of several parasitic diseases. This protocol is cheap and simple producing stable color for a long time without showing any interference in the presence of high phospholipid concentration required for enzyme reactivation.Prepare 10 milliliters of the reactivation buffer solution, containing 20 millimolar M-E-S at PH 6.5, 3.5%volume by volume glycerol, two millimolar D-T-T, and 0.05%D-D-M. Prepare 10 milliliters of the reaction mixer, containing 200 millimolar tris hydrochloride at PH eight. Eight millimolar magnesium chloride, 333 millimolar potassium chloride, and 67 millimolar sodium chloride.To prepare 30 milligrams per milliliter liposomes for enzyme reactivation. First, add 10 milliliters of 20 millimolar of tris hydrochloride at PH eight, with one millimolar D-T-T to 0.3 grams of L alpha phosphatidylcholine from soy bean. Put the liposome on ice and sonicate with one second post-intervals for one minute.Pause for one minute, and repeat until the solution becomes transparent yellow. Aliquot the liposomes in 100 microliters, freeze the liquid nitrogen and store at negative 80 degrees Celsius until used. To reactivate the enzyme, thaw the liposome solution at room temperature.Mix 40 microliters of the liposome solution with 22.5 microliters of 20%D-D-M. Keep the mixture at 55 degrees Celsius for 15 minutes. And allow it to cool to room temperature.Then, add 36.5 microliters of the reactivation buffer solution.Mix. And add one microliter of concentrated protein to make a total concentration of 0.13 milligrams per milliliter. Next, transfer 20 microliters of the reactivated enzyme to 1480 microliters of the reaction mixture.Mix gently and use immediately. First, dissolve the compounds in D-M-S-O to make stock solutions of 50 millimolar in 200 microliters. For soluble compounds dilute the stock solution with water to one milliliter in micro tubes, to give two, 10, and 100 micromolar.For each dilution, vortex the compound solution for proper mixing. After that, check for compound divergation, and using a multichannel pipet, dispense 75 microliters of the reaction mixture into each well in a 96-well plate. Add 75 microliters of each compound and water as control in triplicate, and mix by pipetting up and down five times.Measure each well at 300 volts using a microplate nephelometer. To prepare the arsenite citrate solution, weight five grams of sodium arsenite and five grams of trisodium citrate dihydrate, dissolve both compounds into 100 milliliters of water. Then, add five milliliters of glacial acetic acid.Mix, and add water to 250 milliliters, store at room temperature protected from light. Next, prepare solution A by adding 10 milliliters of ice cold 0.5 molar hydrochloric acid to 0.3 grams of ascorbic acid Dissolve the ascorbic acid by vortexing. Prepare solution B by adding one milliliter of ice cold water to 70 milligrams of ammonium tetrathiomolybdate tetrahydrat, and vortex to dissolve.Store solution A and B on ice. To prepare the phosphate standard with a concentration of zero, 62.5, 250 and 500 micromolar for calibration. Add zero microliters, 12.5 microliters, 50 microliters, and 100 microliters of 5 millimolar disodium phosphate dihydrate to four micro tubes containing 370 microliters of the reaction mixture.Top up to one milliliter with water. Now add one milliliter of solution B to 10 milliliters of solution A, mix by hand and store the solution on ice. Using a multichannel pipet, add 40 microliters of zero, 62.5, 250, and 500 micromolar phosphate standard to the tube scripts in triplicate.Then add 25 microliters of compound solution and 15 microliters of M-P-P-A solution mixture of each tube. Except the tubes containing phosphate standard. Seal the tube strips with an adhesive sealing sheet.Cut the sealing sheet to separate each tube strip. Next, pre incubate the sample for five minutes at 71 degrees Celsius. Place the sample on the heating block with 20 second intervals between each strip.For each strip, open the adhesive sealing. Using a multichannel pipet, add 100 microliters of two molar sodium pyrophosphate dibasic. And mix by pipetting up and down for five times.Seal the tube strip again using the same sealing. Incubate again at 71 degrees Celsius for five minutes. After that, place the samples on the cooling apparatus with 20 second interval between each strip.Let them cool for five minutes and then centrifuge each strip briefly to decant the water drops under the sealing sheet. Put the strip back to the cooling apparatus, remove the sealing and let them cool for another five minutes. Then add 60 microliters of solution A and B, mix by pipetting up and down for five times, and keep the tube strips on the cooling apparatus for 10 more minutes.In a fume hood, add 90 microliters of the arsenite citrate solution and keep at room temperature for at least 30 minutes. To produce a stable blue color. Dispense 180 microliters of each reaction mixture into a clear 96-well polystyrene microplate.Use a microplate spectrophotometer to measure the absorbance of each well at 860 nanometers. In this protocol, eight compounds were tested together with I-D-P, a common inhibitor of pyro phosphatase as a positive control. After the addition of solution A and B, and arsenite citrate, a blue color in 30 minutes of incubation at room temperature.All three concentrations of non inhibiting compounds, displayed the same blue color intensity. As well as E-1 to E-3 without any inhibitor. The plot of enzymatic activity against the concentration of each tested compound, shows that the compound with inhibition activity formed a nonlinear curve fitting.The plot of I-D-P as a positive control, clearly shows a decrease in activity at higher concentrations. The inhibition curve for compounds one, five, six, seven, and eight. Shows half maximal inhibitory concentrations at 1.7 micromolar, 21.4 micromolar, 58.8 micromolar, 239.0 micromolar, and greater than 500 micromolar, respectively.Remember to prepare, the M-P-P-A solution. Just before the beginning of the assay.

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Biochemistry

6.5K visualizações.  University of Helsinki. Nosso protocolo funciona como uma ferramenta para encontrar compostos que inibem a des atividade da pirofosfatase ligada à membrana. E esses inibidores podem ser usados no tratamento de várias doenças parasitárias. Este protocolo é barato e simples produzindo cores estáveis por um longo tempo sem mostrar qualquer interferência na presença de alta concentração fosfolipídid necessária para a reativação da enzima.Prepare 10 mililitros da solução tampão de reativação, co...