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: Mg2+ is required to chelate the pyrophosphate as the substrate of mPPase, K+ is required to increase the enzyme activity as TmPPase is a potassium dependent mPPase, and Na+ is needed for the enzyme activity during sodium ion translocation by TmPPase.</li>
	<li>Prepare 30 mg/mL liposomes for enzyme reactivation.
	<ol>
		<li>Add 10 mL of 20 mM Tris-HCl pH 8.0 with 1 mM DTT to 0.3 g of L-&#945;-phosphatidylcholine from soybean to 10 mL of 20 mM Tris-HCl pH 8.0 with 1 mM DTT.</li>
		<li>Put the liposome on ice, and sonicate with 1 second pulse interval for 1 minute, pause for 1 minute, and repeat until the solution becomes transparent yellow.</li>
		<li>Aliquot the liposomes, freeze in liquid nitrogen and store at -80 &#176;C until used.</li>
	</ol>
	</li>
	<li>Reactivate the enzyme.
	<ol>
		<li>Mix 40 &#181;L of the liposomes solution with 22.5 &#181;L of 20% DDM.</li>
		<li>Heat the mixture at 55 &#176;C for 15 min and allow it to cool to room temperature.</li>
		<li>Add 36.5 &#181;L of the reactivation buffer solution, mix, and add 1 &#181;L of concentrated protein (13 mg/mL) to make a total concentration of 0.13 mg/mL. 
		NOTE: Protein is usually frozen in 10 &#181;L aliquots after purification and thawed on ice before use.</li>
	</ol>
	</li>
	<li>Take 20 &#181;L of the reactivated enzyme and add to 1,480 &#181;L of the reaction mixture, then mix gently. 
	NOTE: The addition of the reactivated enzyme to the reaction mixture should be performed just before it is used.</li>
</ol>
2. Compound preparation
<ol>
	<li>Dissolve the compounds in dimethyl sulfoxide (DMSO) to make stock solutions of 25&#8722;100 mM in 50&#8722;200 &#181;L, based on the availability of the compounds. 
	NOTE: All compounds used here (Figure 2A) have been published previously9. If the compound solubility is low, the stock concentration can be adjusted accordingly.</li>
	<li>Prepare three different concentrations of each compound in water. 
	NOTE: The final concentrations in the reaction mixture will be 1, 5, and 50 micromolar or 1, 5, and 20 micromolar for soluble and sparingly soluble compounds, respectively.
	<ol>
		<li>Dilute the stock solution with water to 1 mL in microtubes to give 2 &#181;M, 10 &#181;M and 100 &#181;M for soluble compounds, or alternatively 2 &#181;M, 10 &#181;M and 40 &#181;M for sparingly soluble compounds.</li>
		<li>Vortex the compound solution instantly after dilution of the stock solution for proper mixing.</li>
	</ol>
	</li>
	<li>Check for compound aggregation using a nephelometer. 
	NOTE: This was studied as triplicates in three concentrations (1 &#181;M, 5 &#181;M and 20 &#181;M) and normalized to the blank in a 96 well plate.
	<ol>
		<li>Dispense 75 &#181;L of the reaction mixture into each well using a multichannel pipette.</li>
		<li>Add 75 &#181;L of each compound (for the blank, use 75 &#181;L of water instead) and mix by pipetting up and down 5&#215;.</li>
		<li>Measure each well at 300 V using a microplate nephelometer.</li>
	</ol>
	</li>
</ol>
3. Reagents for the assay preparation
<ol>
	<li>Prepare the arsenite-citrate solution.
	<ol>
		<li>Weigh 5 g of sodium arsenite and 5 g of trisodium citrate dihydrate. 
		CAUTION: Sodium arsenite is toxic, thus use proper protective equipment and handle with special care. As precaution, do not handle before all necessary safety precautions have been read and understood. Handle only in a fume hood in order not to inhale dust/vapors of the compound or its solution(s). If inhaled, move to fresh air and obtain medical attention. Wear appropriate chemical safety goggles, protective gloves and clothing to avoid ingestion and eye/skin contact. If swallowed, call immediately a poison center or doctor/physician. If it gets on the skin or in the eye(s), wash with plenty of water and obtain medical attention.</li>
		<li>Dissolve into 100 mL of water.</li>
		<li>Add 5 mL of glacial acetic acid, mix, and add water to 250 mL.</li>
		<li>Store at room temperature protected from light. 
		NOTE: The solution is stable for more than a year.</li>
	</ol>
	</li>
	<li>Prepare solution A and solution B.
	<ol>
		<li>For solution A, add 10 mL of ice cold 0.5 M HCl to 0.3 g of ascorbic acid. Dissolve the ascorbic acid by vortexing.</li>
		<li>For solution B, add 1 mL of ice cold water to 70 mg of ammonium heptamolybdate tetrahydrate and vortex to dissolve. 
		NOTE: Store both solutions on ice until use. For the consistency of the assay result, both solutions can be stored on ice for a maximum of one&#160;week.</li>
	</ol>
	</li>
	<li>Prepare the phosphate (Pi) standard with the concentration of 0 &#181;M, 62.5 &#181;M, 250 &#181;M and 500 &#181;M for calibration.
	<ol>
		<li>Add 0 &#181;L, 25 &#181;L, 50 &#181;L, and 100 &#181;L of 5 mM Na2HPO4 dihydrate to four microtubes containing 370 &#181;L of the reaction mixture.</li>
		<li>Top up to 1 mL with water.</li>
	</ol>
	</li>
</ol>
4. Activity assay for one 96 well plate
NOTE: See Figure 1 for the schematic workflow of the assay.
<ol>
	<li>Add 1 mL of solution B to 10 mL of solution A, mix by vortexing and store the solution on ice. 
	NOTE: This solution should be transparent and yellow. Keep solution A + B on ice for at least 30 min prior to use. However, use the solution within 3 h as it will go bad after long-term storage.</li>
	<li>Add 40 &#181;L of 0 &#181;M, 62.5 &#181;M, 250 &#181;M and 500 &#181;M Pi standard to the tube strips in triplicate using a multichannel pipette. 
	NOTE: The reaction mixture with no Pi added will be used as a blank.</li>
	<li>Add 25 &#181;L of compound solution to the tube strips using a multichannel pipette. 
	NOTE: Each compound has three different concentrations in triplicate which is enough for initial estimation of the half maximal inhibitory concentration (IC50). For a more accurate IC50 determination, eight different compound concentrations can be used. For the uninhibited enzyme the compound solution is replaced with equal amount of water. As positive controls 2.5 &#181;M, 25 &#181;M, and 250 &#181;M of imidodiphosphate (IDP) sodium salt were used.</li>
	<li>Add 15 &#181;L of mPPase solution mixture to the tube strips (except to the tubes containing Pi standard) using a multichannel pipette.</li>
	<li>Seal the tube strips with an adhesive sealing sheet. Cut the sealing sheet to separate each tube strip.</li>
	<li>Pre-incubate the samples for 5 min at 71 &#176;C. Place the samples on the heating block with 20 s interval between each strip in order to minimize the time consumption during the subsequent steps.</li>
	<li>For each strip, open the adhesive sealing. Add 10 &#181;L of 2 mM sodium pyrophosphate dibasic using a multichannel pipette and mix by pipetting up and down for 5&#215;. Seal the tube strip again using the same sealing. 
	NOTE: This step might initially be difficult to accomplish in 20 s; however, it will become easier after some assays.</li>
	<li>Incubate at 71 &#176;C for 5 min.</li>
	<li>Place the samples on the cooling apparatus with 20 s interval between each strip. Let them cool for 10 min but centrifuge each strip briefly after 5 min of cooling, to decant water drops under the sealing sheet, then put it back to the cooling apparatus and remove the sealing. 
	NOTE: The cooling apparatus can simply be made by placing a 96 well PCR plate on a polystyrene Petri dish (size 150 mm &#215; 15 mm) filled with water and frozen for at least 1 h. The apparatus should be taken out from the freezer about 5 min prior to the beginning of the assay. Do not take out the cooling apparatus right before sample cooling as it will freeze the reaction mixture and hinder color development.</li>
	<li>After 10 min of cooling, add 60 &#181;L of solution A + B, mix by pipetting up and down for 5&#215; and keep the tube strips on the cooling apparatus for 10 min.</li>
	<li>Add 90 &#181;L of the arsenite-citrate solution and keep at room temperature for at least 30 min to produce a stable blue color. 
	CAUTION: Due to its toxicity all solutions containing sodium arsenite should be handled with extra care at all time. Thus, the addition of arsenite-citrate solution should be done in a fume hood.</li>
	<li>Dispense 180 &#181;L of each reaction mixture into a clear 96 well polystyrene microplate.</li>
	<li>Measure the absorbance of each well at 860 nm using a microplate spectrophotometer.</li>
</ol>
5. Result analysis
<ol>
	<li>Average the triplicates of each sample and the Pi standards. Then subtract with the blank to eliminate the background signal.</li>
	<li>Make a calibration curve by plotting the absorbance (A860) values against the amount of Pi standard (nmol) and perform a linear regression to obtain the trendline function using the following formula: 
	<img alt="Equation 1" src="/files/ftp_upload/60619/60619equ01a.jpg" /></li>
	<li>Calculate the phosphate amount (nmol) released from the enzymatic reaction based on the linear regression formula above.</li>
	<li>Calculate the specific activity using the following formula: 
	<img alt="Equation 2" src="/files/ftp_upload/60619/60619equ02a.jpg" /> 
	where nPi is the amount of phosphate released from the reaction (nmol), t is the reaction time (min), and mTmPPase is the amount of the pure TmPPase used in the assay (mg).</li>
	<li>Calculate the percent activity for each inhibitor concentration using the following formula: 
	<img alt="Equation 3" src="/files/ftp_upload/60619/60619equ03a.jpg" /> 
	where SAi is the specific activity of a sample with inhibitor and SAun is the specific activity of the uninhibited sample.</li>
	<li>Calculate the logIC50 (estimate) and IC50 (estimate) with a nonlinear regression fit from the four-parameter dose-response curve using the following formula: 
	<img alt="Equation 4" src="/files/ftp_upload/60619/60619equ04a.jpg" /> 
	where X is log of concentration ( &#181;M), Y is activity (%), Top and Bottom are plateaus in the same unit as Y (100% and 0%, respectively), logIC50 has the same log units as X, and HillSlope = slope factor or hill slope, which is unitless. 
	NOTE: Software (Table of Materials) is used for the fitting. Use the concentration of 0.01 &#181;M (instead of 0.00 &#181;M) for the sample without inhibitor as the logarithm of zero is not defined.</li>
</ol>

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	<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>

The authors have nothing to disclose.

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...

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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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Biochemistry

6.5K Views.  University of Helsinki. 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.

Video: Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

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 excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages for identifying biologically active chemical structures that can modify complex phenotypes such as lifespan. Described here is a simple protocol for producing hundreds of 96-well culture plates with fairly consistent numbers of C. elegans in each well. Next, we specified how to use these cultures to screen thousands of chemicals for effects on the lifespan of the nematode C. elegans. This protocol makes use of temperature sensitive sterile strains, agar plate conditions, and simple animal handling to facilitate the rapid and high throughput production of synchronized animal cultures for screening.

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

Here we describe a simple protocol for rapidly producing hundreds of nematode growth media agar, 96-well culture plates with consistent numbers of Caenorhhabditis elegans per well. These cultures are useful for the phenotypic screening of whole organisms. We focus here on using these cultures to screen chemicals for pro-longevity effects.

Caenorhabditis elegans is a useful organism for testing chemical effects on physiology. Whole organism small molecule screens offer significant advantages ...

Kinase Inhibitor Screening In Self-assembled Human Protein Microarrays

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with clinical implications. Several methodologies have been reported to accomplish such screening. However, each has its own limitations (e.g., the screening of only ATP analogues, restriction to using purified kinase domains, significant costs associated with testing more than a few kinases at a time, and lack of flexibility in screening protein kinases with novel mutations). Here, a new protocol that overcomes some of these limitations and can be used for the unbiased screening of kinase inhibitors is presented. A strength of this method is its ability to compare the activity of kinase inhibitors across multiple proteins, either between different kinases or different variants of the same kinase. Self-assembled protein microarrays generated through the expression of protein kinases by a human-based in vitro transcription and translation system (IVTT) are employed. The proteins displayed on the microarray are active, allowing for measurement of the effects of kinase inhibitors. The following procedure describes the protocol steps in detail, from the microarray generation and screening to the data analysis.

A detailed protocol for the generation of self-assembled human protein microarrays for the screening of kinase inhibitors is presented.

The screening of kinase inhibitors is crucial for better understanding properties of a drug and for the identification of potentially new targets with ...

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

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 not occur spontaneously. These proteins are crucial for essentially all aspects of cellular life, including metabolism, cell division, responses to environmental changes and movement. The protocol presented here describes a nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay that has been adapted to semi-high throughput screening of small molecule ATPase inhibitors. The assay has been applied to cardiac and skeletal muscle myosin II's, two actin-based molecular motor ATPases, as a proof of principle. The hydrolysis of ATP is coupled to the oxidation of NADH by enzymatic reactions in the assay. First, the ADP generated by the ATPase is regenerated to ATP by pyruvate kinase (PK). PK catalyzes the transition of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH), which catalyzes the oxidation of NADH in parallel. Thus, the decrease in ATP concentration is directly correlated to the decrease in NADH concentration, which is followed by change to the intrinsic fluorescence of NADH. As long as PEP is available in the reaction system, the ADP concentration remains very low, avoiding inhibition of the ATPase enzyme by its own product. Moreover, the ATP concentration remains nearly constant, yielding linear time courses. The fluorescence is monitored continuously, which allows for easy estimation of the quality of data and helps to filter out potential artifacts (e.g., arising from compound precipitation or thermal changes).

A nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has been adapted to semihigh throughput screening of small molecule myosin inhibitors. This kinetic assay is run in a 384-well microplate format with total reaction volumes of only 20 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

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

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their&#160;cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.

X-Ray Crystallography to Study the Oligomeric State Transition of the Thermotoga maritima M42 Aminopeptidase TmPep1050

This protocol has been developed to study the dimer-dodecamer transition of TmPep1050, an M42 aminopeptidase, at the structural level. It is a straightforward pipeline starting from protein purification to X-ray data processing. Crystallogenesis, data set indexation, and molecular replacement have been emphasized through a case of study, TmPep1050H60A H307A variant.

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion ...

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione (GSH) conjugation. In addition, GSTs are regulators of mitogen-activated protein kinases (MAPKs) involved in apoptotic pathways. Overexpression of GSTs is correlated with decreased therapeutic efficacy among patients undergoing chemotherapy with electrophilic alkylating agents. Using GST inhibitors may be a potential solution to reverse this tendency and augment treatment potency. Achieving this goal requires the discovery of such compounds, with an accurate, quick, and easy enzyme assay. A spectrophotometric protocol using 1-chloro-2,4-dinitrobenzene (CDNB) as the substrate is the most employed method in the literature. However, already described GST inhibition experiments do not provide a protocol detailing each stage of an optimal inhibition assay, such as the measurement of the Michaelis-Menten constant (Km) for CDNB or indication of the employed enzyme concentration, crucial parameters to assess the inhibition potency of a tested compound. Hence, with this protocol, we describe each step of an optimized spectrophotometric GST enzyme assay, to screen libraries of potential inhibitors. We explain the calculation of both the half-maximal inhibitory concentration (IC50) and the constant of inhibition (Ki)&#8212;two characteristics used to measure the potency of an enzyme inhibitor. The method described can be implemented using a pool of GSTs extracted from cells or pure recombinant human GSTs, namely GST alpha 1 (GSTA1), GST mu 1 (GSTM1) or GST pi 1 (GSTP1). However, this protocol cannot be applied to GST theta 1 (GSTT1), as CDNB is not a substrate for this isoform. This method was used to test the inhibition potency of curcumin using GSTs from equine liver. Curcumin is a molecule exhibiting anti-cancer properties and showed affinity towards GST isoforms after in silico docking predictions. We demonstrated that curcumin is a potent competitive GST inhibitor, with an IC50 of 31.6 &#177; 3.6 &#181;M and a Ki of 23.2 &#177; 3.2 &#181;M. Curcumin has potential to be combined with electrophilic chemotherapy medication to improve its efficacy.

Glutathione S-transferases (GSTs) are detoxification enzymes involved in the metabolism of numerous chemotherapeutic drugs. Overexpression of GSTs is correlated with cancer chemotherapy resistance. One way to counter this phenotype is to use inhibitors. This protocol describes a method using a spectrophotometric assay to screen for potential GST inhibitors.

Glutathione S-transferases (GSTs) are metabolic enzymes responsible for the elimination of endogenous or exogenous electrophilic compounds by glutathione ...

NMR-Based Fragment Screening in a Minimum Sample but Maximum Automation Mode

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest advantage of NMR-based fragment screening is its ability not only to detect binders over 7-8 orders of magnitude of affinity but also to monitor purity and chemical quality of the fragments and thus to produce high quality hits and minimal false positives or false negatives. A prerequisite within the FBS is to perform initial and periodic quality control of the fragment library, determining solubility and chemical integrity of the fragments in relevant buffers, and establishing multiple libraries to cover diverse scaffolds to accommodate various macromolecule target classes (proteins/RNA/DNA). Further, an extensive NMR-based screening protocol optimization with respect to sample quantities, speed of acquisition and analysis at the level of biological construct/fragment-space, in condition-space (buffer, additives, ions, pH, and temperature) and in ligand-space (ligand analogues, ligand concentration) is required. At least in academia, these screening efforts have so far been undertaken manually in a very limited fashion, leading to limited availability of screening infrastructure not only in the drug development process but also in the context of chemical probe development. In order to meet the requirements economically, advanced workflows are presented. They take advantage of the latest state-of-the-art advanced hardware, with which the liquid sample collection can be filled in a temperature-controlled fashion into the NMR-tubes in an automated manner. 1H/19F NMR ligand-based spectra are then collected at a given temperature. High-throughput sample changer (HT sample changer) can handle more than 500 samples in temperature-controlled blocks. This together with advanced software tools speeds up data acquisition and analysis. Further, application of screening routines on protein and RNA samples are described to make aware of the established protocols for a broad user base in biomacromolecular research.

Fragment-based screening by NMR is a robust method to rapidly identify small molecule binders to biomacromolecules (DNA, RNA, or proteins). Protocols describing automation-based sample preparation, NMR experiments &#38; acquisition conditions, and analysis workflows are presented. The technique allows for optimal exploitation of both 1H and 19F NMR-active nuclei for detection.

Fragment-based screening (FBS) is a well-validated and accepted concept within the drug discovery process both in academia and industry. The greatest ...

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer composition). The utility of TSA, and specifically of nano-Differential Scanning Fluorimetry (nano-DSF), has been established over the years, both for finding conditions that help stabilize a specific protein and for looking at ligand binding by monitoring changes in the apparent Tm. This paper presents an efficient screening of the Diamond-SGC-iNEXT Poised (DSi-Poised) fragment library (768 compounds) by the use of nano-DSF, monitoring Tm to identify potential fragment binding. The prerequisites regarding protein quality and concentration for performing nano-DSF experiments are briefly outlined followed by a step-by-step protocol that uses a nano-liter robotic dispenser commonly used in structural biology laboratories for preparing the required samples in 96-well plates. The protocol describes how the reagent mixtures are transferred to the capillaries needed for nano-DSF measurements. In addition, this paper provides protocols to measure thermal denaturation (monitoring intrinsic tryptophan fluorescence) and aggregation (monitoring light back-scattering) and the subsequent steps for data transfer and analysis. Finally, screening experiments with three different protein targets are discussed to illustrate the use of this procedure in the context of lead discovery campaigns. The overall principle of the method described can be easily transferred to other fragment libraries or adapted to other instruments.

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Thermal shift assays (TSAs) examine how the melting temperature (Tm) of a target protein changes in response to changes in its environment (e.g., buffer ...

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) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

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