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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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. 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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. 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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-&amp;alpha;-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 ...

screening-for-thermotoga-maritima-membrane-bound-pyrophosphatase

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6.6K Views.  University of Helsinki. Ons protocol werkt als een hulpmiddel om verbindingen te vinden die de activiteit van membraangebonden pyrofosfatase remmen. En deze remmers kunnen worden gebruikt bij de behandeling van verschillende parasitaire ziekten. Dit protocol is goedkoop en eenvoudig produceren stabiele kleur voor een lange tijd zonder enige interferentie in de aanwezigheid van hoge fosfolipiden concentratie die nodig is voor enzym reactivering.Bereid 10 milliliter van de reactiveringsbufferoplossing, met 20 m...

Video: Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

Bioengineering

Assessing Cellular Target Engagement by SHP2 (PTPN11) Phosphatase Inhibitors

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase (RTK)-driven cell signaling, promoting cell survival and proliferation. In addition, SHP2 is recruited by immune check point receptors to inhibit B and T cell activation. Aberrant SHP2 function has been implicated in the development, progression, and metastasis of many cancers. Indeed, small molecule SHP2 inhibitors have recently entered clinical trials for the treatment of solid tumors with Ras/Raf/ERK pathway activation, including tumors with some oncogenic Ras mutations. However, the current class of SHP2 inhibitors is not effective against the SHP2 oncogenic variants that occur frequently in leukemias, and the development of specific small molecules that target oncogenic SHP2 is the subject of current research. A common problem with most drug discovery campaigns involving cytosolic proteins like SHP2&#160;is that the primary assays that drive chemical discovery are often in vitro assays that do not report the cellular target engagement of candidate compounds. To provide a platform for measuring cellular target engagement, we developed both wild-type and mutant SHP2 cellular thermal shift assays. These assays reliably detect target engagement of SHP2 inhibitors in cells. Here, we provide a comprehensive protocol of this assay, which provides a valuable tool for the assessment and characterization of SHP2 inhibitors.

Assessing Cellular Target Engagement by SHP2 PTPN11 Phosphatase Inhibitors

The ability to assess target engagement by candidate inhibitors in intact cells is crucial for drug discovery. This protocol describes a 384 well format cellular thermal shift assay that reliably detects cellular target engagement of inhibitors targeting either wild-type SHP2 or its oncogenic variants.

The Src-homology 2 (SH2) domain-containing phosphatase 2 (SHP2), encoded by the PTPN11 proto-oncogene, is a key mediator of receptor tyrosine kinase ...

Cancer Research

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 ...

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive patients, organ transplant recipients and pregnant women, resulting in the severe health condition, Toxoplasmosis. Another major protozoan, Neospora caninum, which bears many similarities to Toxoplasma gondii, causes serious diseases in animals, as does Encephalomyelitis and Myositis-Polyradiculitis in dogs and cows, resulting in stillborn calves. All these exhibited similar nucleoside triphosphate hydrolases (NTPase). Neospora caninum has a NcNTPase, while Toxoplasma gondii has a TgNTPase-I. The enzymes are thought to play crucial roles in propagation and survival. In order to establish compounds and/or extracts preventing protozoan infection, we targeted these enzymes for drug discovery. The next step was to establish a novel, highly sensitive, and highly accurate assay by combining a conventional biochemical enzyme assay with a fluorescent assay to determine ADP content. We also validated that the novel assay fulfills the criteria to carry out high-throughput screening (HTS) in the two protozoan enzymes. We performed HTS, identified 19 compounds and six extracts from two synthetic compound libraries and an extract library derived from marine bacteria, respectively. In this study, a detailed explanation has been introduced on how to carry out HTS, including information about the preparation of reagents, devices, robot arm, etc.

Nucleoside Triphosphate Hydrolases Assay in Toxoplasm gondii and Neospora caninum for High-Throughput Screening using a Robot Arm

Toxoplasma gondii and Neospora caninum infections are found in humans and animals and lead to serious health issues. The two parasites share similar nucleoside triphosphate hydrolases and play important roles in propagation and survival. We established a high-standard assay of the enzymes requiring robot arm usage.

Protozoan parasites infect humans and many warm-blooded animals. Toxoplasma gondii, a major protozoan parasite, is commonly found in HIV-positive ...

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) ...

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 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 ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Immunology and Infection

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies have been proposed and implemented for the identification of new mediators of TCR signaling, which would improve the understanding of T-cell processes, including activation and thymic selection. We describe a screening assay that enables the identification of molecules that influence TCR signaling based on the activation of developing thymocytes. Strong TCR signals cause developing thymocytes to activate apoptotic machinery in a process known as negative selection. Through the application of kinase inhibitors, those with targets that affect TCR signaling are able to override the process of negative selection. The method detailed in this paper can be used to identify inhibitors of canonical kinases with established roles in the TCR signaling pathways and also inhibitors of new kinases yet to be established in the TCR signaling pathways. The screening strategy here can be applied to screens of higher throughput for the identification of novel druggable targets in TCR signaling.

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

This paper uses a flow-cytometry-based assay to screen libraries of chemical inhibitors for the identification of inhibitors and their targets that influence T-cell receptor signaling. The methods described here can also be expanded for high-throughput screenings.

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...

Screening Bioactive Nanoparticles in Phagocytic Immune Cells for Inhibitors of Toll-like Receptor Signaling

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have been limited compounds available so far to attenuate TLR signaling and there have been no clinically approved TLR inhibitors (except the anti-malarial drug hydroxychloroquine) in clinical use. In light of rapid advances in nanotechnology, manipulation of immune responsiveness using nano-devices may provide a new strategy to treat these diseases. Herein, we present a high throughput screening method for quickly identifying novel bioactive nanoparticles that inhibit TLR signaling in phagocytic immune cells. This screening platform is built on THP-1 cell-based reporter cells with colorimetric and luciferase assays. The reporter cells are engineered from the human THP-1 monocytic cell line by stable integration of two inducible reporter constructs. One expresses a secreted embryonic alkaline phosphatase (SEAP) gene under the control of a promoter inducible by the transcription factors NF-&#954;B and AP-1, and the other expresses a secreted luciferase reporter gene under the control of promoters inducible by interferon regulatory factors (IRFs).Upon TLR stimulation, the reporter cells activate transcription factors and subsequently produce SEAP and/or luciferase, which can be detected using their corresponding substrate reagents. Using a library of peptide-gold nanoparticle (GNP) hybrids established in our previous studies as an example, we identified one peptide-GNP hybrid that could effectively inhibit the two arms of TLR4 signaling cascade triggered by its prototypical ligand, lipopolysaccharide (LPS). The findings were validated by standard biochemical techniques including immunoblotting. Further analysis established that this lead hybrid had a broad inhibitory spectrum, acting on multiple TLR pathways, including TLR2, 3, 4, and 5. This experimental approach allows a rapid assessment of whether a nanoparticle (or other therapeutic compounds) can modulate specific TLR signaling in phagocytic immune cells.

Toll-like receptor (TLR) signaling plays an important role in the pathophysiology of many human inflammatory diseases, and regulating TLR responses by bioactive nanoparticles is anticipated to be beneficial in many inflammatory conditions. THP-1 cell-based reporter cells provide a versatile and robust screening platform for identifying novel inhibitors of TLR signaling.

Pharmacological regulation of Toll-like receptor (TLR) responses holds great promise in the treatment of many inflammatory diseases. However, there have ...

Medicine

Multi-enzyme Screening Using a High-throughput Genetic Enzyme Screening System

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a large-scale metagenomic library1. We previously proposed a genetic enzyme screening system (GESS) that uses dimethylphenol regulator activated by phenol or p-nitrophenol. Since a vast amount of natural enzymatic reactions produce these phenolic compounds from phenol deriving substrates, this single genetic screening system can be theoretically applied to screen over 200 different enzymes in the BRENDA database. Despite the general applicability of GESS, applying the screening process requires a specific procedure to reach the maximum flow cytometry signals. Here, we detail the developed screening process, which includes metagenome preprocessing with GESS and the operation of a flow cytometry sorter. Three different phenolic substrates (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate) with GESS were used to screen and to identify three different enzymes (lipase, cellulase, and alkaline phosphatase), respectively. The selected metagenomic enzyme activities were confirmed only with the flow cytometry but DNA sequencing and diverse in vitro analysis can be used for further gene identification.

This work presents a method of high-throughput screening using a universal genetic enzyme screening system that can be theoretically applied to over 200 enzymes. Here, the single screening system identifies three different enzymes (lipase, cellulase, and alkaline phosphatase) by simply changing the substrate used (p-nitrophenyl acetate, p-nitrophenyl-&#946;-D-cellobioside, and phenyl phosphate).

The recent development of a high-throughput single-cell assay technique enables the screening of novel enzymes based on functional activities from a ...

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

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