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

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

<ol>
	<li>Terstappen, G. C., Reggiani, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+silico+research+in+drug+discovery.">In silico research in drug discovery.</a> Trends in Pharmacological Sciences. 22 (1), 23-26 (2001).</li><li>Rask-Andersen, M., Almen, M. S., Schioth, H. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trends+in+the+exploitation+of+novel+drug+targets.">Trends in the exploitation of novel drug targets.</a> Nature Reviews Drug Discovery. 10 (8), 579-590 (2011).</li><li>Shah, N. R., Vidilaseris, K., Xhaard, H., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integral+membrane+pyrophosphatases:+a+novel+drug+target+for+human+pathogens.">Integral membrane pyrophosphatases: a novel drug target for human pathogens.</a> AIMS Biophysics. 3 (1), 171-194 (2016).</li><li>Baykov, A. A., Malinen, A. M., Luoto, H. H., Lahti, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyrophosphate-Fueled+Na++and+H++Transport+in+Prokaryotes.">Pyrophosphate-Fueled Na+ and H+ Transport in Prokaryotes.</a> Microbiology and Molecular Biology Reviews. 77 (2), 267-276 (2013).</li><li>Serrano, A., Perez-Castineira, J. R., Baltscheffsky, M., Baltscheffsky, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=H+-PPases:+yesterday,+today+and+tomorrow.">H+-PPases: yesterday, today and tomorrow.</a> IUBMB Life. 59 (2), 76-83 (2007).</li><li>Liu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+vacuolar-H+-pyrophosphatase+(TgVP1)+is+required+for+microneme+secretion,+host+cell+invasion,+and+extracellular+survival+of+Toxoplasma+gondii.">A vacuolar-H+-pyrophosphatase (TgVP1) is required for microneme secretion, host cell invasion, and extracellular survival of Toxoplasma gondii.</a> Molecular Microbiology. 93 (4), 698-712 (2014).</li><li>Lemercier, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+pyrophosphatase+regulating+polyphosphate+metabolism+in+acidocalcisomes+is+essential+for+Trypanosoma+brucei+virulence+in+mice.">A pyrophosphatase regulating polyphosphate metabolism in acidocalcisomes is essential for Trypanosoma brucei virulence in mice.</a> Journal of Biological Chemistry. 279 (5), 3420-3425 (2004).</li><li>Belogurov, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane-bound+pyrophosphatase+of+Thermotoga+maritima+requires+sodium+for+activity.">Membrane-bound pyrophosphatase of Thermotoga maritima requires sodium for activity.</a> Biochemistry. 44 (6), 2088-2096 (2005).</li><li>Vidilaseris, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Asymmetry+in+catalysis+by+Thermotoga+maritima+membrane-bound+pyrophosphatase+demonstrated+by+a+nonphosphorus+allosteric+inhibitor.">Asymmetry in catalysis by Thermotoga maritima membrane-bound pyrophosphatase demonstrated by a nonphosphorus allosteric inhibitor.</a> Science Advances. 5 (5), (2019).</li><li>Lin, S. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+a+membrane-embedded+H+-translocating+pyrophosphatase.">Crystal structure of a membrane-embedded H+-translocating pyrophosphatase.</a> Nature. 484 (7394), 399-403 (2012).</li><li>Fiske, C. H., Subbarow, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+colorimetric+determination+of+phosphorus.">The colorimetric determination of phosphorus.</a> Journal of Biological Chemistry. 66 (2), 375-400 (1925).</li><li>Nagul, E. A., McKelvie, I. D., Worsfold, P., Kolev, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molybdenum+blue+reaction+for+the+determination+of+orthophosphate+revisited:+Opening+the+black+box.">The molybdenum blue reaction for the determination of orthophosphate revisited: Opening the black box.</a> Analytica Chimica Acta. 890, 60-82 (2015).</li><li>Kellosalo, J., Kajander, T., Palmgren, M. G., Lopez-Marques, R. L., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterologous+expression+and+purification+of+membrane-bound+pyrophosphatases.">Heterologous expression and purification of membrane-bound pyrophosphatases.</a> Protein Expression and Purification. 79 (1), 25-34 (2011).</li><li>Vidilaseris, K., Kellosalo, J., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+high-throughput+method+for+orthophosphate+determination+of+thermostable+membrane-bound+pyrophosphatase+activity.">A high-throughput method for orthophosphate determination of thermostable membrane-bound pyrophosphatase activity.</a> Analytical Methods. 10 (6), 646-651 (2018).</li><li>He, Z. Q., Honeycutt, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+modified+molybdenum+blue+method+for+orthophosphate+determination+suitable+for+investigating+enzymatic+hydrolysis+of+organic+phosphates.">A modified molybdenum blue method for orthophosphate determination suitable for investigating enzymatic hydrolysis of organic phosphates.</a> Communications in Soil Science and Plant Analysis. 36 (9-10), 1373-1383 (2005).</li><li>Martin, B., Pallen, C. J., Wang, J. H., Graves, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+fluorinated+tyrosine+phosphates+to+probe+the+substrate+specificity+of+the+low+molecular+weight+phosphatase+activity+of+calcineurin.">Use of fluorinated tyrosine phosphates to probe the substrate specificity of the low molecular weight phosphatase activity of calcineurin.</a> Journal of Biological Chemistry. 260 (28), 14932-14937 (1985).</li><li>Strauss, J., Wilkinson, C., Vidilaseris, K., Harborne, S. P. D., Goldman, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+strategy+to+determine+the+dependence+of+membrane-bound+pyrophosphatases+on+K++as+a+cofactor.">A simple strategy to determine the dependence of membrane-bound pyrophosphatases on K+ as a cofactor.</a> Methods in Enzymology. 607, 131-156 (2018).</li></ol>

Here we report a detailed protocol for simple screening of inhibitors for membrane-bound pyrophosphatase from T. maritima in a 96 well plate format based on Vidilaseris et al.14. This protocol is inexpensive and based on 12-phosphomolybdic acid, which is formed from orthophosphate and molybdate under acidic conditions and reduced to phosphomolybdenum species with a distinct blue color12. This method is preferred over other protocols, such as the more sensitive mala...

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

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

screening for thermotoga maritima membrane-bound pyrophosphatase inhibitors

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

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

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

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

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

Screening for Thermotoga maritima Membrane-Bound Pyrophosphatase Inhibitors

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

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

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6.5K Views.  University of Helsinki. Our protocol works as a tool to find compounds that inhibits de-activity of membrane bound pyrophosphatase. And this inhibitors may be used in the treatment of several parasitic diseases. This protocol is cheap and simple producing stable color for a long time without showing any interference in the presence of high phospholipid concentration required for enzyme reactivation.Prepare 10 milliliters of the reactivation buffer solution, containing 20 millimolar M-E-S at PH 6.5, 3.5%volume...

Video: 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...

x-ray crystallography to study the oligomeric state transition of the thermotoga maritima m42 aminopeptidase tmpep1050

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...

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

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Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

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High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

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

an in vitro assay for screening interferons as potent inhibitors of zika virus growth

Six hours after seeding a 12-well plate with na&#239;ve Vero cells, add cytokines of interest at indicated concentrations in biological duplicates. 12 hours post-treatment, perform Zika virus infection, and include negative control wells without infection. Four hours after infection, replace the viral inoculum with cytokine-treated medium, and then incubate the cells at 37 degree Celsius for an additional 44 hours. At 48 hours post-infection, count viral plaques using a phase-contrast...

An In Vitro Assay for Screening Interferons as Potent Inhibitors of Zika Virus Growth

The video demonstrates an in vitro assay for screening interferons with antiviral activity against the Zika virus, a human pathogen. Within the host cell, the Zika virus undergoes multiplication upon internalization, infecting neighboring cells and creating distinct zones or viral plaques. Type I interferon, acting as an antiviral agent, hinders virus multiplication, thereby preventing the formation of viral plaques in the host cell...

a semi-high-throughput adaptation of the nadh-coupled atpase assay for screening small molecule inhibitors

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

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...

bacterial inner-membrane display for screening a library of antibody fragments

Bacterial Inner-membrane Display for Screening a Library of Antibody Fragments

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner...

a high-throughput cre-lox activated viral membrane fusion assay to identify inhibitors of hiv-1 viral membrane fusion

A High-throughput Cre-Lox Activated Viral Membrane Fusion Assay to Identify Inhibitors of HIV-1 Viral Membrane Fusion

We describe a cell-based assay to report on HIV-1 fusion via the expression of green fluorescent protein detectable by flow cytometry or fluorescence microscopy. It can be used to test inhibitors of viral entry (specifically at the fusion step) in cell-free and cell-to-cell infection...

assays for validating histone acetyltransferase inhibitors

Assays for Validating Histone Acetyltransferase Inhibitors

Inhibitors of histone acetyltransferases (HATs, also known as lysine acetyltransferases), such as CBP/p300, are potential therapeutics for treating cancer. However, rigorous methods for validating these inhibitors are needed. Three in vitro methods for validation include HAT assays with recombinant acetyltransferases, immunoblotting for histone acetylation in cell culture, and...

natural killer (nk) and car-nk cell expansion method using membrane bound-il-21-modified b cell line

Natural Killer NK and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

Here, we present a method to expand peripheral blood natural killer (PBNK), NK cells from liver tissues, and chimeric antigen receptor (CAR)-NK cells derived from peripheral blood mononuclear cells (PBMCs) or cord blood (CB). This protocol demonstrates the expansion of NK and CAR-NK cells using 221-mIL-21 feeder cells in addition to the optimized purity of expanded NK...

Natural Killer (NK) and CAR-NK Cell Expansion Method using Membrane Bound-IL-21-Modified B Cell Line

high-throughput antiviral assays to screen for inhibitors of zika virus replication

High-throughput Antiviral Assays to Screen for Inhibitors of Zika Virus Replication

In this work, we describe the protocols used in replicon-based and viral enzyme-based assays to screen for inhibitors of Zika virus replication in a high-throughput screening...

green fluorescent protein-based expression screening of membrane proteins in escherichia coli

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

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

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

The malachite green assay protocol is a simple and cost-effective method to discover heat shock protein 90 (Hsp90) suppressors, as well as other inhibitor compounds against ATP-dependent...

identification of egfr and ras inhibitors using caenorhabditis elegans

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

The genetically tractable nematode Caenorhabditis elegans can be used as a simple and inexpensive model for drug discovery. Described here is a protocol to identify anticancer therapeutics that inhibit the downstream signaling of RAS and EGFR...

Identification of EGFR and RAS Inhibitors using Caenorhabditis elegans

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 ...

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in the cytoplasm. Commonly used methods for the display and screening of recombinant antibody libraries do not incorporate intracellular protein folding quality control, and, thus, the antigen-binding capability and cytoplasmic folding and solubility of antibodies engineered using these methods often must be engineered separately. Here, we describe a protocol to screen a recombinant library of single-chain variable fragment (scFv) antibodies for antigen-binding and proper cytoplasmic folding simultaneously. The method harnesses the intrinsic intracellular folding quality control mechanism of the Escherichia coli twin-arginine translocation (Tat) pathway to display an scFv library on the E. coli inner membrane. The Tat pathway ensures that only soluble, well-folded proteins are transported out of the cytoplasm and displayed on the inner membrane, thereby eliminating poorly folded scFvs prior to interrogation for antigen-binding. Following removal of the outer membrane, the scFvs displayed on the inner membrane are panned against a target antigen immobilized on magnetic beads to isolate scFvs that bind to the target antigen. An enzyme-linked immunosorbent assay (ELISA)-based secondary screen is used to identify the most promising scFvs for additional characterization. Antigen-binding and cytoplasmic solubility can be improved with subsequent rounds of mutagenesis and screening to engineer antibodies with high affinity and high cytoplasmic solubility for intracellular applications.

We provide a method to simultaneously screen a library of antibody fragments for binding affinity and cytoplasmic solubility by using the Escherichia coli twin-arginine translocation pathway, which has an inherent quality control mechanism for intracellular protein folding, to display the antibody fragments on the inner membrane.

Antibodies engineered for intracellular function must not only have affinity for their target antigen, but must also be soluble and correctly folded in ...

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. ...

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 ...

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.

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 ...

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) ...