Name: a semi-high-throughput adaptation of the nadh-coupled atpase assay for screening small molecule inhibitors
Uploaded: 2019-08-17T14:00:00
Description: Watch this Scientific Journal Video about A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors at JoVE.com

This work was supported by a grant from the National Institute of Neurological Disorders and Stroke and National Institute on Drug Abuse NS096833 (CAM).

1. Preparing stock solutions and reagents
<ol>
	<li>Prepare dithiothreitol (DTT) stock solution by dissolving crystalline DTT in distilled water to a final concentration of 1000 mM. Adjust the pH to 7.0 with 1 M NaOH solution. Aliquot and store at -20 &#176;C.</li>
	<li>Prepare ATP stock solution by dissolving crystalline ATP in distilled water to a final concentration of 100 mM. Adjust the pH to 7.0 with 1 M NaOH solution. Aliquot and store at -20 &#176;C.</li>
	<li>Prepare 10x NADH buffer containing 70 mM 3-(N-morpho...

<ol>
	<li>Heissler, S. M., Sellers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+Adaptations+of+Myosins+for+Their+Diverse+Cellular+Functions.">Kinetic Adaptations of Myosins for Their Diverse Cellular Functions.</a> Traffic. 17 (8), 839-859 (2016).</li><li>Hartman, M. A., Spudich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myosin+superfamily+at+a+glance.">The myosin superfamily at a glance.</a> Journal of Cell Science. 125 (Pt 7), 1627-1632 (2012).</li><li>Berg, J. S., Powell, B. C., Cheney, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+millennial+myosin+census.">A millennial myosin census.</a> Molecular Biology of the Cell. 12 (4), 780-794 (2001).</li><li>Sebe-Pedros, A., Grau-Bove, X., Richards, T. A., Ruiz-Trillo, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+classification+of+myosins,+a+paneukaryotic+whole-genome+approach.">Evolution and classification of myosins, a paneukaryotic whole-genome approach.</a> Genome Biology and Evolution. 6 (2), 290-305 (2014).</li><li>Newell-Litwa, K. A., Horwitz, R., Lamers, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-muscle+myosin+II+in+disease:+mechanisms+and+therapeutic+opportunities.">Non-muscle myosin II in disease: mechanisms and therapeutic opportunities.</a> Disease Models &amp; Mechanisms. 8 (12), 1495-1515 (2015).</li><li>He, Y. M., Gu, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Research+progress+of+myosin+heavy+chain+genes+in+human+genetic+diseases.">Research progress of myosin heavy chain genes in human genetic diseases.</a> Yi Chuan. 39 (10), 877-887 (2017).</li><li>Rauscher, A. A., Gyimesi, M., Kovacs, M., Malnasi-Csizmadia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeting+Myosin+by+Blebbistatin+Derivatives:+Optimization+and+Pharmacological+Potential.">Targeting Myosin by Blebbistatin Derivatives: Optimization and Pharmacological Potential.</a> Trends in Biochemical Sciences. 43 (9), 700-713 (2018).</li><li>Straight, A. F., 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=Dissecting+temporal+and+spatial+control+of+cytokinesis+with+a+myosin+II+Inhibitor.">Dissecting temporal and spatial control of cytokinesis with a myosin II Inhibitor.</a> Science. 299 (5613), 1743-1747 (2003).</li><li>Sirigu, S., 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=Highly+selective+inhibition+of+myosin+motors+provides+the+basis+of+potential+therapeutic+application.">Highly selective inhibition of myosin motors provides the basis of potential therapeutic application.</a> Proceedings of the National Academy of Sciences of the United States of America. 113 (47), E7448-E7455 (2016).</li><li>Green, E. 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=A+small-molecule+inhibitor+of+sarcomere+contractility+suppresses+hypertrophic+cardiomyopathy+in+mice.">A small-molecule inhibitor of sarcomere contractility suppresses hypertrophic cardiomyopathy in mice.</a> Science. 351 (6273), 617-621 (2016).</li><li>Morgan, B. P., 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=Discovery+of+omecamtiv+mecarbil+the+first,+selective,+small+molecule+activator+of+cardiac+Myosin.">Discovery of omecamtiv mecarbil the first, selective, small molecule activator of cardiac Myosin.</a> ACS Medicinal Chemistry Letters. 1 (9), 472-477 (2010).</li><li>Kepiro, 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=para-Nitroblebbistatin,+the+non-cytotoxic+and+photostable+myosin+II+inhibitor.">para-Nitroblebbistatin, the non-cytotoxic and photostable myosin II inhibitor.</a> Angewandte Chemie International Edition. 53 (31), 8211-8215 (2014).</li><li>Varkuti, B. H., 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+highly+soluble,+non-phototoxic,+non-fluorescent+blebbistatin+derivative.">A highly soluble, non-phototoxic, non-fluorescent blebbistatin derivative.</a> Scientific Reports. 6, 26141 (2016).</li><li>Verhasselt, S., 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=Discovery+of+(S)-3'-hydroxyblebbistatin+and+(S)-3'-aminoblebbistatin:+polar+myosin+II+inhibitors+with+superior+research+tool+properties.">Discovery of (S)-3'-hydroxyblebbistatin and (S)-3'-aminoblebbistatin: polar myosin II inhibitors with superior research tool properties.</a> Organic and Biomolecular Chemistry. 15 (9), 2104-2118 (2017).</li><li>Verhasselt, S., Roman, B. I., Bracke, M. E., Stevens, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+synthesis+and+comparative+analysis+of+the+tool+properties+of+new+and+existing+D-ring+modified+(S)-blebbistatin+analogs.">Improved synthesis and comparative analysis of the tool properties of new and existing D-ring modified (S)-blebbistatin analogs.</a> European Journal of Medicinal Chemistry. 136, 85-103 (2017).</li><li>Warren, G. B., Toon, P. A., Birdsall, N. J., Lee, A. G., Metcalfe, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstitution+of+a+calcium+pump+using+defined+membrane+components.">Reconstitution of a calcium pump using defined membrane components.</a> Proceedings of the National Academy of Sciences of the United States of America. 71 (3), 622-626 (1974).</li><li>Kiianitsa, K., Solinger, J. A., Heyer, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad54+protein+exerts+diverse+modes+of+ATPase+activity+on+duplex+DNA+partially+and+fully+covered+with+Rad51+protein.">Rad54 protein exerts diverse modes of ATPase activity on duplex DNA partially and fully covered with Rad51 protein.</a> Journal of Biological Chemistry. 277 (48), 46205-46215 (2002).</li><li>Hanzelmann, P., Schindelin, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+Basis+of+ATP+Hydrolysis+and+Intersubunit+Signaling+in+the+AAA++ATPase+p97.">Structural Basis of ATP Hydrolysis and Intersubunit Signaling in the AAA+ ATPase p97.</a> Structure. 24 (1), 127-139 (2016).</li><li>Hackney, D. D., Jiang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assays+for+kinesin+microtubule-stimulated+ATPase+activity.">Assays for kinesin microtubule-stimulated ATPase activity.</a> Methods in Molecular Biology. 164, 65-71 (2001).</li><li>Kiianitsa, K., Solinger, J. A., Heyer, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADH-coupled+microplate+photometric+assay+for+kinetic+studies+of+ATP-hydrolyzing+enzymes+with+low+and+high+specific+activities.">NADH-coupled microplate photometric assay for kinetic studies of ATP-hydrolyzing enzymes with low and high specific activities.</a> Analytical Biochemistry. 321 (2), 266-271 (2003).</li><li>Carter, S. G., Karl, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inorganic+phosphate+assay+with+malachite+green:+an+improvement+and+evaluation.">Inorganic phosphate assay with malachite green: an improvement and evaluation.</a> Journal of Biochemical and Biophysical Methods. 7 (1), 7-13 (1982).</li><li>Henkel, R. D., VandeBerg, J. L., Walsh, R. 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+microassay+for+ATPase.">A microassay for ATPase.</a> Analytical Biochemistry. 169 (2), 312-318 (1988).</li><li>Rowlands, M. 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=High-throughput+screening+assay+for+inhibitors+of+heat-shock+protein+90+ATPase+activity.">High-throughput screening assay for inhibitors of heat-shock protein 90 ATPase activity.</a> Analytical Biochemistry. 327 (2), 176-183 (2004).</li><li>Rule, C. S., Patrick, M., Sandkvist, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+In+Vitro+ATPase+Activity+for+Enzymatic+Characterization.">Measuring In Vitro ATPase Activity for Enzymatic Characterization.</a> Journal of Visualized Experiments. (114), 54305 (2016).</li><li>Pardee, J. D., Spudich, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+of+muscle+actin.">Purification of muscle actin.</a> Methods in Cell Biology. 24, 271-289 (1982).</li><li>Zhang, J. H., Chung, T. D., Oldenburg, K. R. <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+Statistical+Parameter+for+Use+in+Evaluation+and+Validation+of+High+Throughput+Screening+Assays.">A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays.</a> Journal of Biomolecular Screening. 4 (2), 67-73 (1999).</li><li>Kovacs, M., Toth, J., Hetenyi, C., Malnasi-Csizmadia, A., Sellers, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+blebbistatin+inhibition+of+myosin+II.">Mechanism of blebbistatin inhibition of myosin II.</a> Chem Journal of Biological Chemistry. 279 (34), 35557-35563 (2004).</li><li>Allingham, J. S., Smith, R., Rayment, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structural+basis+of+blebbistatin+inhibition+and+specificity+for+myosin+II.">The structural basis of blebbistatin inhibition and specificity for myosin II.</a> Nature Structural &amp; Molecular Biology. 12 (4), 378-379 (2005).</li><li>Kettlun, A. 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=Purification+and+Characterization+of+2+Isoapyrases+from+Solanum-Tuberosum+Var+Ultimus.">Purification and Characterization of 2 Isoapyrases from Solanum-Tuberosum Var Ultimus.</a> Phytochemistry. 31 (11), 3691-3696 (1992).</li><li>Hulme, E. C., Trevethick, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand+binding+assays+at+equilibrium:+validation+and+interpretation.">Ligand binding assays at equilibrium: validation and interpretation.</a> British Journal of Pharmacology. 161 (6), 1219-1237 (2010).</li><li>Motulsky, H. J., Neubig, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+binding+data.">Analyzing binding data.</a> Current Protocols in Neuroscience. 52 (1), 7.5.1-7.5.65 (2010).</li><li>Sehgal, P., Olesen, C., Moller, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATPase+Activity+Measurements+by+an+Enzyme-Coupled+Spectrophotometric+Assay.">ATPase Activity Measurements by an Enzyme-Coupled Spectrophotometric Assay.</a> Methods in Molecular Biology. 1377, 105-109 (2016).</li><li>Solinger, J. A., Lutz, G., Sugiyama, T., Kowalczykowski, S. C., Heyer, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rad54+protein+stimulates+heteroduplex+DNA+formation+in+the+synaptic+phase+of+DNA+strand+exchange+via+specific+interactions+with+the+presynaptic+Rad51+nucleoprotein+filament.">Rad54 protein stimulates heteroduplex DNA formation in the synaptic phase of DNA strand exchange via specific interactions with the presynaptic Rad51 nucleoprotein filament.</a> Journal of Molecular Biology. 307 (5), 1207-1221 (2001).</li><li>Banik, U., Roy, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+continuous+fluorimetric+assay+for+ATPase+activity.">A continuous fluorimetric assay for ATPase activity.</a> Biochemistry Journal. 266 (2), 611-614 (1990).</li><li>Xiao, Y. X., Yang, W. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KIFC1:+a+promising+chemotherapy+target+for+cancer+treatment?.">KIFC1: a promising chemotherapy target for cancer treatment?.</a> Oncotarget. 7 (30), 48656-48670 (2016).</li><li>See, S. 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=Cytoplasmic+Dynein+Antagonists+with+Improved+Potency+and+Isoform+Selectivity.">Cytoplasmic Dynein Antagonists with Improved Potency and Isoform Selectivity.</a> ACS Chemical Biology. 11 (1), 53-60 (2016).</li><li>Datta, A., Brosh, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Insights+Into+DNA+Helicases+as+Druggable+Targets+for+Cancer+Therapy.">New Insights Into DNA Helicases as Druggable Targets for Cancer Therapy.</a> Frontiers in Molecular Biosciences. 5, 59 (2018).</li></ol>

Critical steps in the protocol
Optimize plate layout by running several plates with negative control only (ATPase reaction with no inhibitor). Inspect the results carefully for patterns in reaction rates. For example, these may arise from edge effects and/or imperfections in the hydrophilic surface coating of &#34;non-binding&#34; plates. If a pattern is observed, change plate type and/or plate layout to minimize the artifacts. For example, a typical dose-response curve (16 co...

Myosins are mechanochemical energy transducers that hydrolyze adenosine triphosphate (ATP) to generate directional movement along the filaments of the actin cytoskeleton in eukaryotes1,2. They have both structurally and kinetically adapted to their various intracellular functions, such as the transport of organelles, muscle contraction or the generation of cytoskeletal tension1,2. The myosin superfamily is represented by ~40 myosin genes belonging to ~12 distinct myosin classes in the human genome3,4. Members of the myosin classes play various roles in a highly diverse set of disorders, such as several cancers, neurological disorders, skeletal myopathies, and hypertrophic cardiomyopathy5,6. Given the large number of physiological and pathological functions of these molecular motors, it is not surprising that they are becoming increasingly recognized as drug targets for a variety of conditions7. Significant progress has been made recently in the discovery of new myosin inhibitors8,9,10 and activators11, and to improve the properties of existing ones12,13,14,15.
The nicotinamide adenine dinucleotide (NADH)-coupled ATPase assay has long been used to measure the ATPase activity of various enzymes, such as the sarcoplasmic reticulum Ca2+ pump ATPase16, the DNA repair ATPase Rad5417, the AAA+ ATPase p9718 or the microtubule motor kinesin19. The assay employs an ATP regeneration cycle. The adenosine diphosphate (ADP) generated by the ATPase is regenerated to ATP by pyruvate kinase (PK), which transforms one molecule of phosphoenolpyruvate (PEP) to pyruvate in parallel. Subsequently, pyruvate is reduced to lactate by lactate dehydrogenase (LDH). That, in turn, oxidizes one molecule of NADH to NAD. Therefore, the decrease in NADH concentration as a function of time equals the ATP hydrolysis rate. The ATP regeneration cycle keeps the ATP concentration nearly constant and the ADP concentration low as long as PEP is available. This results in linear time courses, making it simple to determine the initial reaction rates and helps to avoid product inhibition by ADP19. Although the NADH-coupled ATPase assay has already been adapted to a 96-well format20, the high reaction volumes (~150 &#181;L) make it relatively expensive due to the high demand of reagents, rendering it less amenable to rapid screening of large numbers of compounds. Alternative methods, such as the malachite green assay19,21, which relies on the detection of the phosphate produced by the ATPase enzyme, were proven more suitable for miniaturization and high-throughput screening22,23,24. However, an endpoint assay is more likely to be affected by several artifacts (discussed below), which may remain undiscovered in the absence of full-time courses.
Here, the NADH-coupled ATPase assay has been optimized for semi-high throughput screening of small molecule inhibitors. Skeletal and cardiac muscle myosin II&#39;s and the myosin inhibitors blebbistatin8, para-aminoblebbistatin13 and para-nitroblebbistatin12 are used to demonstrate the power of the assay, which relies on NADH fluorescence as a readout. This protocol is amenable to screening projects focused on any ADP producing enzymes.

<table border="0" cellpadding="0" cellspacing="0" style="width:956px;" width="955">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:515px;">384-well Low Flange Black Flat Bottom Polystyrene NBS Microplate</td>
			<td style="width:147px;">Corning</td>
			<td style="width:123px;">3575</td>
			<td style="width:172px;"></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">ATP (Adenosine 5&#8242;-triphosphate disodium salt hydrate)</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Aurora FRD-IB Dispenser</td>
			<td>Aurora Discovery, Inc.</td>
			<td>00017425</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Biomek NXP Multichannel Laboratory Automation Workstation</td>
			<td>Beckman Coulter</td>
			<td>A31841</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blebbistatin</td>
			<td style="width:147px;">AMRI</td>
			<td style="width:123px;">N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BSA (Bovine Serum Albumin, Protease-Free)</td>
			<td>Akron Biotech</td>
			<td>AK1391&#160;</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Centrifuge 5430 R, refrigerated, with Rotor FA-35-6-30</td>
			<td>Eppendorf</td>
			<td>022620663</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Centrifuge 5430, non-refrigerated, with Rotor A-2-MTP</td>
			<td>Eppendorf</td>
			<td>022620568</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO (Dimethyl sulfoxide)</td>
			<td>&#160;Sigma</td>
			<td>D2650</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">DTT (DL-Dithiothreitol)</td>
			<td>&#160;Sigma</td>
			<td>D5545</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">E1 ClipTip Multichannel Pipette; 384-format; 8-channel</td>
			<td>Thermo Scientific</td>
			<td>4672010</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">E1 ClipTip Multichannel Pipette; 96-format; 8-channel</td>
			<td>Thermo Scientific</td>
			<td>4672080</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">EGTA (Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid)</td>
			<td>&#160;Sigma</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">EnVision 2104 Multilabel Plate Reader</td>
			<td>PerkinElmer</td>
			<td>2104-0010</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Glycerol</td>
			<td>&#160;Sigma</td>
			<td>G2025</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">LDH (L-Lactic Dehydrogenase from rabbit muscle)</td>
			<td>Sigma</td>
			<td>L1254</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MgCl2.6H2O (Magnesium chloride hexahydrate)</td>
			<td>&#160;Sigma</td>
			<td>M2670</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Microplate Shaker</td>
			<td>VWR</td>
			<td>&#160; 12620-926&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microplate, 384 well, PP, Small Volume, Deep Well, Natural</td>
			<td>Greiner Bio-One</td>
			<td>784201</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">MOPS (3-(N-Morpholino)propanesulfonic acid)</td>
			<td>&#160;Sigma</td>
			<td>M1254</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Myosin Motor Protein (full length) (Bovine cardiac muscle)</td>
			<td>Cytoskeleton&#160;</td>
			<td>MY03</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Myosin Motor Protein (full length) (Rabbit skeletal muscle)</td>
			<td>Cytoskeleton&#160;</td>
			<td>MY02</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">NADH (&#946;-Nicotinamide adenine dinucleotide, reduced disodium salt hydrate)</td>
			<td>Sigma</td>
			<td>N8129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaN3 (Sodium azide)</td>
			<td>&#160;Sigma</td>
			<td>71289</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">NaOH (Sodium hydroxide)</td>
			<td>&#160;Sigma</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Optical Filter CFP 470/24nm (Emission)</td>
			<td>PerkinElmer</td>
			<td>2100-5850</td>
			<td>Barcode 240</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Optical Filter Fura2 380/10nm (Excitation)</td>
			<td>PerkinElmer</td>
			<td>2100-5390</td>
			<td>Barcode 112</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Optical Module: Beta Lactamase</td>
			<td>PerkinElmer</td>
			<td>2100-4270</td>
			<td>Barcode 418</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">OriginPro 2017 software</td>
			<td>OriginLab</td>
			<td style="width:123px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">para-Aminoblebbistatin</td>
			<td style="width:147px;">AMRI</td>
			<td style="width:123px;">N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">para-Nitroblebbistatin</td>
			<td style="width:147px;">AMRI</td>
			<td style="width:123px;">N/A</td>
			<td>Custom synthesis</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">PEP (Phospho(enol)pyruvic acid monopotassium salt)</td>
			<td>Sigma</td>
			<td>P7127</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">PK (Pyruvate Kinase from rabbit muscle)</td>
			<td>Sigma</td>
			<td>P9136</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:20px;">Rabbit Muscle Acetone Powder</td>
			<td>Pel Freez Biologicals</td>
			<td>41995-2</td>
			<td></td>
		</tr>
	</tbody>
</table>

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).

The typical plate layout map used for screening experiments is shown in Figure 1. The first and last rows are reserved for NADH calibration and positive control (20 &#181;M para-aminoblebbistatin, 0.5% DMSO), respectively. The remaining rows (B to O) are used to test the inhibitory activity of compounds. Here, fifteen-step serial 1:2 dilutions starting from 10 mM compound concentration in DMSO are prepared and transferred from the compound plate to t...

Watch this Scientific Journal Video about A Semi-High-Throughput Adaptation of the NADH-Coupled ATPase Assay for Screening Small Molecule Inhibitors at JoVE.com

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 enzyme.

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

This protocol is applicable to a wide range of screening projects aimed at developing ATPase inhibitors, either as probes for basic research or for potential clinical compounds. This method has been optimized for semi-high-throughput screening applications. It's also been designed to avoid several common artifacts and it doesn't require the handling of hazardous materials.In our laboratory we have been using this assay to develop new and specific non-muscle myosin II inhibitors for the treatment of methamphetamine use disorder. In theory, this method can easily be applied to any enzymes producing ATP. Visual demonstration helps to clarify all the important technical details.To begin, prepare 4, 500 microliters of 20 micromolar diluted actin solution for each 384-well black polystyrene microplate by diluting the concentrated actin stock solution in actin buffer. Mix the solution thoroughly by pipetting up and down 30 times to break actin filaments for reducing viscosity and heterogeneity. Then centrifuge the solution to remove any precipitated protein present.Carefully transfer the supernatant into a clean centrifuge tube. Prepare master enzyme mix in a 15-milliliter conical centrifuge tube for each assay plate by combining 3, 252.9 microliters of myosin buffer for the assay involving skeletal muscle myosin II, 171.4 microliters of LDH solution, and 171.4 microliters of PK solution. Prepare master substrate mix in a 15-milliliter conical centrifuge tube for each plate by combining 162.1 microliters of ATP, 162.1 microliters of PEP, and 324.1 microliters of NADH solution.Do not add actin at this point to avoid aggregation and precipitation. To create seven-step serial one-to-two dilutions of NADH for calibration, prepare eight 1.5-milliliter microcentrifuge tubes. Mix 12.3 microliters of NADH stock solution with 257.7 microliters of myosin buffer in the first tube to make a 250 micromolar solution.Then aliquot 135 microliters of myosin buffer into each of the remaining seven tubes. Transfer 135 microliters of the solution from the first tube into the second and mix by pipetting. Repeat until reaching the seventh tube.Use the last tube as no-NADH control with only buffer. Always include positive and negative controls on compound plates, and NADH dilutions for calibration on assay plates. These controls are extremely important for identifying artifacts during data analysis.Using an eight-channel pipette, transfer 20 microliters of the NADH calibration solutions into the first row in triplicates in an assay plate. Now add 4.2 microliters of skeletal muscle myosin II to the enzyme mix. Vortex briefly.Add myosin to the enzyme mix right before dispensing. Long incubation of diluted myosin solutions may result in loss of activity due to precipitation, and nonspecific binding to plastic surfaces. Load the prepared myosin enzyme mix into one of the sample containers of an automatic dispenser.In the plate, except for the first row, dispense 8.4 microliters of the myosin enzyme mix into each well of the assay plate. Then place the assay plate and the compound plate onto the labware positioners of an automated liquid handling system which is equipped with a 100-nanoliter pin tool head. Then run the appropriate method in the software to transfer 100 nanoliters of solutions from the prepared compound plate to the assay plate.Next, place the assay plate on a microplate shaker to shake for one minute at room temperature at 1, 200 rpm. Add 4, 052 microliters of the centrifuged actin solution to the substrate mix and vortex briefly. To start the enzymatic reaction, dispense 11.6 microliters of actin substrate mix into each well of the assay plate using an automatic dispenser, except the first row.On a microplate shaker, shake the assay plate for one minute at room temperature at 1, 200 rpm. Centrifuge the assay plate at 101 times g for 30 seconds. Use a 380-nanometer, 10-nanometer band width excitation filter, and 470 nanometer, 24-nanometer band width emission filter in conjunction with the 425-nanometer cutoff dichroic mirror for the assays.Optimize the number of flashes, detector gain, plate dimensions and measurement height before running the assays. Run the measurement in high concentration mode. Make sure that the inner temperature of the plate reader has stabilized at 25 degrees Celsius.Load the plate into the plate reader and shake for another 30 seconds to make the shape of the liquid surface similar in each well and allow the plate to reach measurement temperature. Scan the plate in 45-second intervals and record NADH fluorescence for 30 minutes. With the exported data, plot the observed fluorescence intensity against time for each well.Perform simple linear regression to determine the slope and intercept of the fluorescence responses for each well. The slope is proportional to the NADH consumption rate which can be used as a very good approximation of the ATP consumption rate. The intercept is proportional to the NADH concentration at the beginning of the measurement.Then, construct a calibration curve for NADH by plotting the intercepts obtained for the first row of the plate against the concentration of NADH. The intercepts estimate the real fluorescence intensities at the beginning with much more confidence than the average of the raw fluorescence intensity reads at the beginning. Perform simple linear regression to obtain the slope and intercept of the NADH calibration line.Here, the intercepts describes the fluorescence background signal with no NADH present, while the slope corresponds to the extrapolated theoretical fluorescence intensity of a one molar NADH solution. To convert fluorescence changes to ATP consumption rates divide the slope of the fluorescence response obtained for the rest of the wells by the slope of the NADH calibration line. Next, plot the ATP consumption rates against the concentration of the inhibitor.To determine the inhibitory constants, use any appropriate statistical software capable of nonlinear curve fitting, such as Origin 2017. Select nonlinear curve fit to fit the dose-response data to a quadratic equation corresponding to a simple one-to-one binding equilibrium model. Y is the ATP consumption rate.Y-minimum is the ATP consumption rate in the absence of inhibitor. Y-maximum is the theoretical ATP consumption rate at 100%inhibition. t and t are the total concentration of the myosin enzyme and inhibitor, respectively.KI is the inhibitory constant. In this study, skeletal and cardiac muscle myosin II ATPase reactions showed linear fluorescence intensity traces regardless of the myosin used or the presence of inhibitors. Basal and actin-activated ATPase rates showed linear dependency on the concentration of skeletal or cardiac muscle myosin II.A screening window coefficient or Z factor of 0.78 indicated a reliable assay with very well-separated positive and negative controls.Blebbistatin, para-Aminoblebbistatin, and para-Nitroblebbistatin showed regular dose-response curves at or below their reported solubility values. Inhibitory constants for both cardiac and skeletal muscle myosin IIs were determined by fitting a quadratic equation representing a simple equilibrium binding model to the data. Fluorescence intensity traces for blebbistatin obtained in an ATPase assay using skeletal muscle myosin II showed normal linearly decreasing signal depending on the amount of inhibitor present.However, above the solubility of blebbistatin an unexpected increase in the signal was observed most likely due to the formation of brightly florescent blebbistatin crystals. In the case of para-Nitroblebbistatin with normal raw fluorescence intensity traces, the reaction rates obtained above solubility diverged from the determined dose-response curve due to precipitation. By using this method, we have developed new, potent, and selective myosin inhibitors that might be used as scientific tools in basic research or as direct candidates in the future.It's always recommended to use a different ATPase or other functional assay specific to the enzyme of interest to distinguish between real and false positive hits. All proteins bind plastic surfaces. This problem can highly affect the results, especially if the protein is present at low concentrations.Always avoid unnecessary liquid handling, and use non-binding tubes. Alternative ATPase assays usually require the handling of sulfuric acid, toxic substances or radioactive materials. In contrast, the NADH-linked ATPase assay requires reagents with no or very low toxicity only.

a-semi-high-throughput-adaptation-nadh-coupled-atpase-assay-for

Research

JoVE Journal

Biochemistry

Identification of Small Molecule-binding Proteins in a Native Cellular Environment by Live-cell Photoaffinity Labeling

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several target identification methods have been developed and used to successfully elucidate the binding proteins of a variety of small molecules, these techniques have drawbacks that make them unsuitable for detecting certain types of small molecule-target interactions. In particular, non-covalent interactions that depend on native cellular conditions, such as those of membrane proteins whose structures may be perturbed upon cell lysis, are often not amenable to affinity-based target identification methods. Here, we demonstrate a method wherein a probe containing a photolabile group is used to covalently crosslink to the small molecule binding protein within the environment of the live cell, allowing the detection and isolation of the target protein without the need for maintenance of the interaction after cell lysis. This technique is a valuable tool for studying biologically interesting small molecules with unknown mechanisms, both in the context of basic biology as well as drug discovery.

We describe here a method for identification of small molecule-binding proteins using photoaffinity labeling. The advantage of this technique is that binding and covalent labeling of the target proteins occurs within the live cellular environment, removing the risk of disrupting native protein structure and binding conditions upon cell lysis.

Identifying the molecular target(s) of small molecules is a challenging but necessary step towards understanding their mechanism of action. While several ...

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

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

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

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

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

A Simple Method for High Throughput Chemical Screening in Caenorhabditis Elegans

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

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

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.

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.

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

Spectrophotometric Screening for Potential Inhibitors of Cytosolic Glutathione S-Transferases

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

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

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

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed extensive studies of cap-dependent translation and its regulation, high resolution kinetic characterization of this translation pathway is still lacking. Recently, we developed an in vitro assay to measure cap-dependent translation kinetics with single-molecule resolution. The assay is based on fluorescently labeled antibody binding to nascent epitope-tagged polypeptide. By imaging the binding and dissociation of antibodies to and from nascent peptide&#8211;ribosome&#8211;mRNA complexes, the translation progression on individual mRNAs can be tracked. Here, we present a protocol for establishing this assay, including mRNA and PEGylated slide preparations, real-time imaging of translation, and analysis of single molecule trajectories. This assay enables tracking of individual cap-dependent translation events and resolves key translation kinetics, such as initiation and elongation rates. The assay can be widely applied to distinct translation systems and should broadly benefit in vitro studies of cap-dependent translation kinetics and translational control mechanisms.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Development of a Lateral Flow Immunochromatographic Strip for Rapid and Quantitative Detection of Small Molecule Compounds

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, physiological index and clinical biomarker detection. In this protocol, we provide a detailed description of the steps to develop a rapid, sensitive and quantitative lateral-flow immunoassay (using AuNPs as a marker and mAbs as a probe). The procedure describes the preparation and characterization of colloidal gold, synthesis of the AuNP-mAb conjugate, assembly of the immunochromatographic strip, and methodological investigation of the assay. The results showed that the final strips can be further utilized for the rapid and convenient self-diagnosis of a small molecule, which may provide an alternative tool in the rapid and precise analysis of physiological and biological indices.

Membrane-based lateral flow immunochromatographic strips (ICSs) are useful tools for low-cost self-diagnosis and have been efficiently applied to toxin, ...

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) ...

Synthesis of Masarimycin, a Small Molecule Inhibitor of Gram-Positive Bacterial Growth

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. Central to understanding cell growth and division is the knowledge of how PG degradation influences biosynthesis and cell wall assembly. Recently, the metabolic labeling of PG through the introduction of modified sugars or amino acids has been reported. While chemical interrogation of biosynthetic steps with small molecule inhibitors is possible, chemical biology tools to study PG degradation by autolysins are underdeveloped. Bacterial autolysins are a broad class of enzymes that are involved in the tightly coordinated degradation of PG. Here, a detailed protocol is presented for preparing a small molecule probe, masarimycin, which is an inhibitor of N-acetylglucosaminidase LytG in Bacillus subtilis, and cell wall metabolism in Streptococcus pneumoniae. Preparation of the inhibitor via microwave-assisted and classical organic synthesis is provided. Its applicability as a tool to study Gram-positive physiology in biological assays is presented.

A detailed protocol is presented for preparing the bacteriostatic diamide masarimycin, a small molecule probe that inhibits the growth of Bacillus subtilis and Streptococcus pneumoniae by targeting cell wall degradation. Its application as a chemical probe is demonstrated in synergy/antagonism assays and morphological studies with B. subtilis and S. pneumoniae.

Peptidoglycan (PG) in the cell wall of bacteria is a unique macromolecular structure that confers shape, and protection from the surrounding environment. ...