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-morpholino)propanesulfonic acid (MOPS), 10 mM MgCl2, 0.9 mM ethylene glycol-bis(&#946;-aminoethyl ether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA), and 3 mM NaN3. Adjust the pH to 7.0 with 1 M NaOH solution. Store at 4 &#176;C.</li>
	<li>Prepare 1x myosin buffer containing 10 mM MOPS and 0.1 mM EGTA. Adjust the pH to 7.0 with 1 M NaOH solution. Store at 4 &#176;C. Add bovine serum albumin (BSA) and DTT to a final concentration of 0.1% (w/v%) and 1 mM, respectively, before use.</li>
	<li>Prepare 1x actin buffer containing 4 mM MOPS, 0.1 mM EGTA, 2 mM MgCl2, and 3 mM NaN3. Adjust the pH to 7.0 with 1 M NaOH solution. Store at 4 &#176;C. Add BSA and DTT to a final concentration of 0.1% (w/v%) and 1 mM, respectively, before use.</li>
	<li>Prepare NADH stock solution by dissolving crystalline NADH in 10x NADH buffer to a final concentration of 5.5 mM. Aliquot and store at -20 &#176;C.</li>
	<li>Prepare PEP stock solution by dissolving crystalline PEP in 10x NADH buffer to a final concentration of 50 mM. Aliquot and store at -20 &#176;C.</li>
	<li>Prepare LDH stock solution by dissolving lyophilized LDH powder in a mixture of glycerol and 10x NADH buffer (50%:50%) to a final concentration of 2000 U/mL. Centrifuge the solution to remove any undissolved protein present (7,197 x g, 20 &#176;C, 10 min). Transfer the supernatant into a clean centrifuge tube carefully. Aliquot and store at -20 &#176;C.</li>
	<li>Prepare PK stock solution by dissolving lyophilized PK powder in a mixture of glycerol and 10x NADH buffer (50%:50%) to a final concentration of 10000 U/mL. Centrifuge the solution to remove any undissolved protein present (7,197 x g, 20 &#176;C, 10 min). Transfer the supernatant into a clean centrifuge tube carefully. Aliquot and store at -20 &#176;C.</li>
	<li>Reconstitute the lyophilized cardiac and skeletal muscle myosin II samples by adding 100 &#181;L distilled water to obtain 10 mg/mL stock solutions corresponding to ~37.9 &#181;M and ~40.8 &#181;M myosin concentrations (monomeric), respectively. For further details, see manufacturer&#39;s instructions.</li>
	<li>Prepare F-actin from rabbit muscle acetone powder as described by Pardee and Spudich25.</li>
</ol>
2. Measuring ATPase activities and inhibitory effects of small molecule inhibitors
<ol>
	<li>Prepare compound plate.
	<ol>
		<li>Dissolve compounds of interest in high-quality dimethylsulfoxide (DMSO).</li>
		<li>Create fifteen-step serial 1:2 dilutions starting from 10 mM compound concentration in DMSO.</li>
		<li>Transfer the samples to a 384-well polypropylene plate in triplicates (12.5 &#181;L each) using a multichannel pipette. Use two rows on the compound plate for one compound (instead of three columns) to minimize the number of wells potentially affected by edge effects. Use the last three wells in the second row for each compound as negative control (DMSO only). Do not use the first and the last row on the plate for compound dilutions.</li>
		<li>Transfer pure DMSO into the wells of the first row (reserved for NADH calibration).</li>
		<li>Use the last row for positive control. 
		NOTE: Para-aminoblebbistatin at 4 mM concentration in DMSO was used here.</li>
	</ol>
	</li>
	<li>Prepare 4500 &#181;L of 20 &#181;M diluted actin solution for each assay plate (384-well black-wall polystyrene microplate) by diluting actin stock solution in actin buffer. Mix the solution thoroughly by pipetting up and down 30x using a 5 mL pipette to reduce viscosity and heterogeneity by breaking actin filaments. Centrifuge the solution to remove any precipitated protein present (7,197 x g, 20 &#176;C, 10 min). Carefully transfer the supernatant into a clean centrifuge tube.</li>
	<li>Prepare master mix containing LDH and PK enzymes (&#34;enzyme mix&#34;). For each assay plate, combine 171.4 &#181;L of LDH solution, 171.4 &#181;L of PK solution and 3189.3 &#181;L or 3252.9 &#181;L of myosin buffer for assays involving cardiac or skeletal muscle myosin II&#39;s, respectively, in a 15 mL conical centrifuge tube. Do not add any myosin at this point to avoid aggregation and precipitation.</li>
	<li>Prepare master mix containing all substrates (&#34;substrate mix&#34;). For each plate, combine 162.1 &#181;L of ATP, 162.1 &#181;L of PEP and 324.1 &#181;L of NADH solution in a 15 mL conical centrifuge tube. Do not add actin at this point to avoid aggregation and precipitation.</li>
	<li>Create seven-step serial 1:2 dilutions of NADH for calibration starting from 250 &#181;M.
	<ol>
		<li>Mix 12.3 &#181;L of NADH stock solution with 257.7 &#181;L of myosin buffer in a 1.5 mL microcentrifuge tube.</li>
		<li>Aliquot 135 &#181;L of myosin buffer into seven 1.5 mL microcentrifuge tubes.</li>
		<li>Transfer 135 &#181;L of solution from the first tube into the second and mix by pipetting. Repeat until reaching the 7th tube.</li>
		<li>Use the last tube as no-NADH control (buffer only).</li>
	</ol>
	</li>
	<li>Using an 8-channel pipette, transfer 20 &#181;L of the NADH calibration solutions into the first row of the assay plate in triplicates.</li>
	<li>Add 68 &#181;L of cardiac or 4.2 &#181;L of skeletal muscle myosin II to the enzyme mix. Vortex briefly.</li>
	<li>Except the first row, dispense 8.4 &#181;L of&#160; the prepared myosin-enzyme mix into each well of the assay plate using an automated dispenser.</li>
	<li>Transfer 100 nL of solutions from the compound plate to the assay plate containing enzyme mix using an automated liquid handling system equipped with a 100 nL pin tool head.</li>
	<li>Shake the assay plate for 1 min at room temperature at 1200 rpm using a microplate shaker.</li>
	<li>Add 4,052 &#181;L of the centrifuged actin solution to the substrate mix. Vortex briefly.</li>
	<li>Dispense 11.6 &#181;L of actin-substrate mix into each well of the assay plate (except first row) to start the enzymatic reaction using an automated dispenser.</li>
	<li>Shake the assay plate for 1 min at room temperature at 1200 rpm using a microplate shaker.</li>
	<li>Centrifuge the assay plate at 101 x g for 30 s.</li>
	<li>Make sure that the inner temperature of the plate reader has been stabilized at 25 &#730;C. Load the plate and shake for another 30 s. This shaking step is necessary to make the shape of the liquid surface similar in each well and allows time for the plate to reach measurement temperature.</li>
	<li>Record NADH fluorescence for 30 min scanning the plate in 45 s intervals. Use a 380 nm, 10 nm bandwidth excitation filter and a 470 nm, 24 nm bandwidth emission filter in conjunction with a 425 nm cut-off dichroic mirror. Run the measurement in high-concentration mode. Optimize the number of flashes, detector gain, plate dimensions and measurement height before running the assays. 
	NOTE: Final assay conditions are 300 nM cardiac/20 nM skeletal muscle myosin II, 10 &#181;M actin, 40 U/mL LDH, 200 U/mL PK, 220 &#181;M NADH, 1 mM PEP, 1 mM ATP in a buffer containing 10 mM MOPS (pH = 7.0), 2 mM MgCl2, 0.15 mM EGTA, 0.1 mg/mL BSA, 0.5% (v/v) DMSO and 1 mM DTT. The total volume is 20 &#181;L/well. The highest final compound concentration is 50 &#181;M. 20 &#181;M para-aminoblebbistatin in 0.5% DMSO serves as the positive control and 0.5% DMSO alone is the negative control. All measurements are carried out in triplicates.</li>
</ol>
3. Analyzing data
<ol>
	<li>Plot the observed fluorescence intensity against time for each well.</li>
	<li>Perform simple linear regression to determine the slope and intercept of the fluorescence responses for each well. The slope is proportional to the ATP (NADH) consumption rate, while the intercept is proportional to the NADH concentration at the beginning of the measurement (t = 0 s).</li>
	<li>Construct a calibration curve for NADH by plotting the intercepts obtained for the first row of the plate against the concentration of NADH. Make sure that the intercepts depend linearly on the NADH concentration. 
	NOTE: The intercepts estimate the real fluorescence intensities at t = 0 s with much more confidence than the average of the raw fluorescence intensity reads at t &#8776; 0 s.</li>
	<li>Perform simple linear regression to obtain the slope and intercept of the NADH calibration line. 
	NOTE: The intercept describes the fluorescence background signal (no NADH present), while the slope corresponds to the extrapolated/theoretical fluorescence intensity of a 1 M NADH solution in that particular experiment.</li>
	<li>Divide the slope of the fluorescence response obtained for the rest of the wells by the slope of the NADH calibration line to convert fluorescence changes to ATP consumption rates.</li>
	<li>Plot the ATP consumption rates against the concentration of the inhibitor.</li>
	<li>To determine inhibitory constants, use appropriate statistical software to fit the dose-response data to the following quadratic equation corresponding to a simple one-to-one binding equilibrium model: 
	<img alt="Equation 1" src="/files/ftp_upload/60017/60017eq01.jpg" style="opacity: 0.9;" /> 
	where Y is the ATP consumption rate, Ymin is the ATP consumption rate int the absence of inhibitor, Ymax is the theoretical ATP consumption rate at 100% inhibition, KI is the inhibitory constant, [E]t and [I]t are the total concentration of the enzyme (myosin) and inhibitor, respectively.</li>
</ol>

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The authors have nothing to disclose.

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

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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 ...

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Research

JoVE Journal

Biochemistry

9.5K Views.  The Scripps Research Institute. Dit protocol is van toepassing op een breed scala van screening projecten gericht op de ontwikkeling van ATPase remmers, hetzij als sondes voor fundamenteel onderzoek of voor potentiële klinische verbindingen. Deze methode is geoptimaliseerd voor semi-high-throughput screening toepassingen. Het is ook ontworpen om verschillende gemeenschappelijke artefacten te vermijden en het vereist niet de behandeling van gevaarlijke stoffen.In ons laboratorium hebben we deze test gebruikt om nieuw...

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

Use of Stopped-Flow Fluorescence and Labeled Nucleotides to Analyze the ATP Turnover Cycle of Kinesins

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. Kinesins display differences across the superfamily both in ATP turnover and in microtubule interaction. These differences tailor specific kinesins to various functions such as cargo transport, microtubule sliding, microtubule depolymerization and microtubule stabilization. To understand the mechanism of action of a kinesin it is important to understand how the chemical cycle of ATP turnover is coupled to the mechanical cycle of microtubule interaction. To dissect the ATP turnover cycle, one approach is to utilize fluorescently labeled nucleotides to visualize individual steps in the cycle. Determining the kinetics of each nucleotide transition in the ATP turnover cycle allows the rate-limiting step or steps for the complete cycle to be identified. For a kinesin, it is important to know the rate-limiting step, in the absence of microtubules, as this step is generally accelerated several thousand fold when the kinesin interacts with microtubules. The cycle in the absence of microtubules is then compared to that in the presence of microtubules to fully understand a kinesin&#8217;s ATP turnover cycle. The kinetics of individual nucleotide transitions are generally too fast to observe by manually mixing reactants, particularly in the presence of microtubules. A rapid mixing device, such as a stopped-flow fluorimeter, which allows kinetics to be observed on timescales of as little as a few milliseconds, can be used to monitor such transitions. Here, we describe protocols in which rapid mixing of reagents by stopped-flow is used in conjunction with fluorescently labeled nucleotides to dissect the ATP turnover cycle of a kinesin.

Kinesins are characterized by nucleotide-dependent interaction with microtubules: a cycle of ATP turnover coupled to a cycle of microtubule interaction. Here, we describe protocols to analyze the kinetics of individual nucleotide transitions in the ATP turnover cycle of a kinesin using fluorescently labeled nucleotides and stopped-flow fluorescence.

The kinesin superfamily of microtubule associated motor proteins share a characteristic motor domain which both hydrolyses ATP and binds microtubules. ...

Chemistry

Drug-induced Sensitization of Adenylyl Cyclase: Assay Streamlining and Miniaturization for Small Molecule and siRNA Screening Applications

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse and Parkinson&#39;s disease. Acute activation of G&#945;i/o-linked receptors inhibits AC activity, whereas persistent activation of these receptors results in heterologous sensitization of AC and increased levels of intracellular cAMP. Previous studies have demonstrated that this enhancement of AC responsiveness is observed both in vitro and in vivo following the chronic activation of several types of G&#945;i/o-linked receptors including D2 dopamine and &#956; opioid receptors. Although heterologous sensitization of AC was first reported four decades ago, the mechanism(s) that underlie this phenomenon remain largely unknown. The lack of mechanistic data presumably reflects the complexity involved with this adaptive response, suggesting that nonbiased approaches could aid in identifying the molecular pathways involved in heterologous sensitization of AC. Previous studies have implicated kinase and Gb&#947; signaling as overlapping components that regulate the heterologous sensitization of AC. To identify unique and additional overlapping targets associated with sensitization of AC, the development and validation of a scalable cAMP sensitization assay is required for greater throughput. Previous approaches to study sensitization are generally cumbersome involving continuous cell culture maintenance as well as a complex methodology for measuring cAMP accumulation that involves multiple wash steps. Thus, the development of a robust cell-based assay that can be used for high throughput screening (HTS) in a 384&#160;well format would facilitate future studies. Using two D2 dopamine receptor cellular models (i.e.&#160;CHO-D2L and HEK-AC6/D2L), we have converted our 48-well sensitization assay (&#62;20 steps 4-5 days) to a five-step, single day assay in 384-well format. This new format is amenable to small molecule screening, and we demonstrate that this assay design can also be readily used for reverse transfection of siRNA in anticipation of targeted siRNA library screening.

Persistent activation of inhibitory G protein-coupled receptors results in sensitization of adenylyl cyclase signaling. To identify the essential molecular pathways, nonbiased approaches are necessary; however, this strategy requires the development of a scalable cell-based cAMP sensitization assay. Herein, we describe a sensitization assay for small molecule and siRNA screening.

Sensitization of adenylyl cyclase (AC) signaling has been implicated in a variety of neuropsychiatric and neurologic disorders including substance abuse ...

Bioengineering

Click-Chemistry Based Fluorometric Assay for Apolipoprotein N-acyltransferase from Enzyme Characterization to High-Throughput Screening

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral membrane enzymes, resulting in triacylated proteins. The first step in the lipoprotein modification pathway involves the transfer of a diacylglyceryl group from phosphatidylglycerol onto the prolipoprotein, resulting in diacylglyceryl prolipoprotein. In the second step, the signal peptide of prolipoprotein is cleaved, forming an apolipoprotein, which in turn is modified by a third fatty acid derived from a phospholipid. This last step is catalyzed by apolipoprotein N-acyltransferase (Lnt). The lipoprotein modification pathway is essential in most &#947;-proteobacteria, making it a potential target for the development of novel antibacterial agents. Described here is a sensitive assay for Lnt that is compatible with high-throughput screening of small inhibitory molecules. The enzyme and substrates are membrane-embedded molecules; therefore, the development of an in vitro test is not straightforward. This includes the purification of the active enzyme in the presence of detergent, the availability of alkyne-phospholipids and diacylglyceryl peptide substrates, and the reaction conditions in mixed micelles. Furthermore, in order to use the activity test in a high-throughput screening (HTS) setup, direct readout of the reaction product is preferred over coupled enzymatic reactions. In this fluorometric enzyme assay, the alkyne-triacylated peptide product is rendered fluorescent through a click-chemistry reaction and detected in a multiwell plate format. This method is applicable to other acyltransferases that use fatty acid-containing substrates, including phospholipids and acyl-CoA.

Presented here is a sensitive fluorescence assay to monitor apolipoprotein N-acyltransferase activity using diacylglyceryl peptide and alkyne-phospholipids as substrates with click-chemistry.

Lipoproteins from proteobacteria are posttranslationally modified by fatty acids derived from membrane phospholipids by the action of three integral ...

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

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 ...

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

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

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

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

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

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

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

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

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

Development of a Click-Based Assay for Screening HAT1 Enzyme Activity

An Acetyl-Click Chemistry Assay to Measure Histone Acetyltransferase 1 Acetylation

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome assembly. It is required for tumor growth in various systems, making it a potential target for cancer treatment. To facilitate the identification of compounds that can inhibit HAT1 enzymatic activity, we have devised an acetyl-click assay for rapid screening. In this simple assay, we employ recombinant HAT1/Rbap46, which is purified from activated human cells. The method utilizes the acetyl-CoA analog 4-pentynoyl-CoA (4P) in a click-chemistry approach. This involves the enzymatic transfer of an alkyne handle through a HAT1-dependent acylation reaction to a biotinylated H4 N-terminal peptide. The captured peptide is then immobilized on neutravidin plates, followed by click-chemistry functionalization with biotin-azide. Subsequently, streptavidin-peroxidase recruitment is employed to oxidize amplex red, resulting in a quantitative fluorescent output. By introducing chemical inhibitors during the acylation reaction, we can quantify enzymatic inhibition based on a reduction of the fluorescence signal. Importantly, this reaction is scalable, allowing for high throughput screening of potential inhibitors for HAT1 enzymatic activity.

Author Spotlight: Developing Acetyl-Click Assay for HAT1 Inhibitor Screening

Quick and accurate chemical assays to screen for specific inhibitors are an important tool in the drug development arsenal. Here, we present a scalable acetyl-click chemistry assay to measure the inhibition of HAT1 acetylation activity.

HAT1, also known as Histone acetyltransferase 1, plays a crucial role in chromatin synthesis by stabilizing and acetylating nascent H4 before nucleosome ...

High-throughput Screening for Small-molecule Modulators of Inward Rectifier Potassium Channels

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, atrial fibrillation, and pain1,2. For the most part, however, progress toward understanding their therapeutic potential or even basic physiological functions has been slowed by the lack of good pharmacological tools. Indeed, the molecular pharmacology of the inward rectifier family has lagged far behind that of the S4 superfamily of voltage-gated potassium (Kv) channels, for which a number of nanomolar-affinity and highly selective peptide toxin modulators have been discovered3. The bee venom toxin tertiapin and its derivatives are potent inhibitors of Kir1.1 and Kir3 channels4,5, but peptides are of limited use therapeutically as well as experimentally due to their antigenic properties and poor bioavailability, metabolic stability and tissue penetrance. The development of potent and selective small-molecule probes with improved pharmacological properties will be a key to fully understanding the physiology and therapeutic potential of Kir channels.
The Molecular Libraries Probes Production Center Network (MLPCN) supported by the National Institutes of Health (NIH) Common Fund has created opportunities for academic scientists to initiate probe discovery campaigns for molecular targets and signaling pathways in need of better pharmacology6. The MLPCN provides researchers access to industry-scale screening centers and medicinal chemistry and informatics support to develop small-molecule probes to elucidate the function of genes and gene networks. The critical step in gaining entry to the MLPCN is the development of a robust target- or pathway-specific assay that is amenable for high-throughput screening (HTS).
Here, we describe how to develop a fluorescence-based thallium (Tl+) flux assay of Kir channel function for high-throughput compound screening7,8,9,10.The assay is based on the permeability of the K+ channel pore to the K+ congener Tl+. A commercially available fluorescent Tl+ reporter dye is used to detect transmembrane flux of Tl+ through the pore. There are at least three commercially available dyes that are suitable for Tl+ flux assays: BTC, FluoZin-2, and FluxOR7,8. This protocol describes assay development using FluoZin-2. Although originally developed and marketed as a zinc indicator, FluoZin-2 exhibits a robust and dose-dependent increase in fluorescence emission upon Tl+ binding. We began working with FluoZin-2 before FluxOR was available7,8 and have continued to do so9,10. However, the steps in assay development are essentially identical for all three dyes, and users should determine which dye is most appropriate for their specific needs. We also discuss the assay's performance benchmarks that must be reached to be considered for entry to the MLPCN. Since Tl+ readily permeates most K+ channels, the assay should be adaptable to most K+ channel targets.

Methods for developing and validating a quantitative fluorescence assay for measuring the activity of inward rectifier potassium (Kir) channels for high-throughput compound screening is presented.

Specific members of the inward rectifier potassium (Kir) channel family are postulated drug targets for a variety of disorders, including hypertension, ...

Biology

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays focus on transcriptional regulation, being essentially aimed at the identification of promoter-targeting molecules. As a result, post-transcriptional mechanisms are largely uncovered by gene expression targeted drug development. Here we describe a cell-based assay aimed at investigating the role of the 3&#39; untranslated region (3&#8217; UTR) in the modulation of the fate of its mRNA, and at identifying compounds able to modify it. The assay is based on the use of a luciferase reporter construct containing the 3&#8217; UTR of a gene of interest stably integrated into a disease-relevant cell line. The protocol is divided into two parts, with the initial focus on the primary screening aimed at the identification of molecules affecting luciferase activity after 24 hr of treatment. The second part of the protocol describes the counter-screening necessary to discriminate compounds modulating luciferase activity specifically through the 3&#8217; UTR. In addition to the detailed protocol and representative results, we provide important considerations about the assay development and the validation of the hit(s) on the endogenous target. The described cell-based reporter gene assay will allow scientists to identify molecules modulating protein levels via post-transcriptional mechanisms dependent on a 3&#8217; UTR.

Here we describe a cell-based reporter gene assay as a valuable tool to screen chemical libraries for compounds modulating post-transcriptional control mechanisms exerted through 3&#8217; UTR.

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

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

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