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 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 ...

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

Research

JoVE Journal

Biochemistry

9.4K Views.  The Scripps Research Institute. 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 µL per well. The platform should be applicable to virtually any ADP producing enzyme.

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

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 ...

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. ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...