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

Podsumowanie

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.

Streszczenie

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).

Wprowadzenie

Myosins are mechanochemical energy transducers that hydrolyze adenosine triphosphate (ATP) to generate directional movement along the filaments of the actin cytoskeleton in eukaryotes^1,2. 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 tension^1,2. The myosin superfamily is represented by ~40 myosin genes belonging to ~12 distinct myosin classes in the human genome^3,4. 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 cardiomyopathy^5,6. 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 inhibitors^8,9,10 and activators¹¹, and to improve the properties of existing ones^12,13,14,15.

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 µL) 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 assay^19,21, which relies on the detection of the phosphate produced by the ATPase enzyme, were proven more suitable for miniaturization and high-throughput screening^22,23,24. 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'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.

Protokół

1. Preparing stock solutions and reagents

1. 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 °C.
2. 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 °C.
3. Prepare 10x NADH buffer containing 70 mM 3-(N-morpholino)propanesulfonic acid (MOPS), 10 mM MgCl₂, 0.9 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), and 3 mM NaN₃. Adjust the pH to 7.0 with 1 M NaOH solution. Store at 4 °C.
4. 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 °C. Add bovine serum albumin (BSA) and DTT to a final concentration of 0.1% (w/v%) and 1 mM, respectively, before use.
5. 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 °C. Add BSA and DTT to a final concentration of 0.1% (w/v%) and 1 mM, respectively, before use.
6. 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 °C.
7. Prepare PEP stock solution by dissolving crystalline PEP in 10x NADH buffer to a final concentration of 50 mM. Aliquot and store at -20 °C.
8. 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 °C, 10 min). Transfer the supernatant into a clean centrifuge tube carefully. Aliquot and store at -20 °C.
9. 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 °C, 10 min). Transfer the supernatant into a clean centrifuge tube carefully. Aliquot and store at -20 °C.
10. Reconstitute the lyophilized cardiac and skeletal muscle myosin II samples by adding 100 µL distilled water to obtain 10 mg/mL stock solutions corresponding to ~37.9 µM and ~40.8 µM myosin concentrations (monomeric), respectively. For further details, see manufacturer's instructions.
11. Prepare F-actin from rabbit muscle acetone powder as described by Pardee and Spudich²⁵.

2. Measuring ATPase activities and inhibitory effects of small molecule inhibitors

1. Prepare compound plate.
   1. Dissolve compounds of interest in high-quality dimethylsulfoxide (DMSO).
   2. Create fifteen-step serial 1:2 dilutions starting from 10 mM compound concentration in DMSO.
   3. Transfer the samples to a 384-well polypropylene plate in triplicates (12.5 µL 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.
   4. Transfer pure DMSO into the wells of the first row (reserved for NADH calibration).
   5. Use the last row for positive control.
      NOTE: Para-aminoblebbistatin at 4 mM concentration in DMSO was used here.
2. Prepare 4500 µL of 20 µM 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 °C, 10 min). Carefully transfer the supernatant into a clean centrifuge tube.
3. Prepare master mix containing LDH and PK enzymes ("enzyme mix"). For each assay plate, combine 171.4 µL of LDH solution, 171.4 µL of PK solution and 3189.3 µL or 3252.9 µL of myosin buffer for assays involving cardiac or skeletal muscle myosin II's, respectively, in a 15 mL conical centrifuge tube. Do not add any myosin at this point to avoid aggregation and precipitation.
4. Prepare master mix containing all substrates ("substrate mix"). For each plate, combine 162.1 µL of ATP, 162.1 µL of PEP and 324.1 µL of NADH solution in a 15 mL conical centrifuge tube. Do not add actin at this point to avoid aggregation and precipitation.
5. Create seven-step serial 1:2 dilutions of NADH for calibration starting from 250 µM.
   1. Mix 12.3 µL of NADH stock solution with 257.7 µL of myosin buffer in a 1.5 mL microcentrifuge tube.
   2. Aliquot 135 µL of myosin buffer into seven 1.5 mL microcentrifuge tubes.
   3. Transfer 135 µL of solution from the first tube into the second and mix by pipetting. Repeat until reaching the 7^th tube.
   4. Use the last tube as no-NADH control (buffer only).
6. Using an 8-channel pipette, transfer 20 µL of the NADH calibration solutions into the first row of the assay plate in triplicates.
7. Add 68 µL of cardiac or 4.2 µL of skeletal muscle myosin II to the enzyme mix. Vortex briefly.
8. Except the first row, dispense 8.4 µL of  the prepared myosin-enzyme mix into each well of the assay plate using an automated dispenser.
9. 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.
10. Shake the assay plate for 1 min at room temperature at 1200 rpm using a microplate shaker.
11. Add 4,052 µL of the centrifuged actin solution to the substrate mix. Vortex briefly.
12. Dispense 11.6 µL of actin-substrate mix into each well of the assay plate (except first row) to start the enzymatic reaction using an automated dispenser.
13. Shake the assay plate for 1 min at room temperature at 1200 rpm using a microplate shaker.
14. Centrifuge the assay plate at 101 x g for 30 s.
15. Make sure that the inner temperature of the plate reader has been stabilized at 25 ˚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.
16. 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 µM actin, 40 U/mL LDH, 200 U/mL PK, 220 µM 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 µL/well. The highest final compound concentration is 50 µM. 20 µM 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.

3. Analyzing data

1. Plot the observed fluorescence intensity against time for each well.
2. 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).
3. 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 ≈ 0 s.
4. 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.
5. 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.
6. Plot the ATP consumption rates against the concentration of the inhibitor.
7. 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:
   
   where Y is the ATP consumption rate, Y_min is the ATP consumption rate int the absence of inhibitor, Y_max is the theoretical ATP consumption rate at 100% inhibition, K_I is the inhibitory constant, [E]_t and [I]_t are the total concentration of the enzyme (myosin) and inhibitor, respectively.

Wyniki

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 µM 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...

Dyskusje

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 "non-binding" 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...

Materiały

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