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

This protocol is applicable to a wide range of screening projects aimed at developing ATPase inhibitors, either as probes for basic research or for potential clinical compounds. This method has been optimized for semi-high-throughput screening applications. It's also been designed to avoid several common artifacts and it doesn't require the handling of hazardous materials.

In our laboratory we have been using this assay to develop new and specific non-muscle myosin II inhibitors for the treatment of methamphetamine use disorder. In theory, this method can easily be applied to any enzymes producing ATP. Visual demonstration helps to clarify all the important technical details.

To begin, prepare 4, 500 microliters of 20 micromolar diluted actin solution for each 384-well black polystyrene microplate by diluting the concentrated actin stock solution in actin buffer. Mix the solution thoroughly by pipetting up and down 30 times to break actin filaments for reducing viscosity and heterogeneity. Then centrifuge the solution to remove any precipitated protein present.

Carefully transfer the supernatant into a clean centrifuge tube. Prepare master enzyme mix in a 15-milliliter conical centrifuge tube for each assay plate by combining 3, 252.9 microliters of myosin buffer for the assay involving skeletal muscle myosin II, 171.4 microliters of LDH solution, and 171.4 microliters of PK solution. Prepare master substrate mix in a 15-milliliter conical centrifuge tube for each plate by combining 162.1 microliters of ATP, 162.1 microliters of PEP, and 324.1 microliters of NADH solution.

Do not add actin at this point to avoid aggregation and precipitation. To create seven-step serial one-to-two dilutions of NADH for calibration, prepare eight 1.5-milliliter microcentrifuge tubes. Mix 12.3 microliters of NADH stock solution with 257.7 microliters of myosin buffer in the first tube to make a 250 micromolar solution.

Then aliquot 135 microliters of myosin buffer into each of the remaining seven tubes. Transfer 135 microliters of the solution from the first tube into the second and mix by pipetting. Repeat until reaching the seventh tube.

Use the last tube as no-NADH control with only buffer. Always include positive and negative controls on compound plates, and NADH dilutions for calibration on assay plates. These controls are extremely important for identifying artifacts during data analysis.

Using an eight-channel pipette, transfer 20 microliters of the NADH calibration solutions into the first row in triplicates in an assay plate. Now add 4.2 microliters of skeletal muscle myosin II to the enzyme mix. Vortex briefly.

Add myosin to the enzyme mix right before dispensing. Long incubation of diluted myosin solutions may result in loss of activity due to precipitation, and nonspecific binding to plastic surfaces. Load the prepared myosin enzyme mix into one of the sample containers of an automatic dispenser.

In the plate, except for the first row, dispense 8.4 microliters of the myosin enzyme mix into each well of the assay plate. Then place the assay plate and the compound plate onto the labware positioners of an automated liquid handling system which is equipped with a 100-nanoliter pin tool head. Then run the appropriate method in the software to transfer 100 nanoliters of solutions from the prepared compound plate to the assay plate.

Next, place the assay plate on a microplate shaker to shake for one minute at room temperature at 1, 200 rpm. Add 4, 052 microliters of the centrifuged actin solution to the substrate mix and vortex briefly. To start the enzymatic reaction, dispense 11.6 microliters of actin substrate mix into each well of the assay plate using an automatic dispenser, except the first row.

On a microplate shaker, shake the assay plate for one minute at room temperature at 1, 200 rpm. Centrifuge the assay plate at 101 times g for 30 seconds. Use a 380-nanometer, 10-nanometer band width excitation filter, and 470 nanometer, 24-nanometer band width emission filter in conjunction with the 425-nanometer cutoff dichroic mirror for the assays.

Optimize the number of flashes, detector gain, plate dimensions and measurement height before running the assays. Run the measurement in high concentration mode. Make sure that the inner temperature of the plate reader has stabilized at 25 degrees Celsius.

Load the plate into the plate reader and shake for another 30 seconds to make the shape of the liquid surface similar in each well and allow the plate to reach measurement temperature. Scan the plate in 45-second intervals and record NADH fluorescence for 30 minutes. With the exported data, plot the observed fluorescence intensity against time for each well.

Perform simple linear regression to determine the slope and intercept of the fluorescence responses for each well. The slope is proportional to the NADH consumption rate which can be used as a very good approximation of the ATP consumption rate. The intercept is proportional to the NADH concentration at the beginning of the measurement.

Then, construct a calibration curve for NADH by plotting the intercepts obtained for the first row of the plate against the concentration of NADH. The intercepts estimate the real fluorescence intensities at the beginning with much more confidence than the average of the raw fluorescence intensity reads at the beginning. Perform simple linear regression to obtain the slope and intercept of the NADH calibration line.

Here, the intercepts describes the fluorescence background signal with no NADH present, while the slope corresponds to the extrapolated theoretical fluorescence intensity of a one molar NADH solution. To convert fluorescence changes to ATP consumption rates divide the slope of the fluorescence response obtained for the rest of the wells by the slope of the NADH calibration line. Next, plot the ATP consumption rates against the concentration of the inhibitor.

To determine the inhibitory constants, use any appropriate statistical software capable of nonlinear curve fitting, such as Origin 2017. Select nonlinear curve fit to fit the dose-response data to a quadratic equation corresponding to a simple one-to-one binding equilibrium model. Y is the ATP consumption rate.

Y-minimum is the ATP consumption rate in the absence of inhibitor. Y-maximum is the theoretical ATP consumption rate at 100%inhibition. t and t are the total concentration of the myosin enzyme and inhibitor, respectively.

KI is the inhibitory constant. In this study, skeletal and cardiac muscle myosin II ATPase reactions showed linear fluorescence intensity traces regardless of the myosin used or the presence of inhibitors. Basal and actin-activated ATPase rates showed linear dependency on the concentration of skeletal or cardiac muscle myosin II.A screening window coefficient or Z factor of 0.78 indicated a reliable assay with very well-separated positive and negative controls.

Blebbistatin, para-Aminoblebbistatin, and para-Nitroblebbistatin showed regular dose-response curves at or below their reported solubility values. Inhibitory constants for both cardiac and skeletal muscle myosin IIs were determined by fitting a quadratic equation representing a simple equilibrium binding model to the data. Fluorescence intensity traces for blebbistatin obtained in an ATPase assay using skeletal muscle myosin II showed normal linearly decreasing signal depending on the amount of inhibitor present.

However, above the solubility of blebbistatin an unexpected increase in the signal was observed most likely due to the formation of brightly florescent blebbistatin crystals. In the case of para-Nitroblebbistatin with normal raw fluorescence intensity traces, the reaction rates obtained above solubility diverged from the determined dose-response curve due to precipitation. By using this method, we have developed new, potent, and selective myosin inhibitors that might be used as scientific tools in basic research or as direct candidates in the future.

It's always recommended to use a different ATPase or other functional assay specific to the enzyme of interest to distinguish between real and false positive hits. All proteins bind plastic surfaces. This problem can highly affect the results, especially if the protein is present at low concentrations.

Always avoid unnecessary liquid handling, and use non-binding tubes. Alternative ATPase assays usually require the handling of sulfuric acid, toxic substances or radioactive materials. In contrast, the NADH-linked ATPase assay requires reagents with no or very low toxicity only.