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

NMR-based activity assays provide an effective method for identifying, evaluating, and validating inhibitors for a given enzyme. They are particularly well-suited for fragment screening. NMR assays are amenable to the higher compound concentrations required for weaker inhibitors.

In addition, the lack of reporter enzymes makes them less prone to false positives. We use the NMR assays in our lab to identify and characterize the inhibitors of two enzymes from Trichomonas vaginalis. Both enzymes represent new targets for novel anti-trichomonal drugs.

The NMR assays are generally applicable to any area of research that involves enzymes, and they can even be used to study the enzymes inside cells. The most important aspects of the technique are to ensure that the correct reagent volumes are pipetted and the reaction components are thoroughly mixed. To prepare the substrate and test compounds, add 12 microliters of substrate to each of four 1.5-milliliter microfuge tubes and add six microliters of deuterated DMSO to the zero-minute control and 30-minute control tubes.

Add six microliters of the test compound to the first experimental tube and three microliters of the test compound and three microliters of deuterated DMSO to the second experimental tube. Next, add 300 microliters of deuterium oxide to a 15-milliliter tube containing 2.59 milliliters of reaction buffer, followed by 25 microliters of enzyme solution. Gently invert the tube two times to mix reaction stock solution and add 10 microliters of 1.5 molar hydrochloric acid to a 1.5-milliliter tube containing 582 microliters of reaction stock solution.

Then, transfer the entire volume of reaction stock solution and acid to the zero-minute control tube to simultaneously initiate and quench the zero-minute control reaction. Aspirating and dispensing the sample two times in a slow but deliberate manner. Initiate and run the remaining three reactions in staggered fashion, immediately transferring 582 microliters of the reaction stock solution to the first experimental tube and mixing the sample two times as just demonstrated.

A careful aspiration and dispensing ensures a complete mixing during the initiation steps. Be consistent with each sample to ensure differential mixing is not a variable. After 30 seconds, transfer 582 microliters of reaction stock solution to the second experimental tube and mix, followed by the addition of another 582 microliters of reaction stock solution to the 30-minute control tube 30 seconds after initiating the reaction in the second experimental tube.

After 30 minutes, add 10 microliters of 1.5-molar hydrochloric acid to the first experimental tube, repeating the quenching at 30-second intervals for the second experimental and 30-minute control tubes. When all of the reactions have been quenched, transfer 600 microliters of solution from each reaction to NMR tubes. To calculate the percent conversion of substrate for the control spectra, load the samples onto the spectrometer, collect the NMR spectra, and overlay the spectra for the zero and 30-minute controls.

Scale the substrate signal in the zero-minute control to match the 30-minute control and note this percentage. Then, calculate the percent conversion as 100 minus the percentage from the matching of the substrate signals. To calculate the percent conversion of the substrate for the reactions containing the test compound, overlay the spectra for the zero-minute control and the first reaction containing the 500-micromolar test compound and scale the substrate signal in the zero-minute control to match the spectrum with the test compound.

Note this percentage, and use it to calculate the percent conversion as just demonstrated. Then, overlay the spectra for the zero-minute control and the first reaction containing the 250-micromolar test compound, and scale the substrate signal in the zero-minute control to match the spectrum with the test compound. After noting the percentage, use it to calculate the percent conversion.

To calculate the percent reaction and percent inhibition for each test compound concentration, first, calculate the percent reaction as the percent conversion of the test compound, divided by the percent conversion of the control times 100. Then calculate the percent inhibition as 100 minus the percent reaction. To perform a detergent counter screen assay, add 300 microliters of deuterium oxide and 25 microliters of enzyme solution to a 15-milliliter tube containing 2.59 milliliters of reaction buffer.

Gently invert the tube two times to mix the reaction stock solution, and add two microliters of Triton X-100 detergent to 20 milliliters of reaction buffer. Then, add 2.59 milliliters of reaction buffer, containing 0.01%Triton X-100 detergent to a new 15-milliliter conical tube. Then add 300 microliters of deuterium oxide and 25 microliters of enzyme solution to the tube.

Gently invert the tube two times to mix reaction stock solution. Then, determine the percent inhibition for the 100-micromolar and 50-micromolar test compounds as just demonstrated. Using the reaction stock solution without detergent, for one set of four tubes and the reaction stock solution with detergent for a second set of four tubes.

To perform a jump-dilution, transfer 468 microliters of reaction buffer and 60 microliters of deuterium oxide to a tube containing 53.8 microliters of reaction buffer, five microliters of enzyme solution, and 1.2 microliters of DMSO that has been incubating for 30 minutes. Aspirate and dispense the mixture two times in a slow, but deliberate manner. Before transferring 468 microliters of reaction buffer and 60 microliters of deuterium oxide to a tube containing 53.8 microliters of reaction buffer, five microliters of enzyme solution, and 1.2 microliters of test compound that has been incubating for 30 minutes.

Mix the second reaction two times as demonstrated. And immediately transfer 588 microliters of solution from the jump-dilution DMSO control tube to a tube containing 12 microliters of substrate with mixing. Continue transferring 588 microliters from the jump-dilution test compound tube, the undiluted DMSO control tube, and the undiluted test compound tube to each of three 1.5-milliliter tubes, containing 12 microliters of substrate per tube with mixing at 30-second intervals thereafter.

Then wait 30 minutes before quenching the reactions, collecting the NMR spectra, and calculating the percent inhibition for each compound as just demonstrated. Here the results for testing two compounds against AgNH in an initial test compound assay using proton NMR as demonstrated are shown. The enzyme reaction is most easily observed and quantified by the disappearance of the adenosine singlet and doublet resonances at 8.48 and 6.09 parts per million respectively.

And the appearance of an adenine singlet residence at 8.33 parts per million as observed in the 30-minute control spectrum. In this figure, the results for testing a compound at two concentrations against AgNH in a detergent counter-screen assay using proton NMR as demonstrated are shown. Only minimal differences are observed in the intensities of the substrate and product signals using the two conditions, indicating that the observed enzyme inhibition is not an artifact of compound aggregation.

Note that resonances arising from the tested compound and the detergent do not interfere with the substrate or product resonances. The results for testing a compound in a jump-dilution assay against AGNH using proton NMR as demonstrated reveal a reduced intensity of the substrate signal in the 20-micromolar reaction compared to the 200-micromolar reaction, indicating that the inhibition is reversible. A complete sample mixing during the initiation of each reaction and for the jump-dilutions is critical.

A careful and consistent aspiration and dispensing is required. NMR-based assays can also be used to measure the enzyme activity in whole cells, using a solution containing suspended cells in place of the reaction buffer and enzyme. Our anti-trichomonal drug discovery efforts continue to rely on these assays to measure the potency of newly synthesized compounds against the ribohydrolase enzymes.

Even though it is used in only very small quantities, the hydrochloric acid used to quench the reactions is hazardous and should be used with the proper personal protective equipment.