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

Summary

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 μM and 250 μM, dose-response assays for determining IC₅₀ values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

Abstract

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 μM and 250 μM, 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 K_m value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

Introduction

Nuclear magnetic resonance (NMR) spectroscopy is well-established for characterizing and monitoring enzyme reactions^1,2. Differences in chemical shifts and coupling patterns are used to distinguish substrate and product resonances, and relative resonance intensities are used to quantify the percent of reaction. Both the consumption of substrate and the creation of product are directly observed in the NMR spectrum. This contrasts with spectrophotometry or fluorescence spectroscopy, in which the reaction time course is indicated by a change in absorbance attributable to some chemical species being consumed or created. Just as with the other methods, NMR can be used to study enzyme reactions as a function of temperature, pH, or other solution conditions, and the effects of inhibitors can be determined.

More recently, NMR-based enzyme activity assays have been demonstrated for fragment screening^3,4. NMR-based assays are ideally suited to work at the higher concentrations of test compounds (often as high as 1 mM) required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in the NMR experiment can easily resolve resonances from substrate, product, and test compounds. This compares favorably to spectrophotometric assays where 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 advantage makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays⁵.

In our research laboratory, NMR-based activity assays are used to identify and evaluate inhibitors of Trichomonas vaginalis nucleoside ribohydrolases. The T. vaginalis parasite causes the most prevalent non-viral sexually transmitted disease⁶. Increasing resistance to existing therapies⁷ is driving the need for novel, mechanism-based treatments, with essential nucleoside salvage pathway enzymes representing prime targets⁸. NMR-based activity assays have been developed for both pyrimidine- and purine-specific enzymes, uridine nucleoside ribohydrolase (UNH)⁹, and adenosine/guanosine preferring nucleoside ribohydrolase (AGNH)¹⁰. The reactions catalyzed by these two enzymes are shown in Figure 1. The NMR assays are being used to screen fragment libraries for chemical starting points, determine IC₅₀ values, and weed out aggregation-based or covalent binding inhibitors¹¹. The same assays are also being translated to assess enzyme activity in whole cells¹².

Detailed protocols are provided for initial compound assays at 500 μM and 250 μM, dose-response assays for determining IC₅₀ values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The protocols are generally applicable to any enzyme in which substrate and product resonances can be observed and distinguished by NMR spectroscopy. Three assumptions have been made for simplicity. First, the substrate is not specified. For NMR-based activity assays to be useful, the final concentration of substrate should be no more than 2-3x the K_m value⁴. In the examples shown, the final concentrations of adenosine and 5-fluorouridine are 100 μM (K_m = 54 μM) and 50 μM (K_m = 15 μM), respectively. In the protocols, achieving these concentrations corresponds to 12 μL of 5 mM adenosine or 12 μL of 2.5 mM 5-fluorouridine.

Second, the amount of enzyme provided for in the protocols, 5 μL, was chosen to correspond to the amount required to result in approximately 75% conversion of substrate to product in 30 min. This quantity typically represents a large dilution from a purified enzyme stock, and the dilution must be determined in advance for each enzyme. Purified AGNH and UNH enzyme stock solutions are stored at -80 °C in aliquots that provide enough enzyme for several thousand reactions. Thus, the dilution factor ideally only needs to be determined or validated every few months. Third, the specific 1D NMR experiment is not specified. In the representative results, ¹H NMR is shown for AGNH¹⁰ and ¹⁹F NMR is shown for UNH⁹, with the NMR experiment described in the corresponding references. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation. Finally, it should be pointed out that the experimental approach described does not adhere to the strict requirements of quantitative NMR (qNMR)^13,14. In the protocol, a percent reaction is determined using the relative changes in intensity of the same resonance in each spectrum, rather than by determining absolute concentrations. This approach eliminates the need for data acquisition and processing modifications as well as internal or external standards, which are required for qNMR.

Protocol

1. Initial test compound assays at 500 μM and 250 μM

1. Prepare substrate and test compound for reactions.
   1. Prepare stock solutions of substrate (adenosine or 5-fluorouridine) in water and 50 mM test compound in deuterated dimethyl sulfoxide (DMSO). Refer to the introduction section for concentrations of substrate solution to use.
   2. Add 12 μL of substrate (adenosine or 5-fluorouridine ) to each of four 1.5 mL microfuge tubes, 1–4.
   3. Add 6 μL of deuterated DMSO to tubes 1 (0 min control) and 4 (30 min control). Add 6 μL of test compound to tube 2. Add 3 μL of test compound and 3 μL of deuterated DMSO to tube 3.
2. Prepare sufficient reaction stock solution.
   NOTE: The stock solution is for five reactions that each contain 517 μL of buffer, 60 μL of deuterium oxide, and 5 μL of enzyme solution (AGNH or UNH). Refer to the introduction section for concentrations of enzyme solution to use.
   1. Add 2.59 mL of reaction buffer (50 mM potassium phosphate, 0.3 M KCl, pH 6.5) to a 15 mL conical tube. Add 300 μL of deuterium oxide to the conical tube. Add 25 μL of enzyme solution (AGNH or UNH) to the conical.
   2. Gently invert the conical tube twice to mix.
3. Simultaneously initiate and quench the 0 min control reaction. Transfer 582 μL of the reaction stock solution to a clean microfuge tube. Add 10 μL of 1.5 M HCl to this microfuge tube. Transfer the combined 592 μL to microfuge tube 1. Aspirate and dispense the sample twice in a slow but deliberate fashion.
4. Initiate and run the remaining three reactions in staggered fashion. At time 0 min, transfer 582 μL of the reaction stock solution to microfuge tube 2. Aspirate and dispense the sample twice in a slow but deliberate fashion. Repeat at 30 s intervals for microfuge tubes 3 and 4. Wait 30 min.
5. Quench the reactions. At time 30 min, add 10 μL of 1.5 M HCl to microfuge 2. Repeat at 30 s intervals for microfuge tubes 3 and 4. Transfer 600 μL of solution from each microfuge to NMR tubes.
6. Acquire a 1D NMR spectrum on each sample. Process the data to ensure correct phasing and flat baselines.
7. Calculate the percent conversion of substrate for control spectra.
   1. Overlay the spectra for 0 min and 30 min controls. Scale the substrate signal in the 0 min control to match the 30 min control. Note this percentage.
   2. Calculate the percent conversion as (100 – percentage determined in step 1.7.1).
8. Calculate the percent conversion of substrate for reactions containing test compound.
   1. Overlay spectra for the 0 min control and first reaction containing the 500 μM test compound. Scale the substrate signal in the 0 min control to match the spectrum with test compound. Note this percentage. Calculate percent conversion as (100 – percentage determined).
   2. Repeat for the second reaction containing the 250 μM test compound.
9. Calculate the percent reaction and percent inhibition for each test compound concentration.
   1. Calculate the percent reaction as (1.8.1/1.7.2) x 100.
   2. Calculate the percent inhibition as (100 – percentage determined in step 1.9.1).

2. Determination of IC₅₀ values

1. Prepare substrate and test compound for reactions.
   1. Prepare stock solutions of substrate (adenosine or 5-fluorouridine) in water and 10 mM test compound in deuterated DMSO. Refer to the introduction section for concentrations of substrate solution to use.
2. Prepare serial dilutions of 10 mM test compound (in deuterated DMSO).
   1. Add 36 μL deuterated DMSO to five 1.5 mL microfuge tubes, 1–5.
   2. Add 12 μL 10 mM test compound to tube 1 and tap lightly to mix. Test compound is now 2.5 mM.
   3. Transfer 12 μL from tube 1 to tube 2 and tap lightly to mix. Test compound is now 0.63 mM. Transfer 12 μL from tube 2 to tube 3 and tap lightly to mix. Test compound is now 0.16 mM. Transfer 12 μL from tube 3 to tube 4 and tap lightly to mix. Test compound is now 0.04 mM. Transfer 12 μL from tube 4 to tube 5 and tap lightly to mix. Test compound is now 0.01 mM.
3. Prepare 14 1.5 mL microfuge tubes for reactions.
   1. Add 12 μL of substrate (adenosine or 5-fluorouridine) to each of 14 1.5 mL microfuge tubes, 1-14.
   2. Add 12 μL of deuterated DMSO to tubes 1 (0 min control) and 14 (30 min control).
   3. Add 12 μL of 10 mM test compound to tubes 2 and 3. Add 12 μL of 2.5 mM test compound to tubes 4 and 5. Add 12 μL of 0.63 mM test compound to tubes 6 and 7. Add 12 μL of 0.16 mM test compound to tubes 8 and 9. Add 12 μL of 0.04 mM test compound to tubes 10 and 11. Add 12 μL of 0.01 mM test compound to tubes 12 and 13.
4. Prepare sufficient reaction stock solution to run 15 reactions that contain 511 μL of buffer, 60 μL of deuterium oxide, and 5 μL of enzyme solution (AGNH or UNH) each. Refer to the introduction section for concentrations of enzyme solution to use.
   1. Add 7.67 mL of reaction buffer (50 mM potassium phosphate, 0.3 M KCl, pH = 6.5) to a 15 mL conical tube. Add 900 μL of deuterium oxide to the conical tube. Add 75 μL of enzyme solution (AGNH or UNH) to the conical.
   2. Gently invert the conical tube 2x to mix.
5. Follow the steps outlined in steps 1.3–1.9 to (using 576 μL of reaction stock solution) to determine percent reaction for each test compound concentration.
6. Calculate IC₅₀ value using GraphPad Prism.
   1. Create a data table correlating the log of reaction test compound concentrations (200 μM, 50 μM, 12.5 μM, 3.13 μM, 0.78 μM, 0.20 μM) with percent reaction (two values each).
   2. Use a nonlinear curve fit to determine the IC₅₀ value and standard error.

3. Detergent Counter Screen Assays at 100 μM and 50 μM

1. Prepare substrate and test compound for reactions.
   1. Prepare stock solutions of substrate (adenosine or 5-fluorouridine) in water and 10 mM test compound in deuterated DMSO. Refer to the introduction section for concentrations of substrate solution to use.
   2. Add 12 μL of substrate (adenosine or 5-fluorouridine) to each of eight 1.5 mL microfuge tubes, 1–8.
   3. Add 6 μL of deuterated DMSO to tubes 1 and 5 (0 min controls) and 4 and 8 (30 min controls). Add 6 μL of test compound to tubes 2 and 6. Add 3 μL of test compound and 3 μL of deuterated DMSO to tubes 3 and 7.
2. Prepare sufficient reaction stock solution WITHOUT detergent to run five reactions that contain 517 μL of buffer, 60 μL of deuterium oxide, and 5 μL of enzyme solution (AGNH or UNH) each. Refer to the introduction section for concentrations of enzyme solution to use.
   1. Add 2.59 mL of reaction buffer (50 mM potassium phosphate, 0.3 M KCl, pH = 6.5) to a 15 mL conical tube. Add 300 μL of deuterium oxide to the conical tube. Add 25 μL of enzyme solution (AGNH or UNH) to the conical.
   2. Gently invert the conical tube 2x to mix.
3. Prepare sufficient reaction stock solution WITH 0.01% v/v Triton X-100 detergent to run five reactions that contain 517 μL of buffer, 60 μL of deuterium oxide, and 5 μL of enzyme solution (AGNH or UNH) each.
   1. Add 2 μL of Triton X-100 detergent to 20 mL of reaction buffer (50 mM potassium phosphate, 0.3 M KCl, pH = 6.5). Detergent solutions must be used within 1 day.
   2. Add 2.59 mL of reaction buffer containing 0.01% Triton X-100 detergent to a 15 mL conical tube. Add 300 μL of deuterium oxide to the conical tube. Add 25 μL of enzyme solution (AGNH or UNH) to the conical.
   3. Gently invert the conical tube 2x to mix.
4. For tubes 1–4, using the reaction stock solution WITHOUT detergent, follow the steps outlined in steps 1.3–1.9 to determine the percent inhibition for each test compound concentration.
5. For tubes 5–8, using the reaction stock solution WITH 0.01% v/v Triton X-100 detergent, follow the steps outlined in steps 1.3–1.9 to determine the percent inhibition for each test compound concentration.

4. Jump-dilution counter screen assays

1. Prepare the substrate and test compounds for reactions
   1. Prepare stock solutions of substrate (adenosine or 5-fluorouridine) in water and 10 mM test compound in deuterated DMSO. Refer to the introduction section for concentrations of substrate solution to use.
   2. Add 53.8 μL of reaction buffer (50 mM potassium phosphate, 0.3 M KCl, pH = 6.5) to two 1.5 mL microfuge tubes (each), 1 and 2. Add 5 μL of enzyme (AGNH or UNH) to tubes 1 and 2.
   3. Add 511 μL of reaction buffer to two 1.5 mL microfuge tubes, 3 and 4 (each). Add 60 μL of deuterium oxide to tubes 3 and 4. Add 5 μL of enzyme (AGNH or UNH) to tubes 3 and 4.
   4. Add 1.2 μL of deuterated DMSO to tube 1 (30 min control). Add 1.2 μL of test compound to tube 2. Add 12 μL of deuterated DMSO to tube 3 (30 min control). Add 12 μL of test compound to tube 4. Incubate for 30 min.
2. Prepare two 1.5 mL microfuge tubes with dilution solution.
   1. Add 468 μL of reaction buffer to each of two 1.5 mL microfuge tubes, 5 and 6. Add 60 μL of deuterium oxide to each tube (5 and 6).
3. Prepare four 1.5 mL microfuge tubes for the reactions.
   1. Add 12 μL of substrate (adenosine or 5-fluorouridine) to each of four 1.5 mL microfuge tubes, 7–10.
4. Perform jump-dilutions and initiate the reactions.
   1. Transfer 528 μL of solution from tube 5 to tube 1. Aspirate and dispense the sample twice in a slow but deliberate fashion. Transfer 528 μL of solution from tube 6 to tube 2. Aspirate and dispense the sample twice in a slow but deliberate fashion.
   2. At time 0 min, transfer 588 μL of solution from tube 1 to tube 7. Aspirate and dispense the sample 2x in a slow but deliberate fashion. At 30 s intervals, transfer 588 μL of solution from tube 2 to tube 8, tube 3 to tube 9, and tube 4 to tube 10. Aspirate and dispense the sample twice in a slow but deliberate fashion. Wait 30 min.
5. For tubes 7–10, follow the steps outlined in steps 1.5–1.9 to determine the percent inhibition for each test compound concentration.

5. Compound assays in E. coli whole cells

1. Prepare 10 mL overnight culture of E. coli on day preceding experiments.
2. Prepare E. coli cells for NMR experiments.
   1. Centrifuge the cells in 1 mL aliquots for 10 min at 15,000 x g.
   2. Discard the supernatant and resuspend each aliquot of cells in 1 mL of reaction buffer (50 mM potassium phosphate, 0.3 M KCl, pH = 6.5) by vortexing.
3. Follow sections 1 or 2 for the desired assay with the following changes:
   1. Substitute 280 μL of buffer in the reaction stock solution with 280 μL of resuspended cells.
   2. Substitute 5 μL of enzyme (AGNH or UNH) in the reaction stock solution with 5 μL of buffer.

Results

Figure 2 shows the results for testing two compounds against AGNH using ¹H NMR following section 1. The enzyme reaction is most easily observed and quantified by the disappearance of adenosine singlet and doublet resonances at 8.48 ppm and 6.09 ppm, respectively, and the appearance of an adenine singlet resonance at 8.33 ppm as observed in the 30 min control spectrum. In the presence of 500 μM compound 1, no product is formed as evidenced by the lack of an adenine resonance at 8.3...

Discussion

The protocols described are generally applicable to many enzymes, provided that the substrates and/or products have resolvable signals in the NMR spectrum. However, it is critical that the concentration of substrate is close to its K_m value and high enough to be detected in an NMR experiment within a reasonable timeframe. A substrate concentration no higher than 2-3x the K_m value is optimal for detecting competitive, noncompetitive, and uncompetitive inhibitors⁴

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
AGNH	Purified in-house	N/A	TVAG_213720
UNH	Purified in-house	N/A	TVAG_092730
Adenosine	Sigma	A9251
5-Fluorouridine	Sigma	F5130
Dimethyl sulfoxide-D6	Cambridge Isotope Labs	DLM-10-100	D, 99.9%
Potassium phosphate monobasic	Sigma	P0662
Potassium phosphate dibasic	Sigma	P3786
Potassium chloride	Sigma	P9541
Deuterium oxide	Cambridge Isotope Labs	DLM-4-100	D, 99.9%
Hydrochloric acid	Fisher Chemical	A144212
Triton X-100	Sigma	X100
3-(Trimethylsilyl)propionic-2,2,3,3,-d4 acid sodium salt (TSP)	Sigma	269913	D, 98%
2,2,2-Trifluoroethanol-1,1-d2	Sigma	612197	D, 99.5%
Pipette	Gilson	F123602	PIPETMAN Classic P1000
Pipette	Gilson	F123601	PIPETMAN Classic P200
Pipette	Gilson	F123600	PIPETMAN Classic P20
Microfuge tubes	Fisher Scientific	05-408-129
Conical tubes	Corning	352099
Microcentrifuge	Eppendorf	5418
Vortex mixer	Fisher Scientific	02215365
NMR tubes	Norell	502-7	Or as appropriate for the NMR
NMR spectrometer	Bruker	N/A	AvanceIII500
Prism software	GraphPad	N/A	Version 5.04