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 cr....

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 deuter....

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