We thank Dr. Dean Brown for providing compounds from the AstraZeneca fragment library and Dr. David Parkin for providing the AGNH and UNH enzymes. Research reported in this publication was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health under Award Number R15AI128585 to B. J. S. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.Research was also supported by a Horace G. McDonell Summer Research Fellowship awarded to S. N. M., a Landesberg Family Fellowship awarded to J. A. G., and Faculty Development Grants and Frederick Bettelheim Research Award from Adelphi University to B. J. S.

1. Initial test compound assays at 500 &#956;M and 250 &#956;M
<ol>
	<li>Prepare substrate and test compound for reactions.
	<ol>
		<li>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.</li>
		<li>Add 12 &#956;L of substrate (adenosine or 5-fluorouridine ) to each of four 1.5 mL microfuge tubes, 1&#8211;4.</li>
		<li>Add 6 &#956;L of deuterated DMSO to tubes 1 (0 min control) and 4 (30 min control). Add 6 &#956;L of test compound to tube 2. Add 3 &#956;L of test compound and 3 &#956;L of deuterated DMSO to tube 3.</li>
	</ol>
	</li>
	<li>Prepare sufficient reaction stock solution. 
	NOTE: The stock solution is for five reactions that each contain 517 &#956;L of buffer, 60 &#956;L of deuterium oxide, and 5 &#956;L of enzyme solution (AGNH or UNH). Refer to the introduction section for concentrations of enzyme solution to use.
	<ol>
		<li>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 &#956;L of deuterium oxide to the conical tube. Add 25 &#956;L of enzyme solution (AGNH or UNH) to the conical.</li>
		<li>Gently invert the conical tube twice to mix.</li>
	</ol>
	</li>
	<li>Simultaneously initiate and quench the 0 min control reaction. Transfer 582 &#956;L of the reaction stock solution to a clean microfuge tube. Add 10 &#956;L of 1.5 M HCl to this microfuge tube. Transfer the combined 592 &#956;L to microfuge tube 1. Aspirate and dispense the sample twice in a slow but deliberate fashion.</li>
	<li>Initiate and run the remaining three reactions in staggered fashion. At time 0 min, transfer 582 &#956;L 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.</li>
	<li>Quench the reactions. At time 30 min, add 10 &#956;L of 1.5 M HCl to microfuge 2. Repeat at 30 s intervals for microfuge tubes 3 and 4. Transfer 600 &#956;L of solution from each microfuge to NMR tubes.</li>
	<li>Acquire a 1D NMR spectrum on each sample. Process the data to ensure correct phasing and flat baselines.</li>
	<li>Calculate the percent conversion of substrate for control spectra.
	<ol>
		<li>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.</li>
		<li>Calculate the percent conversion as (100 &#8211; percentage determined in step 1.7.1).</li>
	</ol>
	</li>
	<li>Calculate the percent conversion of substrate for reactions containing test compound.
	<ol>
		<li>Overlay spectra for the 0 min control and first reaction containing the 500 &#956;M 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 &#8211; percentage determined).</li>
		<li>Repeat for the second reaction containing the 250 &#956;M test compound.</li>
	</ol>
	</li>
	<li>Calculate the percent reaction and percent inhibition for each test compound concentration.
	<ol>
		<li>Calculate the percent reaction as (1.8.1/1.7.2) x 100.</li>
		<li>Calculate the percent inhibition as (100 &#8211; percentage determined in step 1.9.1).</li>
	</ol>
	</li>
</ol>
2. Determination of IC50 values
<ol>
	<li>Prepare substrate and test compound for reactions.
	<ol>
		<li>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.</li>
	</ol>
	</li>
	<li>Prepare serial dilutions of 10 mM test compound (in deuterated DMSO).
	<ol>
		<li>Add 36 &#956;L deuterated DMSO to five 1.5 mL microfuge tubes, 1&#8211;5.</li>
		<li>Add 12 &#956;L 10 mM test compound to tube 1 and tap lightly to mix. Test compound is now 2.5 mM.</li>
		<li>Transfer 12 &#956;L from tube 1 to tube 2 and tap lightly to mix. Test compound is now 0.63 mM. Transfer 12 &#956;L from tube 2 to tube 3 and tap lightly to mix. Test compound is now 0.16 mM. Transfer 12 &#956;L from tube 3 to tube 4 and tap lightly to mix. Test compound is now 0.04 mM. Transfer 12 &#956;L from tube 4 to tube 5 and tap lightly to mix. Test compound is now 0.01 mM.</li>
	</ol>
	</li>
	<li>Prepare 14 1.5 mL microfuge tubes for reactions.
	<ol>
		<li>Add 12 &#956;L of substrate (adenosine or 5-fluorouridine) to each of 14 1.5 mL microfuge tubes, 1-14.</li>
		<li>Add 12 &#956;L of deuterated DMSO to tubes 1 (0 min control) and 14 (30 min control).</li>
		<li>Add 12 &#956;L of 10 mM test compound to tubes 2 and 3. Add 12 &#956;L of 2.5 mM test compound to tubes 4 and 5. Add 12 &#956;L of 0.63 mM test compound to tubes 6 and 7. Add 12 &#956;L of 0.16 mM test compound to tubes 8 and 9. Add 12 &#956;L of 0.04 mM test compound to tubes 10 and 11. Add 12 &#956;L of 0.01 mM test compound to tubes 12 and 13.</li>
	</ol>
	</li>
	<li>Prepare sufficient reaction stock solution to run 15 reactions that contain 511 &#956;L of buffer, 60 &#956;L of deuterium oxide, and 5 &#956;L of enzyme solution (AGNH or UNH) each. Refer to the introduction section for concentrations of enzyme solution to use.
	<ol>
		<li>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 &#956;L of deuterium oxide to the conical tube. Add 75 &#956;L of enzyme solution (AGNH or UNH) to the conical.</li>
		<li>Gently invert the conical tube 2x to mix.</li>
	</ol>
	</li>
	<li>Follow the steps outlined in steps 1.3&#8211;1.9 to (using 576 &#956;L of reaction stock solution) to determine percent reaction for each test compound concentration.</li>
	<li>Calculate IC50 value using GraphPad Prism.
	<ol>
		<li>Create a data table correlating the log of reaction test compound concentrations (200 &#956;M, 50 &#956;M, 12.5 &#956;M, 3.13 &#956;M, 0.78 &#956;M, 0.20 &#956;M) with percent reaction (two values each).</li>
		<li>Use a nonlinear curve fit to determine the IC50 value and standard error.</li>
	</ol>
	</li>
</ol>
3. Detergent Counter Screen Assays at 100 &#956;M and 50 &#956;M
<ol>
	<li>Prepare substrate and test compound for reactions.
	<ol>
		<li>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.</li>
		<li>Add 12 &#956;L of substrate (adenosine or 5-fluorouridine) to each of eight 1.5 mL microfuge tubes, 1&#8211;8.</li>
		<li>Add 6 &#956;L of deuterated DMSO to tubes 1 and 5 (0 min controls) and 4 and 8 (30 min controls). Add 6 &#956;L of test compound to tubes 2 and 6. Add 3 &#956;L of test compound and 3 &#956;L of deuterated DMSO to tubes 3 and 7.</li>
	</ol>
	</li>
	<li>Prepare sufficient reaction stock solution WITHOUT detergent to run five reactions that contain 517 &#956;L of buffer, 60 &#956;L of deuterium oxide, and 5 &#956;L of enzyme solution (AGNH or UNH) each. Refer to the introduction section for concentrations of enzyme solution to use.
	<ol>
		<li>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 &#956;L of deuterium oxide to the conical tube. Add 25 &#956;L of enzyme solution (AGNH or UNH) to the conical.</li>
		<li>Gently invert the conical tube 2x to mix.</li>
	</ol>
	</li>
	<li>Prepare sufficient reaction stock solution WITH 0.01% v/v Triton X-100 detergent to run five reactions that contain 517 &#956;L of buffer, 60 &#956;L of deuterium oxide, and 5 &#956;L of enzyme solution (AGNH or UNH) each.
	<ol>
		<li>Add 2 &#956;L 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.</li>
		<li>Add 2.59 mL of reaction buffer containing 0.01% Triton X-100 detergent to a 15 mL conical tube. Add 300 &#956;L of deuterium oxide to the conical tube. Add 25 &#956;L of enzyme solution (AGNH or UNH) to the conical.</li>
		<li>Gently invert the conical tube 2x to mix.</li>
	</ol>
	</li>
	<li>For tubes 1&#8211;4, using the reaction stock solution WITHOUT detergent, follow the steps outlined in steps 1.3&#8211;1.9 to determine the percent inhibition for each test compound concentration.</li>
	<li>For tubes 5&#8211;8, using the reaction stock solution WITH 0.01% v/v Triton X-100 detergent, follow the steps outlined in steps 1.3&#8211;1.9 to determine the percent inhibition for each test compound concentration.</li>
</ol>
4. Jump-dilution counter screen assays
<ol>
	<li>Prepare the substrate and test compounds for reactions
	<ol>
		<li>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.</li>
		<li>Add 53.8 &#956;L 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 &#956;L of enzyme (AGNH or UNH) to tubes 1 and 2.</li>
		<li>Add 511 &#956;L of reaction buffer to two 1.5 mL microfuge tubes, 3 and 4 (each). Add 60 &#956;L of deuterium oxide to tubes 3 and 4. Add 5 &#956;L of enzyme (AGNH or UNH) to tubes 3 and 4.</li>
		<li>Add 1.2 &#956;L of deuterated DMSO to tube 1 (30 min control). Add 1.2 &#956;L of test compound to tube 2. Add 12 &#956;L of deuterated DMSO to tube 3 (30 min control). Add 12 &#956;L of test compound to tube 4. Incubate for 30 min.</li>
	</ol>
	</li>
	<li>Prepare two 1.5 mL microfuge tubes with dilution solution.
	<ol>
		<li>Add 468 &#956;L of reaction buffer to each of two 1.5 mL microfuge tubes, 5 and 6. Add 60 &#956;L of deuterium oxide to each tube (5 and 6).</li>
	</ol>
	</li>
	<li>Prepare four 1.5 mL microfuge tubes for the reactions.
	<ol>
		<li>Add 12 &#956;L of substrate (adenosine or 5-fluorouridine) to each of four 1.5 mL microfuge tubes, 7&#8211;10.</li>
	</ol>
	</li>
	<li>Perform jump-dilutions and initiate the reactions.
	<ol>
		<li>Transfer 528 &#956;L of solution from tube 5 to tube 1. Aspirate and dispense the sample twice in a slow but deliberate fashion. Transfer 528 &#956;L of solution from tube 6 to tube 2. Aspirate and dispense the sample twice in a slow but deliberate fashion.</li>
		<li>At time 0 min, transfer 588 &#956;L 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 &#956;L 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.</li>
	</ol>
	</li>
	<li>For tubes 7&#8211;10, follow the steps outlined in steps 1.5&#8211;1.9 to determine the percent inhibition for each test compound concentration.</li>
</ol>
5. Compound assays in E. coli whole cells
<ol>
	<li>Prepare 10 mL overnight culture of E. coli on day preceding experiments.</li>
	<li>Prepare E. coli cells for NMR experiments.
	<ol>
		<li>Centrifuge the cells in 1 mL aliquots for 10 min at 15,000 x g.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Follow sections 1 or 2 for the desired assay with the following changes:
	<ol>
		<li>Substitute 280 &#956;L of buffer in the reaction stock solution with 280 &#956;L of resuspended cells.</li>
		<li>Substitute 5 &#956;L of enzyme (AGNH or UNH) in the reaction stock solution with 5 &#956;L of buffer.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Biomolecular NMR spectroscopy. , (1995).</li><li>Dalvit, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+NMR+method+for+rapid,+efficient,+and+reliable+biochemical+screening.">A general NMR method for rapid, efficient, and reliable biochemical screening.</a> Journal of the American Chemical Society. 125, 14620-14625 (2003).</li><li>Dalvit, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-+and+substrate-based+19F+NMR+screening:+Principles+and+applications+to+drug+discovery.">Ligand- and substrate-based 19F NMR screening: Principles and applications to drug discovery.</a> Progress in NMR Spectroscopy. 51, 243-271 (2007).</li><li>Stockman, B. 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N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ligand-efficient+inhibitors+of+Trichomonas+vaginalis+adenosine/guanosine+preferring+nucleoside+ribohydrolase.">Ligand-efficient inhibitors of Trichomonas vaginalis adenosine/guanosine preferring nucleoside ribohydrolase.</a> ACS Infectious Diseases. 5, 345-352 (2019).</li><li>Veronesi, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorine+nuclear+magnetic+resonance-based+assay+in+living+mammalian+cells.">Fluorine nuclear magnetic resonance-based assay in living mammalian cells.</a> Analytical Biochemistry. 495, 52-59 (2016).</li><li>Holzgrabe, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+NMR+spectroscopy+in+pharmaceutical+applications.">Quantitative NMR spectroscopy in pharmaceutical applications.</a> Progress in NMR Spectroscopy. 57, 229-240 (2010).</li><li>Bharti, S. 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J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=2-Fluoro-ATP+as+a+versatile+tool+for+19F+NMR-based+activity+screening.">2-Fluoro-ATP as a versatile tool for 19F NMR-based activity screening.</a> Journal of the American Chemical Society. 130, 5870-5871 (2008).</li><li>Aldrich, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ecstasy+and+agony+of+assay+interference+compounds.">The ecstasy and agony of assay interference compounds.</a> ACS Central Science. 3, 143-147 (2017).</li><li>Feng, B. Y., Shoichet, B. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+detergent-based+assay+for+the+detection+of+promiscuous+inhibitors.">A detergent-based assay for the detection of promiscuous inhibitors.</a> Nature Protocols. 1, 550-553 (2006).</li><li>Copeland, R. A., Basavapathruni, A., Moyer, M., Scott, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+enzyme+concentration+and+residence+time+on+apparent+activity+recovery+in+jump+dilution+analysis.">Impact of enzyme concentration and residence time on apparent activity recovery in jump dilution analysis.</a> Analytical Biochemistry. , 206-210 (2011).</li><li>Griveta, J. -. P., Delort, A. -. 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The authors have nothing to disclose.

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 Km value and high enough to be detected in an NMR experiment within a reasonable timeframe. A substrate concentration no higher than 2-3x the Km value is optimal for detecting competitive, noncompetitive, and uncompetitive inhibitors4</...

Nuclear magnetic resonance (NMR) spectroscopy is well-established for characterizing and monitoring enzyme reactions1,2. 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 screening3,4. 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 &#956;M and 250 &#956;M, 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 Km value4. In the examples shown, the final concentrations of adenosine and 5-fluorouridine are 100 &#956;M (Km = 54 &#956;M) and 50 &#956;M (Km = 15 &#956;M), respectively. In the protocols, achieving these concentrations corresponds to 12 &#956;L of 5 mM adenosine or 12 &#956;L of 2.5 mM 5-fluorouridine.
Second, the amount of enzyme provided for in the protocols, 5 &#956;L, 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 &#176;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.

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

nmr-based activity assays for determining compound inhibition, ic50 values, artifactual activity, and whole-cell activity of nucleoside ribohydrolases

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 &#956;M and 250 &#956;M, 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 Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

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 &#956;M compound 1, no product is formed as evidenced by the lack of an adenine resonance a...

Watch this Scientific Journal Video about NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases at JoVE

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

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 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

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

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

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Research

JoVE Journal

Chemistry

9.9K Views.  Adelphi University. Op NMR gebaseerde activiteitstesten bieden een effectieve methode voor het identificeren, evalueren en valideren van remmers voor een bepaald enzym. Ze zijn bijzonder geschikt voor fragmentscreening. NMR-tests zijn vatbaar voor de hogere samengestelde concentraties die nodig zijn voor zwakkere remmers.Bovendien maakt het gebrek aan reporterenzymen ze minder gevoelig voor false positives. We gebruiken de NMR-testen in ons lab om de remmers van twee enzymen uit Trichomonas vaginalis te i...

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

In Vitro Transcription Assays and Their Application in Drug Discovery

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process requires multi-subunit DNA-dependent RNA polymerase (RNAP) and a series of transcription factors that act to modulate the activity of RNAP during gene expression. Sequencing gel electrophoresis of radiolabeled transcripts is used to provide detailed mechanistic information on how transcription proceeds and what parameters can affect it. In this paper we describe the protocol to study how the essential elongation factor NusA regulates transcriptional pausing, as well as a method to identify an antibacterial agent targeting transcription initiation through inhibition of RNAP holoenzyme formation. These methods can be used a as platform for the development of additional approaches to explore the mechanism of action of the transcription factors which still remain unclear, as well as new antibacterial agents targeting transcription which is an underutilized drug target in antibiotic research and development.

In Vitro Transcription Assays and Their Application in Drug Discovery

In this manuscript, we describe a protocol to functionally examine transcription and the inhibitory activity of antibacterial agents targeting bacterial transcription.

In vitro transcription assays have been developed and widely used for many years to study the molecular mechanisms involved in transcription. This process ...

Genetics

Kinetic Screening of Nuclease Activity using Nucleic Acid Probes

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. Nucleases display a variety of vital physiological roles in prokaryotic and eukaryotic organisms, ranging from maintaining genome stability to providing protection against pathogens. Altered nuclease activity has been associated with several pathological conditions including bacterial infections and cancer. To this end, nuclease activity has shown great potential to be exploited as a specific biomarker. However, a robust and reproducible screening method based on this activity remains highly desirable.
Herein, we introduce a method that enables screening for nuclease activity using nucleic acid probes as substrates, with the scope of differentiating between pathological and healthy conditions. This method offers the possibility of designing new probe libraries, with increasing specificity, in an iterative manner. Thus, multiple rounds of screening are necessary to refine the probes&#39; design with enhanced features, taking advantage of the availability of chemically modified nucleic acids. The considerable potential of the proposed technology lies in its flexibility, high reproducibility, and versatility for the screening of nuclease activity associated with disease conditions. It is expected that this technology will allow the development of promising diagnostic tools with a great potential in the clinic.

Altered nuclease activity has been associated with different human conditions, underlying its potential as a biomarker. The modular and easy to implement screening methodology presented in this paper allows the selection of specific nucleic acid probes for harnessing nuclease activity as a biomarker of disease.

Nucleases are a class of enzymes that break down nucleic acids by catalyzing the hydrolysis of the phosphodiester bonds that link the ribose sugars. ...

Biochemistry

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

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).

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 &#181;L per well. The platform should be applicable to virtually any ADP producing enzyme.

ATPase enzymes utilize the free energy stored in adenosine triphosphate to catalyze a wide variety of endergonic biochemical processes in vivo that would ...

High-throughput Screening for Broad-spectrum Chemical Inhibitors of RNA Viruses

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat because of increased worldwide exchanges and human populations penetrating more and more natural ecosystems. A good example of such an emerging situation is chikungunya virus epidemics of 2005-2006 in the Indian Ocean. Recent progresses in our understanding of cellular pathways controlling viral replication suggest that compounds targeting host cell functions, rather than the virus itself, could inhibit a large panel of RNA viruses. Some broad-spectrum antiviral compounds have been identified with host target-oriented assays. However, measuring the inhibition of viral replication in cell cultures using reduction of cytopathic effects as a readout still represents a paramount screening strategy. Such functional screens have been greatly improved by the development of recombinant viruses expressing reporter enzymes capable of bioluminescence such as luciferase. In the present report, we detail a high-throughput screening pipeline, which combines recombinant measles and chikungunya viruses with cellular viability assays, to identify compounds with a broad-spectrum antiviral profile.

In vitro assays to measure virus replication have been greatly improved by the development of recombinant RNA viruses expressing luciferase or other enzymes capable of bioluminescence. Here we detail a high-throughput screening pipeline that combines such recombinant strains of measles and chikungunya viruses to isolate broad-spectrum antivirals from chemical libraries.

RNA viruses are responsible for major human diseases such as flu, bronchitis, dengue, Hepatitis C or measles. They also represent an emerging threat ...

Immunology and Infection

Rapid Screening of HIV Reverse Transcriptase and Integrase Inhibitors

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify new compounds that can inhibit infection by the common drug resistant HIV-1 strains with minimal toxicity. Here we describe an efficient assay that can be used to rapidly determine the cellular cytotoxicity and efficacy of a compound against WT and mutant viral strains.
The desired target cell line is seeded in a 96-well plate and, after a 24 hr incubation, serially dilutions of the compounds to be tested are added. No further manipulations are necessary for cellular cytotoxicity assays; for anti HIV assays a predetermined amount of either a WT or drug resistant HIV-1 vector that expresses luciferase is added to the cells. Cytotoxicity is measured by using an ATP dependent luminescence assay and the impact of the compounds on infectivity is measured by determining the amount of luciferase in the presence or the absence of the putative inhibitors.
This screening assay takes 4 days to complete and multiple compounds can be screened in parallel. Compounds are screened in triplicate and the data are normalized to the infectivity/ATP levels in absence of target compounds. This technique provides a quick and accurate measurement of the efficacy and toxicity of potential anti HIV compounds.

Here we describe cellular cytotoxicity and single round infectivity assays that allow for the rapid and accurate screening of compounds to determine their cellular cytotoxicity (CC50) and IC50 values against WT and drug resistant HIV-1.

Although a number of anti HIV drugs have been approved, there are still problems with toxicity and drug resistance. This demonstrates a need to identify ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread of multi drug-resistant TB (MDR-TB), there is a real urgency to develop new therapeutic strategies against M. tuberculosis infections. Traditionally, compounds are evaluated based on their antibacterial activity under in vitro growth conditions in broth; however, results are often misleading for intracellular pathogens like M. tuberculosis since in-broth phenotypic screening conditions are significantly different from the actual disease conditions within the human body. Screening for inhibitors that work inside macrophages has been traditionally difficult due to the complexity, variability in infection, and slow replication rate of M. tuberculosis. In this study, we report a new approach to rapidly assess the effectiveness of compounds on the viability of M. tuberculosis in a macrophage infection model. Using a combination of a cytotoxicity assay and an in-broth M. tuberculosis viability assay, we were able to create a screening system that generates a comprehensive analysis of compounds of interest. This system is capable of producing quantitative data at a low cost that is within reach of most labs and yet is highly scalable to fit large industrial settings.

System for Efficacy and Cytotoxicity Screening of Inhibitors Targeting Intracellular Mycobacterium tuberculosis

We have developed a modular high-throughput screening system for discovering novel compounds against Mycobacterium tuberculosis, targeting intracellular and in-broth growing bacteria as well as cytotoxicity against the mammalian host cell.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is a leading cause of morbidity and mortality worldwide. With the increased spread ...

Quantitative Structure-Activity Relationship, Activity Prediction, and Molecular Dynamics of Non-nucleotide Reverse Transcriptase Inhibitors

The increasing incidence of HIV-1 drug resistance presents a challenge to the effectiveness of combination antiretroviral therapy, particularly in Southern Africa. The development of resistance to non-nucleotide reverse transcriptase inhibitors (NNRTIs) threatens the long-term success of antiretroviral therapy. In 2019, antimicrobial resistance directly accounted for an estimated 1.27 million deaths globally. This study employed an in-silico approach to investigate NNTRI drugs and their derivatives. Techniques used included density functional theory calculations, molecular docking, enumeration, quantitative structure-activity relationship (QSAR) analysis, molecular dynamics simulation (MDS), and molecular mechanics with generalized Born and surface area methods. The analysis focused on various pyrimidine derivatives and six NNRTI drugs, examining their interactions with the HIV-1 protein (PDB code 1HQU).
A QSAR model was developed to predict the biological activity of the six NNRTIs under investigation. Using 94 pyrimidine derivatives, the QSAR model achieved an R2 OF 0.822 and a Q2 of 0.815, indicating a high level of predictive accuracy.
MDS were conducted to assess the stability of various ligands and their newly developed alternatives, ensuring they remained bound to the protein&#39;s active site over a 200-nanosecond simulation timeframe. Etravirine exhibited root mean square deviation (RMSD) fluctuations of approximately 4.5 &#197;, while its enumerated derivatives showed RMSD fluctuations of 3.5 &#197;. Through molecular docking, MDS, and free energy computations, enumerated Etravirine demonstrated the best performance, with an activity value of 7.373 and a docking score of -10.517 kcal/mol. Furthermore, the calculated free energy of binding for enumerated Etravirine was -89.684 kcal/mol, outperforming other ligands under investigation. The significant improvement suggests that the modified Etravirine holds promising potential as a novel agent in antiretroviral therapy.
The obtained lower RMSD value, the enhanced amino acid interactions, and the highest binding free energy indicate that enumerated Etravirine could serve as a viable alternative for HIV/AIDS treatment.

This study used in-silico strategies to identify Enumerated Etravirine as a promising therapeutic agent for HIV. Our findings on molecular interactions and dynamics support the rational design of novel NNRTIs as possible HIV treatment alternatives.

The increasing incidence of HIV-1 drug resistance presents a challenge to the effectiveness of combination antiretroviral therapy, particularly in ...

Assays for DNA Enzymes Using Synthetic Oligonucleotides

Using Modified Synthetic Oligonucleotides to Assay Nucleic Acid-Metabolizing Enzymes

The availability of a range of modified synthetic oligonucleotides from commercial vendors has allowed the development of sophisticated assays to characterize diverse properties of nucleic acid metabolizing enzymes that can be run in any standard molecular biology lab. The use of fluorescent labels has made these methods accessible to researchers with standard PAGE electrophoresis equipment and a fluorescent-enabled imager, without using radioactive materials or requiring a lab designed for the storage and preparation of radioactive materials, i.e., a Hot Lab. The optional addition of standard modifications such as phosphorylation can simplify assay setup, while the specific incorporation of modified nucleotides that mimic DNA damages or intermediates can be used to probe specific aspects of enzyme behavior. Here, the design and execution of assays to interrogate several aspects of DNA processing by enzymes using commercially available synthetic oligonucleotides are demonstrated. These include the ability of ligases to join or nucleases to degrade different DNA and RNA hybrid structures, differential cofactor usage by the DNA ligase, and evaluation of the DNA-binding capacity of enzymes. Factors to consider when designing synthetic nucleotide substrates are discussed, and a basic set of oligonucleotides that can be used for a range of nucleic acid ligase, polymerase, and nuclease enzyme assays are provided.

Author Spotlight: Characterizing Novel Enzymes from Extremophiles and Common Pathogens to Understand DNA Repair and Replication

Here, a protocol for assaying nucleic acid metabolizing enzymes is presented, using examples of ligase, nuclease, and polymerase enzymes. The assay utilizes fluorescently labeled and unlabeled oligonucleotides that can be combined to form duplexes mimicking RNA and/or DNA damages or pathway intermediates, allowing for the characterization of enzyme behavior.

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

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