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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A., 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=Identification+of+proton-pump+inhibitor+drugs+that+inhibit+Trichomonas+vaginalis+uridine+nucleoside+ribohydrolase.">Identification of proton-pump inhibitor drugs that inhibit Trichomonas vaginalis uridine nucleoside ribohydrolase.</a> Bioorganic &amp; Medicinal Chemistry Letters. 24, 1080-1084 (2014).</li><li>Beck, S., Muellers, S. N., Benzie, A. L., Parkin, D. W., Stockman, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adenosine/guanosine+preferring+nucleoside+ribohydrolase+is+a+distinct,+druggable+antitrichomonal+target.">Adenosine/guanosine preferring nucleoside ribohydrolase is a distinct, druggable antitrichomonal target.</a> Bioorganic &amp; Medicinal Chemistry Letters. 25, 5036-5039 (2015).</li><li>Muellers, S. 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. K., Roy, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+1H+NMR+spectroscopy.">Quantitative 1H NMR spectroscopy.</a> Trends in Analytical Chemistry. 35, 5-26 (2012).</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=Sensitivity+improvement+in+19F+NMR-based+screening+experiments:+Theoretical+considerations+and+experimental+applications.">Sensitivity improvement in 19F NMR-based screening experiments: Theoretical considerations and experimental applications.</a> Journal of the American Chemical Society. 127, 13380-13385 (2005).</li><li>Lambruschini, 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=Development+of+fragment-based+n-FABS+NMR+screening+applied+to+the+membrane+enzyme+FAAH.">Development of fragment-based n-FABS NMR screening applied to the membrane enzyme FAAH.</a> ChemBioChem. 14, 1611-1619 (2013).</li><li>Stockman, B. 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 border="0" cellpadding="0" cellspacing="0" style="width:825px;" width="825">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">AGNH</td>
			<td style="width:180px;">Purified in-house</td>
			<td style="width:180px;">N/A</td>
			<td style="width:215px;">TVAG_213720</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">UNH</td>
			<td style="width:180px;">Purified in-house</td>
			<td style="width:180px;">N/A</td>
			<td>TVAG_092730</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Adenosine</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:180px;">A9251</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">5-Fluorouridine</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:180px;">F5130</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Dimethyl sulfoxide-D6</td>
			<td style="width:180px;">Cambridge Isotope Labs</td>
			<td style="width:180px;">DLM-10-100</td>
			<td>D, 99.9%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Potassium phosphate monobasic</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:180px;">P0662</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Potassium phosphate dibasic</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:180px;">P3786</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Potassium chloride</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:180px;">P9541</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Deuterium oxide</td>
			<td style="width:180px;">Cambridge Isotope Labs</td>
			<td style="width:180px;">DLM-4-100</td>
			<td>D, 99.9%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Hydrochloric acid</td>
			<td style="width:180px;">Fisher Chemical</td>
			<td style="width:180px;">A144212</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Triton X-100</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:180px;">X100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:251px;">3-(Trimethylsilyl)propionic-2,2,3,3,-d4 acid sodium salt (TSP)</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:180px;">269913</td>
			<td>D, 98%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">2,2,2-Trifluoroethanol-1,1-d2</td>
			<td style="width:180px;">Sigma</td>
			<td style="width:180px;">612197</td>
			<td>D, 99.5%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Pipette</td>
			<td style="width:180px;">Gilson</td>
			<td style="width:180px;">F123602</td>
			<td>PIPETMAN Classic P1000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Pipette</td>
			<td style="width:180px;">Gilson</td>
			<td style="width:180px;">F123601</td>
			<td style="width:215px;">PIPETMAN Classic P200</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Pipette</td>
			<td style="width:180px;">Gilson</td>
			<td style="width:180px;">F123600</td>
			<td style="width:215px;">PIPETMAN Classic P20</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Microfuge tubes</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:180px;">05-408-129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Conical tubes</td>
			<td style="width:180px;">Corning</td>
			<td style="width:180px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Microcentrifuge</td>
			<td style="width:180px;">Eppendorf</td>
			<td style="width:180px;">5418</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Vortex mixer</td>
			<td style="width:180px;">Fisher Scientific</td>
			<td style="width:180px;">02215365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">NMR tubes</td>
			<td style="width:180px;">Norell</td>
			<td style="width:180px;">502-7</td>
			<td>Or as appropriate for the NMR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">NMR spectrometer</td>
			<td style="width:180px;">Bruker</td>
			<td style="width:180px;">N/A</td>
			<td>AvanceIII500</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:251px;">Prism software</td>
			<td style="width:180px;">GraphPad</td>
			<td style="width:180px;">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 ...

nmr-based-activity-assays-for-determining-compound-inhibition-ic50

Research

JoVE Journal

Chemistry

9.9K Views.  Adelphi University. 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. 

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

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Nucleoside Triphosphates - From Synthesis to Biochemical Characterization

The traditional strategy for the introduction of chemical functionalities is the use of solid-phase synthesis by appending suitably modified phosphoramidite precursors to the nascent chain. However, the conditions used during the synthesis and the restriction to rather short sequences hamper the applicability of this methodology. On the other hand, modified nucleoside triphosphates are activated building blocks that have been employed for the mild introduction of numerous functional groups into nucleic acids, a strategy that paves the way for the use of modified nucleic acids in a wide-ranging palette of practical applications such as functional tagging and generation of ribozymes and DNAzymes. One of the major challenges resides in the intricacy of the methodology leading to the isolation and characterization of these nucleoside analogues.
	
	In this video article, we present a detailed protocol for the synthesis of these modified analogues using phosphorous(III)-based reagents. In addition, the procedure for their biochemical characterization is divulged, with a special emphasis on primer extension reactions and TdT tailing polymerization. This detailed protocol will be of use for the crafting of modified dNTPs and their further use in chemical biology.

The protocol described herein aims to explain and abridge the numerous obstacles in the way of the intricate route leading to modified nucleoside triphosphates. Consequently, this protocol facilitates both the synthesis of these activated building-blocks and their availability for practical applications.

The  traditional strategy for the introduction of chemical functionalities is the  use of solid-phase synthesis by appending suitably modified ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

A Colorimetric Assay that Specifically Measures Granzyme B Proteolytic Activity: Hydrolysis of Boc-Ala-Ala-Asp-S-Bzl

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is the most studied granzyme in humans and mice and therefore, researchers need specific and reliable tools to study its function and role in pathophysiology. This especially necessitates assays that do not recognize proteases such as caspases or other granzymes that are structurally or functionally related. Here, we apply GzmB&#8217;s preference for cleavage after aspartic acid residues in a colorimetric assay using the peptide thioester Boc-Ala-Ala-Asp-S-Bzl. GzmB is the only mammalian serine protease capable of cleaving this substrate. The substrate is cleaved with similar efficiency by human, mouse and rat GzmB, a property not shared by other commercially available peptide substrates, even some that are advertised as being suitable for this purpose. This protocol is demonstrated using unfractionated lysates from activated NK cells or CTL and is also suitable for recombinant proteases generated in a variety of prokaryotic and eukaryotic systems, provided the correct controls are used. This assay is a highly specific method to ascertain the potential pro-apoptotic activity of cytotoxic molecules in mammalian lymphocytes, and of their recombinant counterparts expressed by a variety of methodologies.

We describe a simple, quantitative colorimetric assay that specifically measures the proteolytic activity of human, mouse or rat Granzyme B (GzmB). This protocol can be easily adapted for determining protease activity of other granule serine proteases by the hydrolysis of other synthetic peptide substrates with an appropriate recognition sequence.

The serine protease Granzyme B (GzmB) mediates target cell apoptosis when released by cytotoxic T lymphocytes (CTL) or natural killer (NK) cells. GzmB is ...

Deacetylation Assays to Unravel the Interplay between Sirtuins (SIRT2) and Specific Protein-substrates

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further supported by recent findings in high-resolution mass spectrometry based proteomics showing that many new proteins and sites within these proteins can be acetylated. However the identity of the enzymes regulating these proteins and sites is often unknown. Among these enzymes, sirtuins, which belong to the class III histone lysine deacetylases, have attracted great interest as enzymes regulating the acetylome under different physiological or pathophysiological conditions. Here we describe methods to link SIRT2, the cytoplasmic sirtuin, with its substrates including both in vitro and in vivo deacetylation assays. These assays can be applied in studies focused on other members of the sirtuin family to unravel the specific role of sirtuins and are necessary in order to establish the regulatory interplay of specific deacetylases with their substrates as a first step to better understand the role of protein acetylation. Furthermore, such assays can be used to distinguish functional acetylation sites on a protein from what may be non-regulatory acetylated lysines, as well as to examine the interplay between a deacetylase and its substrate in a physiological context.

Deacetylation Assays to Unravel the Interplay between Sirtuins SIRT2 and Specific Protein-substrates

This protocol describes the required steps to execute in vitro and in vivo deacetylation assays in order to establish the role of proteins as specific deacetylation substrates for sirtuins and further study the role of reversible - lysine acetylation as a post-translational modification.

Acetylation has emerged as an important post-translational modification (PTM) regulating a plethora of cellular processes and functions. This is further ...

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids (BPCA)

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.
Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC&#39;s characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Characterization, Quantification and Compound-specific Isotopic Analysis of Pyrogenic Carbon Using Benzene Polycarboxylic Acids BPCA

We present the benzene polycarboxylic acid (BPCA) method for assessing pyrogenic carbon (PyC) in the environment. The compound-specific approach uniquely provides simultaneous information about the characteristics, quantity and isotopic composition (13C and 14C) of PyC.

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as ...

HPLC-based Assay to Monitor Extracellular Nucleotide/Nucleoside Metabolism in Human Chronic Lymphocytic Leukemia Cells

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and validated for the quantification of extracellular purine nucleotides and nucleosides produced by purified chronic lymphocytic leukemia (CLL) cells under different culture conditions. The chromatographic separation of adenosine 5&#39;-monophosphate (AMP), adenosine (ADO) and inosine (INO) is performed at RT on a silica-based, reversed-phase column that is used for polar compound retention. The method includes a binary mobile phase, which consists of 7 mM ammonium acetate and acetonitrile with a flow rate of 1.00 ml/min. The eluates are monitored using a Photodiode Array UV detector set at 260 nm. A standard calibration curve is generated to calculate the equation for the analytical quantification of each purine compound. System control, data acquisition and analysis are then performed. Applying this protocol, AMP, INO and ADO elute at 7, 11 and 11.9 min, respectively, and the total run time for each sample is 20 min. This protocol may be applied to different cell types and cell lines (both suspension and adherent), using culture media as matrix. The advantages are easy and fast sample preparation and the requirement of a small amount of supernatant for analysis. Furthermore, the use of a serum-free medium allows skipping the protein precipitation step with acetonitrile that impacts the final concentration of purine compounds. One of the limitations of the method is the requirement of the equilibration column run before each single sample run, making the total run time of the experiment longer and preventing high throughput screening applications.

The protocol described here represents an easy and reproducible method that employs reverse phase high-performance liquid chromatography (RP-HPLC) to measure purine metabolism on chronic lymphocytic leukemia (CLL) cells cultured under different conditions.

This method describes a sensitive, specific, reliable and reproducible reverse phase high-performance liquid chromatography (RP-HPLC) assay developed and ...

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. Detailed and generalized protocols are also presented for testing the catalytic activity of such complexes in arylation and Suzuki-Miyaura cross-coupling reactions. Representative results are shown for the catalytic activity of the four complexes in arylation and Suzuki-Miyaura type reactions. For each of the reactions investigated, at least one of the four complexes successfully catalyzed the reaction, qualifying them as promising candidates for catalysis of many carbon-carbon bond-forming reactions. The protocols presented are general enough to be adapted for the synthesis, purification and catalytic activity testing of new palladium N-heterocyclic carbene complexes.

Palladium N-Heterocyclic Carbene Complexes: Synthesis from Benzimidazolium Salts and Catalytic Activity in Carbon-carbon Bond-forming Reactions

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from benzimidazolium salts. The complexes were tested for catalytic activity in arylation and Suzuki-Miyaura reactions. For each reaction investigated, at least one of the four complexes successfully catalyzed the reaction.

Detailed and generalized protocols are presented for the synthesis and subsequent purification of four palladium N-heterocyclic carbene complexes from ...

Simultaneous Measurement of HDAC1 and HDAC6 Activity in HeLa Cells Using UHPLC-MS

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since the approval of romidepsin, a class I HDAC inhibitor for cancer therapy, and the clinical investigation of HDAC6-specific inhibitors for multiple myeloma. The present method is used to determine the inhibitory activity of test compounds on HDAC1 and HDAC6 in cells. The isoform activity is measured using the ultra-high-performance liquid chromatography &#8211; mass spectrometry (UHPLC-MS) analysis of specific substrates incubated with treated and untreated HeLa cells. The method has the advantage of reflecting the endogenous HDAC activity within the cell environment, in contrast to cell-free biochemical assays conducted on isolated isoforms. Moreover, because it is based on the quantification of synthetic substrates, the method does not require the antibody recognition of endogenous acetylated proteins. It is easily adaptable to several cell lines and an automated process. The method has already proved useful in finding HDAC6-selective compounds in neuroblasts. Representative results are shown here with the standard HDAC inhibitors trichostatin A (non-specific), MS275 (HDAC1-specific), and tubastatin A (HDAC6-specific) using HeLa cells.

The present method serves to identify isoform-specific inhibitors of histone deacetylases (HDAC) in HeLa cells by the UHPLC-MS analysis of multiple substrates. This is an antibody-free method developed to reflect HDAC1 and HDAC6 activity in the living cell environment, in contrast to single-isoform cell-free assays.

The search for new histone deacetylase (HDAC) inhibitors is of increasing interest in drug discovery. Isoform selectivity has been in the spotlight since ...