Jun 20, 2025
Screening compounds that inhibit SARS-CoV-2 nsp3 macrodomain: ADP-Ribose quantification by LC/MS-MS
- Noa Lahav1,2,
- Haim Barr3,2
- 1Weizmann Institute;
- 2ASAP Discovery Consortium;
- 3Weizmann Institute of Science
- ASAP Discovery
Protocol Citation: Noa Lahav, Haim Barr 2025. Screening compounds that inhibit SARS-CoV-2 nsp3 macrodomain: ADP-Ribose quantification by LC/MS-MS . protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5rpydg1b/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 16, 2024
Last Modified: June 20, 2025
Protocol Integer ID: 107677
Keywords: SARS-CoV-2, screening, inhibition, Mac1, Coronaviridae, ADPr, LC/MS-MS, nsp3, macrodomain, treatments for viral infectious disease, viral infectious disease, presence of potential inhibitor, potential inhibitor, inhibitor, mass spectrometry, relative potency of inhibitor, functional biochemical assay, ribose quantification by lc, mac1 ability, sar, screening compound, liquid chromatography, utilizing liquid chromatography
Funders Acknowledgements:
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)
Grant ID: U19AI171399
Disclaimer
The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Abstract
Functional biochemical assay to identify treatments for viral infectious disease that targets SARS-CoV-2 nsp3 macrodomain (MAC1). Utilizing Liquid Chromatography-Mass Spectrometry/Mass Spectrometry (LC-MS/MS) analysis, MAC1 ability to hydrolyze αNAD+ in the presence of potential inhibitors was measured by quantifying ADPR and αNAD+ levels. This assay is characterized by the relative potency of inhibitors as IC50, it is modified from https://doi.org/10.1126/sciadv.abf8711. In this version, we have added the Addgene id.
Materials
Construct used
Assay Reagents (Concentration listed are from Stock Solutions)
- 250 millimolar (mM) HEPES 0.5M buffer soln. pH 7.0Fisher ScientificCatalog #AAJ60064AE (or similar)
- 200 millimolar (mM) Sodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
- 0.5 mg/mL Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7030
- α-Nicotinamide adenine dinucleotideMerck MilliporeSigma (Sigma-Aldrich)Catalog #N6754
- Adenosine 5′-diphosphoribose sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0752
Protocol materials
α-Nicotinamide adenine dinucleotideMerck MilliporeSigma (Sigma-Aldrich)Catalog #N6754
Sodium ChlorideMerck MilliporeSigma (Sigma-Aldrich)Catalog #S9888
Bovine Serum AlbuminMerck MilliporeSigma (Sigma-Aldrich)Catalog #A7030
Adenosine 5′-diphosphoribose sodium saltMerck MilliporeSigma (Sigma-Aldrich)Catalog #A0752
Sars Cov 2 Mac domain PlasmidaddgeneCatalog #204787
HEPES 0.5M buffer soln. pH 7.0Fisher ScientificCatalog #AAJ60064AE
Troubleshooting
Safety warnings
Please be sure to wear proper Personal Protective Equipment (PPE) while performing this experiment.
Before start
Note: Inhibitor compounds stock concentration is 20 mM. Compounds are pre-dispensed into assay plates and stored at -20˚C until use.
SARS-CoV-2 nsp3 expression and purification
The protein used in this assay was expressed and purified according to this protocol:
We used the following plasmid in the protein expression and purification protocol.
Sars Cov 2 Mac domain PlasmidaddgeneCatalog #204787
Protocol
CREATED BY
Korvus Wang
Assay reagents preparation
Assay buffer:
| A | B | D | E | |
| Stock | Final in assay plate | Units | ||
| HEPES pH=7.0 | 1000 | 25 | mM | |
| NaCl | 5000 | 100 | mM | |
| BSA | 10 | 1 | mg/ml |
| A | B | C | D | E | |
| Reagent | Stock | dispensed to plate | Final in assay plate | Units | |
| His-SARS-CoV-2 MAC1 | 183000 | 800 | 200 | nM | |
| Substrate (NAD+) | Dry (disslove fresh for each assay) | 32 | 8 | μM |
Prepare 96 well-plate
1m
PRIME the dispenser with Assay Buffer by selecting the PRIME button until the tubes are filled completely.
DISPENSE 75 µL Mac1 assay Buffer to control wells of assay plate and 50 µL Mac1 Buffer to the rest of the plate.
- Note: Control wells are for NAD+ alone and MAC1+NAD+, without compounds
EMPTY the dispenser tubes.
- Discard the liquid discharged from the tubes.
PRIME the dispenser with 4x MAC1 enzyme (800 nM) by selecting the PRIME button until the tubes are filled completely.
DISPENSE 25 µL 4xMac1 to all wells of assay plate besides NAD+ alone control well.
EMPTY the dispenser tubes.
- Discard the liquid discharged from the tubes.
CENTRIFUGE plate 1500 rpm, Room temperature, 00:01:00 to remove bubbles
1m
INCUBATE plate for 00:15:00 atRoom temperature
15m
PRIME the dispenser with 4x NAD+ (32 μM) by selecting the PRIME button until the tubes are filled completely.
DISPENSE 25 µL 4xNAD+ to all wells of assay plate.
EMPTY the dispenser tubes.
- Discard the liquid discharged from the tubes.
CENTRIFUGE plate 1500 rpm, Room temperature, 00:01:00 to remove bubbles
1m
INCUBATE plate on an Orbi-plate shaker at 300 rpm for 02:45:00 atRoom temperature
2h 45m
STOP the reaction by freezing the plate and store in -80 until LC/MS analysis.
LC/MS-MS analysis
Quantitative analysis of ADPR and αNAD+ was performed as was recently published at
Lee, Alpha A et al. “Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response.” bioRxiv : the preprint server for biology 2024.08.19.608619. 21 Aug. 2024, doi:10.1101/2024.08.19.608619. Preprint. https://pmc.ncbi.nlm.nih.gov/articles/PMC11370477/
Protocol references