NMR-Based Activity Assays for Determining Compound Inhibition, IC<sub>50</sub> Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

Brian J. Stockman; Abinash Kaur; Julia  K. Persaud; Maham Mahmood; Samantha  F. Thuilot; Melissa  B. Emilcar; Madison Canestrari; Juliana  A. Gonzalez; Shannon Auletta; Vital Sapojnikov; Wagma Caravan; Samantha  N. Muellers

doi:10.3791/59928

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

NMR-Based Activity Assays for Determining Compound Inhibition, IC<sub>50</sub> Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

JoVE Journal
Chemistry

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JoVE Journal Chemistry

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

9,992 Views

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10:24 min

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June 30, 2019

DOI:

10.3791/59928-v

DOI:

10.3791/59928-v

•

10:24 min

June 30, 2019

•

9985 Views

Brian J. Stockman¹, Abinash Kaur¹, Julia K. Persaud¹, Maham Mahmood¹, Samantha F. Thuilot¹, Melissa B. Emilcar¹, Madison Canestrari¹, Juliana A. Gonzalez¹, Shannon Auletta¹, Vital Sapojnikov¹, Wagma Caravan^1,2, Samantha N. Muellers^1,3

¹Department of Chemistry,Adelphi University ²Department of Chemistry,Washington University in St. Louis ³Department of Chemistry,Boston University

Summary

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 μM and 250 μM, dose-response assays for determining IC₅₀ values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.