1H and 19F NMR-based activity assays are being used increasingly in the pharmaceutical industry to complement high-throughput screening (HTS) methods. They provide orthogonal, uncoupled assays useful for HTS hit triage, and are also perfectly suited to high concentrations of test compounds used in fragment screening. In the undergraduate biochemistry laboratory setting, NMR-based activity assays are a logical extension from prerequisite organic chemistry NMR analyses. Relationships between chemical structure and chemical shift, and between peak area and quantity, are extended to distinguish between substrate and product resonances, and to determine their corresponding concentrations, respectively. Compounds can be quickly tested for inhibition, and the equilibrium constant for a reaction can be determined by integrating peak areas. The project-based biochemistry laboratory course at Adelphi has students work in small groups to purify and characterize a protein of their choosing. NMR spectroscopy is a required component of the characterization. This typically involves developing and validating a 1H NMR-based activity assay. Recent enzymes include amylase, lactate dehydrogenase, and malate dehydrogenase. Student groups that decide to purify a protein that lacks enzymatic activity are required to use NMR spectroscopy to monitor the methanol-induced denaturation of their protein. In the undergraduate research setting, NMR-based activity assays are being developed and used to identify inhibitors of nucleoside ribohydrolases from Trichomonas vaginalis. These enzymes are important components of purine and pyrimidine salvage pathways used by the parasite and represent novel drug targets. One assay uses 19F NMR to monitor the conversion of 5-fluorouridine to 5-fluorouracil and ribose catalyzed by uridine nucleoside ribohydrolase (UNH), while another uses 1H NMR to monitor the conversion of adenosine to adenine and ribose catalyzed by adenosine/guanosine nucleoside ribohydrolase (AGNH). The UNH assay has been used to screen the NIH Clinical Collection and identified several compounds with μM inhibition.

NMR-based activity assays to characterize enzymes in the biochemistry laboratory and in undergraduate research

249th National Meeting (2015)

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