¹⁹F NMR Functional Screening on a Benchtop NMR Instrument: Theoretical Analysis and Efficient Application to Drug...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Drug Discovery

Background:

• Benchtop Nuclear Magnetic Resonance (NMR) instruments offer accessible NMR analysis, crucial for drug discovery.
• However, their widespread adoption is hindered by limited sensitivity and resolution.
• Fluorine-19 NMR (¹⁹F NMR) is a powerful technique for studying molecular interactions.

Purpose of the Study:

• To develop and validate a functional assay using ¹⁹F NMR on a benchtop spectrometer.
• To determine the relative affinity of enzyme inhibitors across a wide potency range.
• To assess the capabilities and limitations of this novel assay for cost-effective drug screening.

Main Methods:

• A ¹⁹F NMR-based functional assay (n-FABS) was established on a commercial 80 MHz NMR spectrometer.
• The assay was applied to the cancer-related enzyme thymidine phosphorylase.
• Inhibitor potencies (IC₅₀ values) were determined for various compounds.

Main Results:

• The n-FABS assay reliably determined relative inhibitor affinities from nanomolar (nM) to hundreds of micromolar (μM) concentrations.
• Accurate IC₅₀ values were obtained for thymidine phosphorylase inhibitors.
• Theoretical evaluation provided in-depth insights into the assay's strengths and limitations.

Conclusions:

• The developed n-FABS assay enables efficient and cost-effective functional assays on benchtop NMR instruments.
• Optimized assays using fluorinated substrates can be designed based on the insights gained.
• This approach broadens the accessibility of NMR-based drug discovery tools.