Magic angle spinning NMR of fluorinated pharmaceuticals in cellular environments
Date
2023
Authors
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Publisher
University of Delaware
Abstract
Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique that provides atomic resolution information about structure and dynamics in biomolecules in vitro and in vivo. Solution, as well as, solid-state magic angle spinning (MAS) and dynamic nuclear polarization (DNP) enhanced NMR have successfully analyzed proteins and small molecules in a cellular environment. While understanding the native fold of proteins is critical for drug design, determining the selectivity and bioavailability of pharmaceuticals in cellular environments is also important. 19F incorporation into pharmaceuticals is becoming more common because 19F increases the bioavailability of the drug. Since 19F does not naturally occur in cell cultures, it is distinguishable from the large cellular background making fluorinated drugs a promising target for solid-state in-cell NMR studies. ☐ The cryopreservation of cells during in-cell NMR experiments would allow for more signal averaging. We assessed the cell viability during cryopreservation under various conditions. Cells were cryopreserved and stored in temperatures ranging from -20 to -80 °C. Cells that were cryopreserved as stocks or pellets saw little decrease in cell viability at temperatures below -60 °C. Pelleted cells stored at 4 °C remain about 50% viable for at least 4 days. It was found that packing cells in a MAS NMR rotor at 68,000 x g does not increase the number of cells packed as compared to 5,000 x g. In fact, spinning the cells at 68,000 x g damages the cells, which is seen by a lag in their growth curve. ☐ Flutamide (FLA), typically used to treat prostate cancer, has an aryl-CF3 (Ar-CF3) moiety and its target protein, androgen receptor (AR), is naturally occurring in human embryonic kidney 293 (HEK 293) cells. We have explored the conditions for MAS NMR characterization of FLA in mammalian cells. It was found that FLA is insoluble at 500 µM in complete DMEM with 0.5% DMSO. This limits how much FLA can be used during cell incubation. However, FLA does not have a significant impact on cell viability. This allows for cells to be incubated with FLA for 16 hours, thereby maximizing the natural uptake of FLA. ☐ HEK 293 cells were electroporated (EP) with FLA and 19F-NMR spectra were collected on the in-cell sample. The FLA signals in the in-cell sample were shifted as compared to those of powder FLA and FLA in buffer. While the cells did not survive this particular experiment, this novel result represents the first proof of concept 19F solid-state NMR spectrum of a fluorinated pharmaceutical in a cellular environment.
Description
Keywords
Flutamide, In-cell NMR, Dynamic nuclear polarization, Nuclear magnetic resonance