Abstract
In-cell NMR can investigate protein conformational changes at atomic resolution, such as those changes induced by drug binding or chemical modifications, directly in living human cells, and therefore has great potential in the context of drug development as it can provide an early assessment of drug potency. NMR bioreactors can greatly improve the cell sample stability over time and, more importantly, allow for recording in-cell NMR data in real time to monitor the evolution of intracellular processes, thus providing unique insights into the kinetics of drug-target interactions. However, current implementations are limited by low cell viability at >24 h times, the reduced sensitivity compared to “static” experiments and the lack of protocols for automated and quantitative analysis of large amounts of data. Here, we report an improved bioreactor design which maintains human cells alive and metabolically active for up to 72 h, and a semiautomated workflow for quantitative analysis of real-time in-cell NMR data relying on Multivariate Curve Resolution. We apply this setup to monitor protein–ligand interactions and protein oxidation in real time. High-quality concentration profiles can be obtained from noisy 1D and 2D NMR data with high temporal resolution, allowing further analysis by fitting with kinetic models. This unique approach can therefore be applied to investigate complex kinetic behaviors of macromolecules in a cellular setting, and could be extended in principle to any real-time NMR application in live cells.
Highlights
In-cell Nuclear Magnetic Resonance (NMR) can investigate protein conformational changes at atomic resolution, such as those changes induced by drug binding or chemical modifications, directly in living human cells, and has great potential in the context of drug development as it can provide an early assessment of drug potency
HEK293T cells were suspended in a 1.5% solution of low-gelling agarose, cooled down in FPLC capillary tubing to form threads, which were packed into the flow unit NMR tube, where growth medium was supplemented at a constant flow rate through an inlet placed at the bottom of the tube
To study the kinetics of cellular processes via NMR, which provides values of intensity and chemical shift averaged over all cells within the active volume, it is important to ensure that the cells are experiencing a uniform microenvironment
Summary
The NMR bioreactor setup was optimized to maximize cell viability for long periods of time, while simultaneously allowing high cell numbers in the NMR spectrometer. The slow binding of AAZ to intracellular CA2 is approximated by a single exponential function better than the fast binding of MZA and the ebselen-dependent SOD1 oxidation (Figure 5a) Both CA2 binding curves can be fitted with the kinetic model described previously,[16] in which the bound species initially increases at a constant rate due to the membrane diffusion being the rate-determining step. SOD1 oxidation appears to follow yet another mechanism, which is best modeled with a biexponential equation, suggesting that ebselen may promote SOD1 disulfide bond formation through different coexisting pathways (Figure 5c) These observations show that, with properly designed sets of experiments, real-time in-cell NMR can provide detailed information on the mechanism of the observed biological processes
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