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<p>The cellular environment is a complex and crowded space, with organelles, compartments and multitudes of molecules engaged in intricate networks of communication that modulate binary protein-ligand/protein interactions. As a result, it is becoming increasingly appreciated that evaluations of protein-drug binding should be carried out in the native cellular environment. Here, we present a proof-of-concept study where we measured the lifetime (1/<i>k</i><sub>off</sub>) of a protein-drug complex in human cells by <sup>19</sup>F NMR spectroscopy using fluorinated Cyclophilin A (CypA) bound to Cyclosporine A (CsA). Harnessing the exceptional detection sensitivity of the trifluoromethyl group attached at the para position of Phe60 in CypA, high-quality 2D <sup>19</sup>F-<sup>19</sup>F exchange spectra were obtained in cells. Essentially identical <i>k</i><sub>off</sub> values were observed in cells and in vitro, suggesting that the overall impact of the cellular environment on the lifetime of tfmF60 CypA/CsA complex is minimal. Using similar approaches for quantifying protein-drug lifetimes in the native cellular environment paves the way for efficiently screening drug libraries in human cells by <sup>19</sup>F NMR spectroscopy.</p>

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Quantifying protein-drug lifetimes in human cells by 19F NMR spectroscopy

  • 期刊论文

  • 发表日期：

    2025年3月24日

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  • Wenkai Zhu,
  • Fatema Bhinderwala,
  • Sarah Rambo,
  • Angela M. Gronenborn

摘要

The cellular environment is a complex and crowded space, with organelles, compartments and multitudes of molecules engaged in intricate networks of communication that modulate binary protein-ligand/protein interactions. As a result, it is becoming increasingly appreciated that evaluations of protein-drug binding should be carried out in the native cellular environment. Here, we present a proof-of-concept study where we measured the lifetime (1/koff) of a protein-drug complex in human cells by 19F NMR spectroscopy using fluorinated Cyclophilin A (CypA) bound to Cyclosporine A (CsA). Harnessing the exceptional detection sensitivity of the trifluoromethyl group attached at the para position of Phe60 in CypA, high-quality 2D 19F-19F exchange spectra were obtained in cells. Essentially identical koff values were observed in cells and in vitro, suggesting that the overall impact of the cellular environment on the lifetime of tfmF60 CypA/CsA complex is minimal. Using similar approaches for quantifying protein-drug lifetimes in the native cellular environment paves the way for efficiently screening drug libraries in human cells by 19F NMR spectroscopy.