Insights into the interactions between a drug and a membrane protein target by fluorine cross-polarization magic angle spinning NMR

Martin P. Boland, David A. Middleton

    Research output: Contribution to journalArticlepeer-review


    The fluorinated anti-psychotic drug trifluoperazine (TFP) has been shown to be a K+-competitive inhibitor of gastric H+/K +-ATPase, a membrane-embedded therapeutic target for peptic ulcer disease. This paper describes how variable contact time 19F cross-polarization magic angle spinning (VCT-CP/MAS) NMR has been used to probe the inhibitory interactions between TFP and H+/K+-ATPase in native gastric membranes. The 19F CP/MAS spectra for TFP in H +/K+-ATPase enriched (GI) gastric membranes and in control membranes containing less than 5 nmol of the protein indicated that the drug associates with the membranes independently of the presence of H +/K+-ATPase. The 19F peak intensities in the VCT-CP/MAS experiment confirmed that TFP undergoes slow dissociation (k off <100 s-1) from binding sites in GI membranes, and more rapid dissociation (koff <100 s-1) from control membranes. The spectra showed that up to 40% of bound TFP was displaced from GI membranes by 100 mM K+ and by the K+-competitive inhibitor TMPIP, but TFP was not displaced from the control membranes. Hence the spectra of TFP in GI membranes represent the drug bound to the K +-competitive inhibitory site of H+/K+-ATPase and to other non-specific sites. The affinity of TFP for the K +-competitive site (KD = 4 mM) was determined from a binding curve of 19F peak intensity versus TFP concentration after correction for non-specific binding. The XD was much higher than the IC50 for ATPase inhibition (8 μM), which suggests that the substantial non-specific binding of TFP to the membranes contributes to ATPase inhibition. This novel approach to probing ligand binding can be applied to a wide range of membrane-embedded pharmaceutical targets, such as G-protein coupled receptors and ion channels, regardless of the size of the protein or strength of binding. Copyright © 2004 John Wiley & Sons, Ltd.
    Original languageEnglish
    Pages (from-to)204-211
    Number of pages7
    JournalMagnetic Resonance in Chemistry
    Issue number2
    Publication statusPublished - Feb 2004


    • Cross-polarization
    • H +/K+-ATPase
    • Ligand screening
    • Magic angle spinning
    • NMR
    • Trifluoperazine


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