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

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 19 F 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 19 F 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 19 F 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 ( k off < 100 s −1 ) from control membranes. The spectra showed that up to 40% of bound TFP was displaced from GI membranes by 100 m M 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 ( K D = 4 m M ) was determined from a binding curve of 19 F peak intensity versus TFP concentration after correction for non‐specific binding. The K D was much higher than the IC 50 for ATPase inhibition (8 µ M ), 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.