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

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 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 (K(D) = 4 mM) 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 microM), 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.