Boland Martin P, Middleton David A
Department of Biomolecular Sciences, University of Manchester Institute of Science and Technology, P.O. Box 88, Manchester M60 1QD, UK.
Magn Reson Chem. 2004 Feb;42(2):204-11. doi: 10.1002/mrc.1319.
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.
含氟抗精神病药物三氟拉嗪（TFP）已被证明是胃H⁺/K⁺-ATP酶的K⁺竞争性抑制剂，胃H⁺/K⁺-ATP酶是消化性溃疡疾病的一个膜嵌入治疗靶点。本文描述了可变接触时间¹⁹F交叉极化魔角旋转（VCT-CP/MAS）核磁共振如何用于探测TFP与天然胃膜中H⁺/K⁺-ATP酶之间的抑制性相互作用。在富含H⁺/K⁺-ATP酶的（GI）胃膜以及蛋白质含量低于5 nmol的对照膜中，TFP的¹⁹F CP/MAS光谱表明，该药物与膜的结合独立于H⁺/K⁺-ATP酶的存在。VCT-CP/MAS实验中的¹⁹F峰强度证实，TFP从GI膜中的结合位点缓慢解离（kₒff < 100 s⁻¹），而从对照膜中解离更快（kₒff < 100 s⁻¹）。光谱显示，高达40%结合的TFP被100 mM K⁺和K⁺竞争性抑制剂TMPIP从GI膜中取代，但TFP未从对照膜中被取代。因此，GI膜中TFP的光谱代表了与H⁺/K⁺-ATP酶的K⁺竞争性抑制位点以及其他非特异性位点结合的药物。在校正非特异性结合后，根据¹⁹F峰强度与TFP浓度的结合曲线确定了TFP对K⁺竞争性位点的亲和力（Kᴅ = 4 mM）。该Kᴅ远高于ATP酶抑制的IC₅₀（8 μM），这表明TFP与膜的大量非特异性结合有助于ATP酶抑制。这种探测配体结合的新方法可应用于广泛的膜嵌入药物靶点，如G蛋白偶联受体和离子通道，而不论蛋白质的大小或结合强度如何。
