Insights into the interactions between a drug and a membrane protein target by fluorine cross-polarization magic angle spinning NMR
✍ Scribed by Martin P. Boland; David A. Middleton
- Book ID
- 102529369
- Publisher
- John Wiley and Sons
- Year
- 2004
- Tongue
- English
- Weight
- 248 KB
- Volume
- 42
- Category
- Article
- ISSN
- 0749-1581
- DOI
- 10.1002/mrc.1319
No coin nor oath required. For personal study only.
✦ Synopsis
Abstract
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 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.