pubs.acs.org/jmc Published on Web 12/21/2009 r 2009 American Chemical Society J. Med. Chem. 2010, 53, 867–875 867 DOI: 10.1021/jm901537q Using NMR Solvent Water Relaxation to Investigate Metalloenzyme-Ligand Binding Interactions Ivanhoe K. H. Leung, Emily Flashman, Kar Kheng Yeoh, Christopher J. Schofield, and Timothy D. W. Claridge* Department of Chemistry, University of Oxford, Chemistry Research Laboratory, 12 Mansfield Road, Oxford OX1 3TA, United Kingdom Received October 16, 2009 This report demonstrates that solvent water relaxation measurements can be used for quantitative screening of ligand binding and for mechanistic investigations of enzymes containing paramagnetic metal centers by using conventional NMR instrumentation at high field. The method was exemplified using prolyl hydroxylase domain containing enzyme 2 (PHD2), a human enzyme involved in hypoxic sensing, with Mn(II) substituting for Fe(II) at the active site. K D values were determined for inhibitors that hinder access of water to the paramagnetic center. This technique is also useful for investigating the mechanism of suitable metalloenzymes, including order of ligand binding and modes of inhibition. Introduction NMR spectroscopy is widely used for the study of small molecule-protein interactions; saturation transfer difference (STD a ), 1 transferred NOE, 2 and chemical shift perturbation techniques have emerged over recent years to play useful roles in ligand screening. 3-6 Although widely employed, these techniques may suffer from low intrinsic sensitivity and can require careful optimization of experimental conditions. For instance, STD enhancements can be rather weak and these analyses typically require a large excess of ligand, which may result in nonspecific binding or cause problems if the ligands are of limited solubility. The transferred NOE method re- quires a delicate ligand-protein ratio, and the results can be difficult to interpret in cases of relatively large ligands such as for peptide-protein or polysaccharide-protein interactions. Chemical shift perturbation using 1 H- 15 N HSQC requires access to 15 N labeled protein and can be difficult to apply in systems larger than 25 kDa. 7 These factors may limit the applicability of these methods as primary screening tools. Relaxation enhancement has been used as a tool to study paramagnetic metalloprotein-ligand interactions since the early 1960s; 8 the technique is based on the fact that the magnetic moment of an electron is 658 times greater than that of a proton nuclear spin. 9 Therefore, in the presence of a paramagnetic metalloprotein, the longitudinal and transverse relaxation rates of nuclei within a binding ligand may be enhanced, while for nonbinders the relaxation rates will remain unchanged. The changes in relaxation behavior de- pend on factors including the applied magnetic field (B 0 ), molecular correlation times, accessibility of the active site, and the properties of the paramagnetic metal itself. 8,10-12 The same paramagnetic relaxation enhancement (PRE) effect can be observed through solvent water proton relaxation rates. Various groups have made use of this effect to study para- magnetic metal binding interactions with proteins and other macromolecules by monitoring changes in the bulk water relaxation rate. 13-17 Recently, a novel competition method has been proposed for screening small molecules binding to paramagnetic me- talloproteins by employing water as the reporter molecule and monitoring changes in the bulk water proton longitudinal relaxation rate (R 1 ). 18 In the absence of any ligand, providing that access is possible, water molecules may chelate to the paramagnetic metal and undergo exchange with water mole- cules in the bulk solvent matrix. This leads to a net increase in the bulk water relaxation rate. The binding of ligands to the active site will normally hinder or prevent access of water molecules to the paramagnetic center and so decrease the observed bulk water relaxation rate. 19 Because the exchange of water will not normally be directly reliant on the exchange kinetics of the reversibly forming protein-ligand complex (as in the case for STD-NMR), this technique does not suffer from the limits imposed by other ligand-based NMR screen- ing methods. 18 For example, while the association of very strongly binding ligands is often difficult to detect directly with ligand-observed methods, alterations in water accessi- bility may still arise under these conditions and remain readily detectable through water relaxation measurements. Thus, provided the enzyme under investigation contains a paramag- netic center at the binding site, this present approach can overcome the difficulties and limitations often associated with other ligand based NMR screening techniques. The effect of paramagnetic relaxation is maximized at low field, generally between 0.01 to 100 MHz; 18 hence the original application of this solvent-based method for ligand screening was focused toward the use of specialist low field NMR instruments or fast field-cycling relaxometers. 18,20 While it was demonstrated that it is possible to conduct screen- ing experiments at high field (400 MHz) on very small sample volumes (∼2 μL) using an inhibitor with a K D in the *To whom correspondence should be addressed. Phone: þ44 (0)1865 275 658. Fax: þ44 (0)1865 285 002. E-mail: tim.claridge@chem.ox.ac. uk. a Abbreviations: 2OG, 2-oxoglutarate; NOG, N-oxalylglycine; PHD, prolyl hydroxylase; STD, saturation transfer difference; PRE, para- magnetic relaxation enhancement; HIF, hypoxia inducible factor; ESI- MS, electrospray ionization mass spectrometry; ILOEs, interligand NOEs; CODD, C-terminal oxygen-dependent degradation domain; NODD, N-terminal oxygen-dependent degradation domain; BNS, bi- cyclic naphthalenylsulfonyl compound; BIQ, bicyclic isoquinolinyl compound.