[PubMed] [Google Scholar] 26. ESI-MS has been used by our group as a simple and rapid primary screening method for 2OG oxygenase inhibitors. However, because non-covalent protein-ligand complexes may not usually survive the transition from answer phase to gas phase,32 there is a need for complementary solution-based screening techniques. NMR spectroscopy is an established technique for the study of protein-ligand binding interactions. Ligand detection methods such as saturation transfer difference (STD)33 and water ligand observed gradient spectroscopy (waterLOGSY)34 are widely used for ligand screening because they do not require isotopically labelled protein and are relatively quick and sensitive. However, many ligand-based NMR methods suffer from limited detection ranges of binding affinities, false positives arising from non-specific binding35 and complications with binding constant determination that arise from, for instance, ligand rebinding events and dependencies on ligand spin relaxation rates and saturation occasions.36,37 NMR reporter screening methods are useful alternatives for the site-specific detection GW6471 of both high- and low-affinity ligands. By observing changes in the NMR parameter(s) (such as chemical shift or relaxation rate) associated with a reporter ligand, it is possible to obtain qualitative and quantitative information around the binding of ligands that compete with the reporter ligand for the target protein (Physique 1). Several nuclei, including proton,38-40 phosphorus,41 fluorine42 and carbon43 have been proposed for reporter screening by NMR. Open in a separate window Figure 1 Schematic representation for the NMR reporter screening method. When the reporter ligand is bound to the protein, the corresponding 1H NMR resonance is broadened and its intensity lowered. In the presence of a competitive inhibitor, the reporter ligand is displaced from the protein binding site and its signal intensity is recovered. We reasoned that 2OG is a potentially good reporter ligand candidate for generic NMR binding assays for 2OG oxygenases because they all utilise it as co-substrate. Using unlabelled and commercially available [1,2,3,4-13C4]-labelled 2OG ([13C]-2OG), we have optimised the experimental conditions and demonstrated the feasibility of applying the reporter ligand method for high-throughput screening and binding constant (form was present. It should be noted that a caveat of our method is that it does not employ the native metal ion, although the binding affinity of ligands, including 2OG, is not, at least substantially, affected by the use of ZnII as the active site metal GW6471 (see below; Supplemental Figure S2). Both PHD2 and FIH form stable complexes with 2OG that saturate at a ~1:1 ratio (Supplemental Figure S3).23,31 Under these conditions, the 1H NMR spectra were dominated by the protein resonances (Supplemental Figure S4). In order to observe the reporter ligand (2OG) signal, the Carr-Purcell-Meiboom-Gill (CPMG)44-46 sequence was applied to attenuate any broad (protein) resonances (Supplemental Figure S4). In particular, the recently proposed Periodic Refocusing Of J Evolution by Coherence Transfer (PROJECT) method,47 which uses an additional 90reversible inter-conversions of simple building blocks in the presence of a target protein template (Supplemental Figure S24). Previously, we have successfully applied protein-directed DCC to identify ligands produced by reversible boronate ester formation that led to novel nanomolar inhibitors for PHD2.81,82 This work involved the use of a boronic acid scaffold, which binds in the 2OG binding pocket and is a weak inhibitor of PHD2. However, upon addition of appropriate diols, boronate esters form reversibly, GW6471 which can bind tightly to PHD2 (Supplemental Figure S25).82 As a proof-of-principle study, the boronic acid scaffold and the Rabbit Polyclonal to SIRPB1 reported diol hits were subjected to the NMR reporter analyses using CPMG-edited 1H NMR (Supplemental Figure S25).82 At 50 M concentration, the boronic acid appeared to cause GW6471 ~50% 2OG displacement (Figure 5), confirming it is a weak binder to PHD2. A slightly higher concentration (150 M) of diols (than the boronic acid scaffold) was used GW6471 to ensure the generation of the boronate ester species. In the absence of the boronic acid, none of the diols appeared to displace 2OG from PHD2 (Figure 5). However, in the presence of the boronic acid and the diols, a significant improvement in 2OG displacement was observed (~75% to ~85% 2OG displacement; Figure 5), in agreement with the results obtained from non-denaturing electrospray ionisation mass spectrometry (ESI-MS).82 As negative controls, in the presence of the boronic acid scaffold and butane-2,3-diol, no improvement in 2OG displacement was observed (Figure 5), and in the presence of the boronic acid scaffold and catechol, only a moderate improvement in 2OG displacement was observed (~60%; Figure 5), again in.