Chemical shifts perturbations

Fragment screening by NMR was applied recently in the search of non-peptidic small molecule inhibitors. Two scaffolds (13) and (14), which bind the enzyme at the S1-S3 and the S2 binding site respectively, as shown by chemical shift perturbation, were linked together to yield competitive inhibitors such as (15) with micromolar IC50 values [158]. There have been no reports of non-peptidic inhibitors with potency and pharmacokinetics similar to the peptidic or peptidomimetic inhibitors described above. 

A simple NMR technique, and arguably the most widely used and effective for hit validation, is the chemical shift perturbation method. In this approach, a reference spectrum of isotopically labeled target is recorded in absence and presence of a given test ligand (or a mixture of test ligands). Commonly, differences in chemical shift between free and bound protein target are observed in 2D [15N, 1H and/or 2D [13C, H] correlation spectra of a protein (or nucleic acid) upon titration of a ligand... 

Fig. 2 Chemical shift perturbation and chemical shift mapping, (a) Portions of the [15N, 1H]-HSQC spectra of Bcf-xL recorded in absence (black) and in presence of each of the four molecules (in colors). Resonance assignments for amino acid residues that exhibit large shifts are reported, (b) Structure of Bc1-Xl in complex with the BH3 peptide from Bak (PDB code 1BXL) showing the chemical shift changes in Bcl-xL upon ligand binding (blue, large shits yellow, no shifts the Bak peptide is reported in cyan). Adapted from [48]...

Other studies by Chen et al.35 have focused on how O-GlcNAcylation and O-phosphorylation can induce relevant differences in the structural and functional behaviour of the /V-terminus of Murine Estrigen Receptor b (mER-b). By using NOE data, chemical shift perturbations of NH protons and molecular dynamics simulations, subtle differences in the secondary structure of the characterized peptides were identified. 

Fig. 18.1 Fragment combination strategies. Left In SAR by NMR [5], small molecular fragments that bind to the target are identified using protein-detected experiments. In the follow-up step, compounds bound in close proximity to one another are identified by chemical shift perturbation and intermolecular NOE experiments, and distance information is used to design and synthesize a linked compound. Center In fragment...

Aqueous solubility of compounds is a critical issue, since NMR screens must be run at relatively high compound concentrations, anywhere from millimolar for NOE, chemical shift perturbation or affinity NMR methods [3, 5, 103-108] to ca. 50 pM for saturation-... 

Figure 12.4A shows the interaction of the first CUE domain of Cue2 interacting with ubiquitin, which might serve as a general model for the interaction mode of other UBA-like domains. The CUE domain binds to the Ile-44 patch of ubiquitin, in accordance with the chemical shift perturbation results of the UBA ubiquitin interaction [52], On the side of the CUE domain, residues of the first and third helix participate in this interaction surface. These residues include the Phe-Pro and Leu-Leu motifs, which had been predicted to be important for ubiquitin binding, based on comparative sequence analysis of CUE-A and CUE-B domains [62]. Positions in close contact with ubiquitin are also indicated in the alignment of Figure 12.3. The two available structures of the CUE ubiquitin complexes offer little expla-...

The C-terminal domain (85 amino acid residues, not completely denatured at 90 °C) of the so-called a subunit of the RNAP from the extremely thermophilic eubacterium T. thermophilus (Tt) has been expressed uniformly N/ C-labelled and structurally characterized by the NMR spectroscopy. The tertiary structure of the domain, comprising a helical turn and four helices, was found to be almost identical to that of the corresponding domain from the mesophilic E. coli, despite 32% sequence homology. The interaction of the Tt domain with a variety of DNAs at 37 °C and 50 °C was investigated by chemical shift perturbation of the NMR signals and the DNA binding site was localized. ... 

The coupling of the GPC spin column/ESI-MS screening results with NMR (2D HSQC) is a powerful method for confirming that the non-covalent binders identified by the MS experiments truly bind at the predicted active site by observing NMR chemical shift perturbations in the vicinity of the protein active site [1, 15]. In contrast, the absence of chemical shift perturbations or a random distribution of chemical shift changes on the protein surface would imply a lack of an interaction of the compound with the protein or potentially the existence of non-specific binding. The development of the GPC spin column/MS/NMR assay... 

Protein chemical shift perturbation — Unshifted Shifted 32... 

McCoy, M. A. and Wyss, D. F., Spatial localization of ligand binding sites from electron current density surfaces calculated from NMR chemical shift perturbations. JAm Chem Soc, 2002,... 

Rajesh, S., Sakamoto, T., Iwamoto-Sugai, M., Shibata, T., Kohno, T., and Ito, Y. (1999) Ubiquitin binding interface mapping on yeast ubiquitin hydrolase by NMR chemical shift perturbation. Biochemistry 38, 9242-9253. 

Another variant of this methodology involves the analysis of differential chemical shift perturbations of a series of closely related ligands to rapidly determine the precise location of the ligand binding site and the orientation of the ligand in the binding pocket, which provides critical structural information for lead development and optimization [116, 117]. 

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