Deuterated Disaccharides for the Investigation of Protein-Carbohydrate Interactions-Application of Bioaffinity-and STD-NMR

Hellebrandt, Wilfried; Haselhorst, Thomas; Köhli, Thies; Bäuml, Englbert; Peters, Thomas

doi:10.1080/07328300008544115

Cited by 4 publications

(1 citation statement)

References 17 publications

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“…Moreover, the method is limited to studies of proteins having molecular masses <40 kDa. For those target proteins that do not meet these criteria, other NMR screening methods for binding activity, such as saturation-transfer difference spectroscopy ( − ) and diffusion-based experiments ( − ), have been developed. Although these methods do not require large amounts of pure, labeled protein and are not limited by the size of the protein, they do not provide any information about the location of the binding site.…”

Section: Resultsmentioning

confidence: 99%

Identification of Novel Small Molecules That Bind to Two Different Sites on the Surface of Tetanus Toxin C Fragment

Cosman

Lightstone

Krishnan

et al. 2002

Chem. Res. Toxicol.

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A combination of computational methods, electrospray ionization mass spectroscopy (ESI-MS), and NMR spectroscopy has been used to identify novel small molecules that bind to two adjacent sites on the surface of the C fragment of tetanus toxin (TetC). One of these sites, Site-1, binds gangliosides present on the surface of motor neurons, while Site-2 is a highly conserved deep cleft in the structures of the tetanus (TeNT) and botulinum (BoNT) neurotoxins. ESI-MS was used to experimentally determine which of the top 11 computationally predicted Site-2 candidates bind to TetC. Each of the six molecules that tested positive was further screened, individually and as mixtures, for binding to TetC in aqueous solutions by NMR. A trNOESY competition assay was developed that used doxorubicin as a marker for Site-1 to provide insight into whether the predicted Site-2 ligands bound to a different site. Of the six predicted Site-2 ligands tested, only four were observed to bind. Naphthofluorescein-di-beta-galactopyranoside was insoluble under conditions compatible with TetC. Sarcosine-Arg-Gly-Asp-Ser-Pro did not appear to bind, but its binding affinity may have been outside the range detectable by the trNOESY experiment. Of the remaining four, three [3-(N-maleimidopropionyl)biocytin, lavendustin A, and Try-Glu-Try] bind in the same site, presumably the predicted Site-2. The fourth ligand, Ser-Gln-Asn-Tyr-Pro-Ile-Val, binds in a third site that differs from Site-1 or predicted Site-2. The results provide a rational, cost- and time-effective strategy for the selection of an optimal set of Site-1 binders and predicted Site-2 binders for use in synthesizing novel bidendate antidotes or detection reagents for clostridial neurotoxins, such as TeNT and BoNT.

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Section: Resultsmentioning

confidence: 99%

Identification of Novel Small Molecules That Bind to Two Different Sites on the Surface of Tetanus Toxin C Fragment

Cosman

Lightstone

Krishnan

et al. 2002

Chem. Res. Toxicol.

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NMRand Drug Discovery

Craik

Clark

2003

Burger's Medicinal Chemistry and Drug Discovery

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In this chapter we give an overview of the two major approaches used in NMR and drug discovery: structure‐based design and NMR‐based screening. Combined with the more traditional uses of NMR in medicinal chemistry, they demonstrate the versatility of NMR as a tool for drug discovery. Structure‐based design is considered in two parts, ligand‐based design and receptor‐based design. In the former, NMR provides information on structure elucidation of drug leads, conformational analysis, charge state, tautomeric equilibria, and molecular dynamics and assists in the development of pharmacophore models. In receptor‐based design NMR allows the structures of macromolecular drug targets to be determined and their interactions with ligands to be elucidated. NMR‐based screening is a newer application of NMR technology in drug discovery but it is showing great promise as a tool for lead generation and optimization. Screening approaches based on chemical‐shift perturbation, magnetization transfer, diffusion, relaxation, NOEs, and spin labels are described. The utility of NMR has been enhanced over the last decade by developments in both instruments and methodology. On the instrumental side, increases in magnetic field strengths and the development of cryoprobes have increased sensitivity. Linkages of NMR to liquid chromatography and/or mass spectrometry have increased versatility. On the methods front there have been a range of new approaches discovered that will enhance the study of larger molecular complexes. Advances in protein expression and labeling have played a major role in stimulating the development of new NMR pulse sequences to extract information from such complexes.

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NMR‐Based Screening and Drug Discovery

Craik

Smith

Clark

2010

Burger's Medicinal Chemistry and Drug Discovery

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NMR spectroscopy is a powerful technique for determining the structures of molecules in solution. It involves placing a sample in a strong homogeneous magnetic field and irradiating it with radiowaves of defined frequency. The emitted signals provide information about the local molecular environments of nuclei in the sample, from which structures can be derived. NMR can also be used for determining interactions between molecules and is particularly useful for determining the nature of binding interactions between ligands and macromolecules. This information is very important in drug design: by understanding how bioactive molecules interact with a target protein or nucleic acid it is, in principle, possible to design ligands with improved affinity and specificity that may make useful drug leads. In addition to this structure‐based approach to drug design NMR is also useful as a screening tool in drug discovery programs to identify ligands that bind to target macromolecules.

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Deuterated Disaccharides for the Investigation of Protein-Carbohydrate Interactions-Application of Bioaffinity-and STD-NMR

Cited by 4 publications

References 17 publications

Identification of Novel Small Molecules That Bind to Two Different Sites on the Surface of Tetanus Toxin C Fragment

Identification of Novel Small Molecules That Bind to Two Different Sites on the Surface of Tetanus Toxin C Fragment

NMRand Drug Discovery

NMR‐Based Screening and Drug Discovery

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