Use of 19F NMR spectroscopy to screen chemical libraries for ligands that bind to proteins.

Identification of compounds from chemical libraries that bind to macromolecules by use of NMR spectroscopy has gained increasing importance during recent years. A simple methodology based on (19)F NMR spectroscopy for the screening of ligands that bind to proteins, which also provides qualitative information about relative binding strengths and the presence of multiple binding sites, is presented here. A library of fluorinated compounds was assembled and investigated for binding to the two bacterial chaperones PapD and FimC, and also to human serum albumin (HSA). It was found that library members which are bound to a target protein could be identified directly from line broadening and/or induced chemical shifts in a single, one-dimensional (19)F NMR spectrum. The results obtained for binding to PapD using (19)F NMR spectroscopy agreed well with independent studies based on surface plasmon resonance, providing support for the versatility and accuracy of the technique. When the library was titrated to a solution of PapD chemical shift and linewidth changes were observed with increasing ligand concentration, which indicated the presence of several binding sites on PapD and enabled the assessment of relative binding strengths for the different ligands. Screening by (19)F NMR spectroscopy should thus be a valuable addition to existing NMR techniques for evaluation of chemical libraries in bioorganic and medicinal chemistry.

[1]  J. Pinkner,et al.  Real-time and equilibrium 19F-NMR studies reveal the role of domain–domain interactions in the folding of the chaperone PapD , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Stephen W. Fesik,et al.  One-Dimensional Relaxation- and Diffusion-Edited NMR Methods for Screening Compounds That Bind to Macromolecules , 1997 .

[3]  M. Sundström,et al.  Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water* , 2000, Journal of biomolecular NMR.

[4]  J. T. Gerig,et al.  Binding of 5-fluoro-L-tryptophan to human serum albumin , 1980 .

[5]  Marina Veronesi,et al.  Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures. , 2002, Combinatorial chemistry & high throughput screening.

[6]  R. Reed.  Location of long chain fatty acid-binding sites of bovine serum albumin by affinity labeling. , 1986, The Journal of biological chemistry.

[7]  J. Hamilton,et al.  Locations of the three primary binding sites for long-chain fatty acids on bovine serum albumin. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[8]  L. Fielding.  Determination of Association Constants (Ka) from Solution NMR Data , 2000 .

[9]  J. T. Gerig.  Fluorine NMR of proteins , 1994 .

[10]  Mengfen Lin,et al.  Diffusion-Edited NMR−Affinity NMR for Direct Observation of Molecular Interactions , 1997 .

[11]  P. Hajduk,et al.  Discovering High-Affinity Ligands for Proteins: SAR by NMR , 1996, Science.

[12]  M. Shapiro,et al.  Screening Mixtures by Affinity NMR , 1997 .

[13]  J. Falke,et al.  Open conformation of a substrate-binding cleft: 19F NMR studies of cleft angle in the D-galactose chemosensory receptor. , 1991, Biochemistry.

[14]  H. Kalbitzer,et al.  Mobility of the N-terminal segment of rabbit skeletal muscle F-actin detected by 1H and 19F nuclear magnetic resonance spectroscopy. , 1996, Biochemistry.

[15]  J. T. Gerig.  Fluorine nuclear magnetic resonance of fluorinated ligands. , 1989, Methods in enzymology.

[16]  Bernd Meyer,et al.  Characterization of Ligand Binding by Saturation Transfer Difference NMR Spectroscopy. , 1999, Angewandte Chemie.

[17]  L. Phillips,et al.  Fluorine 19 NMR studies of the interaction of selectively labeled actin and myosin , 1989 .

[18]  Claudio Dalvit,et al.  Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practical considerations, and range of applicability. , 2003, Journal of the American Chemical Society.

[19]  Lee Fielding,et al.  NMR methods for the determination of protein-ligand dissociation constants. , 2003, Current topics in medicinal chemistry.

[20]  Carl Frieden,et al.  19F NMR spectroscopy of [6-19F]tryptophan-labeled Escherichia coli dihydrofolate reductase: equilibrium folding and ligand binding studies. , 1994, Biochemistry.

[21]  Bruce G. Jenkins,et al.  Detection of site-specific binding and co-binding of ligands to human serum albumin using 19F NMR. , 1990, Molecular pharmacology.

[22]  L. Sklar,et al.  Conjugated polyene fatty acids as fluorescent probes: binding to bovine serum albumin. , 1977, Biochemistry.

[23]  J. Pinkner,et al.  Design and Evaluation of Pilicides: Potential Novel Antibacterial Agents Directed Against Uropathogenic Escherichia coli , 2001, Chembiochem : a European journal of chemical biology.

[24]  Michael J. Shapiro,et al.  NOE Pumping. 2. A High-Throughput Method To Determine Compounds with Binding Affinity to Macromolecules by NMR , 2000 .

[25]  B. Sykes,et al.  19F nuclear magnetic resonance studies of selectively fluorinated derivatives of G- and F-actin. , 1986, Biochemistry.

[26]  Per Falk,et al.  Pilus and nonpilus bacterial adhesins: Assembly and function in cell recognition , 1993, Cell.

[27]  E. Oldfield,et al.  Fluorine-19 nuclear magnetic resonance spectroscopic study of fluorophenylalanine- and fluorotryptophan-labeled avian egg white lysozymes. , 1994, Biochemistry.

[28]  Michael J. Shapiro,et al.  NOE Pumping: A Novel NMR Technique for Identification of Compounds with Binding Affinity to Macromolecules , 1998 .

[29]  J. Pinkner,et al.  Design and parallel solid-phase synthesis of ring-fused 2-pyridinones that target pilus biogenesis in pathogenic bacteria. , 2002, Journal of combinatorial chemistry.

[30]  J. Falke,et al.  Use of 19F NMR to probe protein structure and conformational changes. , 1996, Annual review of biophysics and biomolecular structure.

[31]  J. Emsley,et al.  Fluorine chemical shifts , 1971 .

[32]  M. Shapiro,et al.  Mixture Analysis in Combinatorial Chemistry. Application of Diffusion-Resolved NMR Spectroscopy. , 1996, The Journal of organic chemistry.

[33]  Jens Klein,et al.  DETECTING BINDING AFFINITY TO IMMOBILIZED RECEPTOR PROTEINS IN COMPOUND LIBRARIES BY HR-MAS STD NMR , 1999 .

[34]  J. Falke,et al.  19F NMR studies of the D-galactose chemosensory receptor. 2. Ca(II) binding yields a local structural change. , 1991, Biochemistry.

[35]  Ajay,et al.  The SHAPES strategy: an NMR-based approach for lead generation in drug discovery. , 1999, Chemistry & biology.

[36]  B. Sykes,et al.  [13] Fluorine nuclear magnetic resonance studies of proteins , 1978 .

[37]  B. Meyer,et al.  Screening mixtures for biological activity by NMR. , 1997, European journal of biochemistry.