A general framework for development and data analysis of competitive high-throughput screens for small-molecule inhibitors of protein-protein interactions by fluorescence polarization.

Equilibrium binding experiments are widely used for the accurate characterization of binding and competitive binding behavior in biological systems. Modern high-throughput discovery efforts in chemical biology rely heavily upon this principle. Here, we derive exact analytical expressions for general competitive binding models which can also explain a commonly encountered phenomenon in these types of experiments, anticooperative incomplete displacement. We explore the effects of nonspecific binding behavior and parameter misestimation. All expressions are derived in terms of total concentrations determined a priori. We discuss a general framework for high-throughput screening assays based on fluorescence polarization and strategies for assay development, sensitivity regimes, data quality control, analysis, and ranking. Theoretical findings are visualized by simulations using realistic parameter sets. Our results are the basis for the discovery of small-molecule inhibitors of the protein-protein interaction between human calcineurin and NFAT transcription factors, as discussed in the subsequent paper (31).

[1]  General numerical treatment of competitive binding kinetics: application to thrombin-dehydrothrombin-hirudin. , 1999 .

[2]  U. Haupts,et al.  Homogeneous fluorescence readouts for miniaturized high-throughput screening: theory and practice. , 1999, Drug discovery today.

[3]  A. Hamilton,et al.  Peptide and protein recognition by designed molecules. , 2000, Chemical reviews.

[4]  S. Ng,et al.  Development of a high-throughput fluorescence polarization assay for Bcl-x(L). , 2002, Analytical biochemistry.

[5]  P. Toogood.  Inhibition of protein-protein association by small molecules: approaches and progress. , 2002, Journal of medicinal chemistry.

[6]  T. Hazlett,et al.  Optical spectroscopy in studies of antibody-hapten interactions. , 2000, Methods.

[7]  Gregory J. Parker,et al.  Development of High Throughput Screening Assays Using Fluorescence Polarization: Nuclear Receptor-Ligand-Binding and Kinase/Phosphatase Assays , 2000, Journal of biomolecular screening.

[8]  D. Jameson,et al.  Quantification of protein-protein interactions using fluorescence polarization. , 1999, Methods.

[9]  James R. Knight,et al.  A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae , 2000, Nature.

[10]  G. Weber.  Polarization of the fluorescence of macromolecules. I. Theory and experimental method. , 1952, The Biochemical journal.

[11]  J. García de la Torre,et al.  Calculation of hydrodynamic properties of globular proteins from their atomic-level structure. , 2000, Biophysical journal.

[12]  S. Ng,et al.  Development of a high-throughput fluorescence polarization assay for Bcl-x(L). , 2002, Analytical biochemistry.

[13]  M. Allen,et al.  High Throughput Fluorescence Polarization: A Homogeneous Alternative to Radioligand Binding for Cell Surface Receptors , 2000, Journal of biomolecular screening.

[14]  T. Pawson,et al.  Protein-protein interactions define specificity in signal transduction. , 2000, Genes & development.

[15]  J M Gauthier,et al.  Protein--protein interaction maps: a lead towards cellular functions. , 2001, Trends in genetics : TIG.

[16]  W. Janzen,et al.  High-throughput screening: advances in assay technologies. , 1997, Current opinion in chemical biology.

[17]  R. Hertzberg,et al.  High-throughput screening: new technology for the 21st century. , 2000, Current opinion in chemical biology.

[18]  Z. X. Wang,et al.  An exact mathematical expression for describing competitive binding of two different ligands to a protein molecule , 1995, FEBS letters.

[19]  S. Jones,et al.  Principles of protein-protein interactions. , 1996, Proceedings of the National Academy of Sciences of the United States of America.

[20]  A. Fersht.  Structure and mechanism in protein science , 1998 .

[21]  T. Creighton.  Proteins: Structures and Molecular Properties , 1986 .

[22]  B Cox,et al.  Application of high-throughput screening techniques to drug discovery. , 2000 .

[23]  Sarah A. Teichmann,et al.  Principles of protein-protein interactions , 2002, ECCB.

[24]  A G Cochran,et al.  Antagonists of protein-protein interactions. , 2000, Chemistry & biology.

[25]  G. Weber.  Polarization of the fluorescence of macromolecules. II. Fluorescent conjugates of ovalbumin and bovine serum albumin. , 1952, The Biochemical journal.

[26]  P. Bork,et al.  Functional organization of the yeast proteome by systematic analysis of protein complexes , 2002, Nature.

[27]  P. Uetz.  Two-hybrid arrays. , 2002, Current opinion in chemical biology.

[28]  G. Wagner,et al.  Selective inhibition of calcineurin-NFAT signaling by blocking protein-protein interaction with small organic molecules. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[29]  M. Vidal,et al.  Combined Functional Genomic Maps of the C. elegans DNA Damage Response , 2002, Science.

[30]  Thomas D. Y. Chung,et al.  A Simple Statistical Parameter for Use in Evaluation and Validation of High Throughput Screening Assays , 1999, Journal of biomolecular screening.

[31]  N. Oppenheimer,et al.  Structure and mechanism , 1989 .

[32]  K Ashman,et al.  Cell Signalling - The Proteomics of It All , 2001, Science's STKE.

[33]  C. Chothia,et al.  The atomic structure of protein-protein recognition sites. , 1999, Journal of molecular biology.

[34]  M. Toth,et al.  Measurement of specific protease activity utilizing fluorescence polarization. , 1997, Analytical biochemistry.

[35]  R. Strausberg,et al.  From Knowing to Controlling: A Path from Genomics to Drugs Using Small Molecule Probes , 2003, Science.

[36]  G. Wagner,et al.  Discovery of small-molecule inhibitors of the NFAT--calcineurin interaction by competitive high-throughput fluorescence polarization screening. , 2004, Biochemistry.

[37]  P. Uetz,et al.  Towards an understanding of complex protein networks. , 2001, Trends in cell biology.