Dissecting glucose signalling with diversity-oriented synthesis and small-molecule microarrays

Small molecules that alter protein function provide a means to modulate biological networks with temporal resolution. Here we demonstrate a potentially general and scalable method of identifying such molecules by application to a particular protein, Ure2p, which represses the transcription factors Gln3p and Nil1p. By probing a high-density microarray of small molecules generated by diversity-oriented synthesis with fluorescently labelled Ure2p, we performed 3,780 protein-binding assays in parallel and identified several compounds that bind Ure2p. One compound, which we call uretupamine, specifically activates a glucose-sensitive transcriptional pathway downstream of Ure2p. Whole-genome transcription profiling and chemical epistasis demonstrate the remarkable Ure2p specificity of uretupamine and its ability to modulate the glucose-sensitive subset of genes downstream of Ure2p. These results demonstrate that diversity-oriented synthesis and small-molecule microarrays can be used to identify small molecules that bind to a protein of interest, and that these small molecules can regulate specific functions of the protein.

[1]  S. Schreiber,et al.  Carbon- and nitrogen-quality signaling to translation are mediated by distinct GATA-type transcription factors , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[2]  P. Clemons,et al.  A one-bead, one-stock solution approach to chemical genetics: part 2. , 2001, Chemistry & biology.

[3]  JOHN W. Moore,et al.  Tetrodotoxin Blockage of Sodium Conductance Increase in Lobster Giant Axons , 1964, The Journal of general physiology.

[4]  J. Satrústegui,et al.  Ammonia accumulation in acetate-growing yeast. , 1983, Biochimica et biophysica acta.

[5]  Michael N. Hall,et al.  The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors , 1999, Nature.

[6]  John A. Tallarico,et al.  A one-bead, one-stock solution approach to chemical genetics: part 1. , 2001, Chemistry & biology.

[7]  S. Schreiber,et al.  Target-oriented and diversity-oriented organic synthesis in drug discovery. , 2000, Science.

[8]  R. Wickner,et al.  [URE3] as an altered URE2 protein: evidence for a prion analog in Saccharomyces cerevisiae. , 1994, Science.

[9]  E. Lander,et al.  Remodeling of yeast genome expression in response to environmental changes. , 2001, Molecular biology of the cell.

[10]  S. Schreiber,et al.  Split--pool synthesis of 1,3-dioxanes leading to arrayed stock solutions of single compounds sufficient for multiple phenotypic and protein-binding assays. , 2001, Journal of the American Chemical Society.

[11]  B. Magasanik,et al.  Interaction of the GATA factor Gln3p with the nitrogen regulator Ure2p in Saccharomyces cerevisiae , 1996, Journal of bacteriology.

[12]  Stuart L. Schreiber,et al.  Printing Small Molecules as Microarrays and Detecting Protein−Ligand Interactions en Masse , 1999 .

[13]  X. Zheng,et al.  Tripartite Regulation of Gln3p by TOR, Ure2p, and Phosphatases* , 2000, The Journal of Biological Chemistry.

[14]  R Andhare,et al.  Nitrogen Catabolite Repression of DAL80Expression Depends on the Relative Levels of Gat1p and Ure2p Production in Saccharomyces cerevisiae * , 2000, The Journal of Biological Chemistry.

[15]  J. Heitman,et al.  The TOR signaling cascade regulates gene expression in response to nutrients. , 1999, Genes & development.

[16]  J. Lehmann,et al.  An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-activated Receptor γ (PPARγ) (*) , 1995, The Journal of Biological Chemistry.

[17]  B. Magasanik,et al.  The URE2 gene product of Saccharomyces cerevisiae plays an important role in the cellular response to the nitrogen source and has homology to glutathione s-transferases , 1991, Molecular and cellular biology.

[18]  S. Schreiber,et al.  Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[19]  H. Mewes,et al.  Toward a catalog of human genes and proteins: sequencing and analysis of 500 novel complete protein coding human cDNAs. , 2001, Genome research.

[20]  S. Schreiber,et al.  Vector algebra in the analysis of genome-wide expression data , 2002, Genome Biology.

[21]  R. Wickner,et al.  Mks1p is a regulator of nitrogen catabolism upstream of Ure2p in Saccharomyces cerevisiae. , 1999, Genetics.

[22]  S. Xu,et al.  Roles of URE2 and GLN3 in the proline utilization pathway in Saccharomyces cerevisiae , 1995, Molecular and cellular biology.

[23]  R. Wickner,et al.  A protein required for prion generation: [URE3] induction requires the Ras-regulated Mks1 protein. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[24]  D. Cheresh.  URE 3 ] as an Altered URE 2 Protein : Evidence for a Prion Analog in Saccharomyces cerevisiae , 2022 .

[25]  Stuart L. Schreiber,et al.  Partitioning the transcriptional program induced by rapamycin among the effectors of the Tor proteins , 2000, Current Biology.

[26]  D. Botstein,et al.  Genomic expression programs in the response of yeast cells to environmental changes. , 2000, Molecular biology of the cell.

[27]  H. Krebs,et al.  Synthesis of Cell Constituents from C2-Units by a Modified Tricarboxylic Acid Cycle , 1957, Nature.

[28]  Stuart L. Schreiber,et al.  Small-Molecule Microarrays: Covalent Attachment and Screening of Alcohol-Containing Small Molecules on Glass Slides , 2000 .