title: Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis.
abstract: Small molecule microarrays were screened to identify a small molecule ligand for Hap3p, a subunit of the yeast Hap2/3/4/5p transcription factor complex. The compound, named haptamide A, was determined to have a KD of 5.03 muM for binding to Hap3p using surface plasmon resonance analysis. Haptamide A also inhibited activation of a GDH1-lacZ reporter gene in a dose-dependent fashion. To explore structure-activity relationships, 11 derivatives of haptamide A were prepared using the same synthetic route that was developed for the original library synthesis. Analysis of dissociation constants and IC50 values for the reporter gene assay revealed a more potent inhibitor, haptamide B, with a KD of 330 nM. Whole-genome transcriptional profiling was used to compare effects of haptamide B with a hap3Delta yeast strain. Treatment with haptamide B, like the deletion mutant, reduced lactate-induced transcription of several genes from wild-type levels. Profiling the genetic "knockout" and the chemical genetic "knockdown" led to the identification of several genes that are regulated by Hap3p under nonfermentative conditions. These results demonstrate that a small molecule discovered using the small molecule microarray binding assay can permeate yeast cells and reach its target transcription factor protein in cells.
authors.affiliation: Department of Chemistry and Chemical Biology, Harvard Biophysics Program, Howard Hughes Medical Institute, Harvard Institute of Chemistry and Cell Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
authors.forename: Angela N
authors.initials: AN
authors.lastname: Koehler
authors.forename: Alykhan F
authors.initials: AF
authors.lastname: Shamji
authors.forename: Stuart L
authors.initials: SL
authors.lastname: Schreiber
mesh_terms: D000577:Amides / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology*; D023081:CCAAT-Binding Factor / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D005656:Fungal Proteins / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D011714:Pyrans / Q000138:chemical synthesis / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014157:Transcription Factors / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D000577:Amides; D023081:CCAAT-Binding Factor; D005656:Fungal Proteins; D011714:Pyrans; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase