Discovery of an inhibitor of a transcription factor using small molecule microarrays and diversity-oriented synthesis.
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.
Koehler
Angela N
AN
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.
Shamji
Alykhan F
AF
Schreiber
Stuart L
SL
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
D000577:Amides; D023081:CCAAT-Binding Factor; D005656:Fungal Proteins; D011714:Pyrans; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase