Discovery of an Inhibitor of a Transcription Factor Using Small Molecule Microarrays and Diversity-Oriented Synthesis

• Published in: Journal of the American Chemical Society Vol. 125; no. 28; pp. 8420 - 8421
• Main Authors: Koehler, Angela N, Shamji, Alykhan F, Schreiber, Stuart L
• Format: Journal Article
• Language: English
• Published: WASHINGTON American Chemical Society 16.07.2003; Amer Chemical Soc
• Subjects: Amides - chemical synthesis; Amides - metabolism; Amides - pharmacology; Biological and medical sciences; CCAAT-Binding Factor - antagonists & inhibitors; CCAAT-Binding Factor - genetics; CCAAT-Binding Factor - metabolism; Chemistry; Chemistry, Multidisciplinary; Combinatorial Chemistry Techniques - methods; Fundamental and applied biological sciences. Psychology; Fungal Proteins - antagonists & inhibitors; Fungal Proteins - genetics; Fungal Proteins - metabolism; Glutathione Transferase - genetics; Glutathione Transferase - metabolism; Kinetics; Molecular and cellular biology; Physical Sciences; Pyrans - chemical synthesis; Pyrans - pharmacology; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae; Science & Technology; Structure-Activity Relationship; Surface Plasmon Resonance; Transcription Factors - antagonists & inhibitors; Transcription Factors - genetics; Transcription Factors - metabolism; ORGANIC-SYNTHESIS; YEAST HAP2; CHEMICAL GENETICS; DRUG DISCOVERY; ONE-BEAD; GENE-EXPRESSION; STOCK SOLUTION APPROACH; hap3; SACCHAROMYCES-CEREVISIAE; CCAAT-BINDING; MITOCHONDRIAL PROTEIN; Genetics
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja0352698

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 K D of 5.03 μM 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 K D of 330 nM. Whole-genome transcriptional profiling was used to compare effects of haptamide B with a hap3Δ 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.