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 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.