title: Discovery of chemical modulators of a conserved translational control pathway by parallel screening in yeast.
abstract: Eukaryotic initiation factor 2 (eIF2) B is a guanine nucleotide exchange factor that plays a central role in translation initiation and its control, especially in response to diverse cellular stresses. In addition, inherited mutations in human eIF2B subunits cause a fatal brain disorder commonly called childhood ataxia with central nervous system hypomyelination or leukoencephalopathy with vanishing white matter. In yeast, inhibiting activity of eIF2B up-regulates expression of the transcriptional activator general control nondepressible (GCN) 4. We report here evaluation of high-throughput screening (HTS) using a yeast-based reporter gene assay, in which strains containing either wild-type or a mutant eIF2B were screened in parallel to identify compounds modifying eIF2B-dependent responses. The goals of the HTS were twofold: first, to discover compounds that restore normal function to mutant eIF2B, which may have therapeutic utility for the fatal human disease; and second, to identify compounds that activate a GCN4 response, which might be useful experimental tools. The HTS assay measured cell growth by absorbance, and activation of gene expression via a beta-galactosidase reporter gene fusion. Because mutant eIF2B activates GCN4 in the absence of stress inducers, the mutant strain was screened for reduction in GCN4 activation. HTS revealed apparent mutant-selective inhibitors but did not reliably predict selectivity as these hits affected both wild-type and mutant strains equally on dose-response confirmation. Wild-type strain results from the HTS identified two GCN4 response activators, both of which were confirmed to be wild-type selective in dose-response testing, suggesting that these compounds may activate GCN4 by a mechanism that down-regulates eIF2B activity.
authors.affiliation: Penn Center for Molecular Discovery, Institute for Medicine and Engineering, and Department of Chemical and Biomolecular Engineering, University of Pennsylvania, Philadelphia, Pennsylvania, USA.
authors.forename: Nuzhat
authors.initials: N
authors.lastname: Motlekar
authors.forename: Rogerio Alves
authors.initials: RA
authors.lastname: de Almeida
authors.forename: Graham D
authors.initials: GD
authors.lastname: Pavitt
authors.forename: Scott L
authors.initials: SL
authors.lastname: Diamond
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Napper
mesh_terms: D004305:Dose-Response Relationship, Drug; D055808:Drug Discovery / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D015723:Gene Library; D006801:Humans; D049449:Luminescence; D008904:Miniaturization; D009154:Mutation / Q000502:physiology; D018384:Oxidative Stress / Q000502:physiology; D015203:Reproducibility of Results; D012371:Robotics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D015398:Signal Transduction / Q000502:physiology; D054852:Small Molecule Libraries; D001616:beta-Galactosidase / Q000378:metabolism
chemical_list: D054852:Small Molecule Libraries; D001616:beta-Galactosidase