[Frontiers in Bioscience E4, 2701-2708, June 1, 2012]

A high-throughput screen to identify inhibitors of SOD1 transcription

Paul D Wright1, Nicholas Wightman1, Mickey Huang2, Alexandra Weiss1, Peter C Sapp1,3, Gregory D Cuny2, Adrian J Ivinson2, Marcie A Glicksman2, Robert J Ferrante4,5, Wayne Matson5, Samantha Matson6,7, Robert H Brown Jr1

1Department of Neurology, University of Massachusetts Medical School, Worcester, MA 01655, United States, 2Laboratory for Drug Discovery in Neurodegeneration, Harvard NeuroDiscovery Center, Harvard Medical School and Brigham and Women's Hospital, Cambridge, MA 02139, United States, 3Howard Hughes Medical Institute, Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, United States, 4Neurological Surgery, Neurology, and Neurobiology Departments, University School of Medicine of Pittsburgh, Pittsburgh, PA 15213, United States, 5Geriatric Research Educational and Clinical Center (00-GR-H), V.A. Pittsburgh Healthcare System, 7180 Highland Drive, Pittsburgh, PA 15206, United States, 6Neurology Service, Massachusetts General Hospital and Harvard Medical School, Boston, MA 02114, United States, 7MassGeneral Institute for Neurodegenerative Disease, Massachusetts General Hospital, Charlestown, MA 02129, United States

TABLE OF CONTENTS

1. Abstract
2. Introduction
3. Materials and methods
3.1. Cell culture
3.2. Compound screening
3.3. Western blotting
3.4. Quantitative PCR
3.5. In vivo studies
4. Results
4.1. Screening 30,000 small molecules for inhibitors of the SOD1 promoter
4.2. Structure Activity Relationship
4.3. Effects of SOD1 inhibition on HeLa cell growth rates and general transcription
4.4. Effects of Compound 7687685 on SOD1 transcription in vivo
5. Discussion
6. Acknowledgments
7. References

1. ABSTRACT

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. Approximately 20% of familial ALS cases are caused by mutations in the Cu/Zn superoxide dismutase (SOD1) gene. Rodents expressing mutant SOD1 transgenes develop progressive, fatal motor neuron disease and disease onset and progression is dependent on the level of SOD1. We investigated the possibility that a reduction in SOD1 protein may be of therapeutic benefit in ALS and screened 30,000 compounds for inhibition of SOD1 transcription. The most effective inhibitor identified was N-{4-(4-(4-methylbenzoyl)-1-piperazinyl)phenyl}-2-thiophenecarboxamide (Compound ID 7687685), which in PC12 cells showed an EC50 of 10.6 microM for inhibition of SOD1 expression and an LD50 >30 microM. This compound was subsequently shown to reduce endogenous SOD1 levels in HeLa cells and to exhibit a modest reduction of SOD1 protein levels in mouse spinal cord tissue. These data suggest that the efficacy of compound 7687685 as an inhibitor of SOD1 gene expression is not likely to be clinically useful, although the strategy reported could be applied broadly to screening for small molecule inhibitors of gene expression.