A high-throughput screen to identify inhibitors of SOD1 transcription

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.

Neuroprotective Small Molecules for the Treatment of Amyotrophic Lateral Sclerosis

“…And, Benmohamed et al (39) developed a cellular model in which the readout is the blocking of mutant SOD1-induced aggregation. Furthermore, Wright et al (40) used the inhibition of SOD1 as their readout. What these methods lack is simplicity.…”

High content analysis in amyotrophic lateral sclerosis

“…In one screening, most of the drugs that were able to reduce the promoter activity also reduced cellular viability while no drug resulted in specific reduction of SOD1 protein 150 . In the second study only 3 compounds showed significant effect in reducing the promoter without affecting cell survival 82 . Compound number 7687685 was the only one able to reduce the amount of SOD1 mRNA and protein also when tested in a human cell line (HeLa cells).…”

“…In particular, many investigators using different methods proved that the reduction of the human SOD1 protein was beneficial in rescuing the ALS phenotype 75–77 , in delaying the onset and/or increasing the life span of animal models of ALS 6,78,79 . Different methods have been applied to achieve direct or indirect reduction of the SOD1 protein through administration of small molecules 80–82 or antibodies 83,84 , or the application of RNA technologies, including microRNAs 85,86 , RNAi 77,79,87–89 and antisense Oligonucleotides (ASO) 90 . Of great interest is the first human clinical trial conducted by Richard Smith and Tim Miller and his team in collaboration with Ionis Pharmaceuticals, involving the intrathecal delivery of an ASO against SOD1.…”

“…In the other assay, they were transfected with a vector expressing a fusion protein of a mutant form of SOD1 and GFP. In two separate studies, 82,150 these assays were used in combination with a HCA approach to screen various libraries for more than 100,000 compounds for their ability to: 1) reduce SOD1 promoter activity or 2) increase the degradation of the protein, following the assumption that reducing the cellular amount of SOD1 would ameliorate the ALS phenotype. Interestingly, despite the large number of chemicals screened the studies reported very few positive hits.…”

Regulation of CuZnSOD and Its Redox Signaling Potential: Implications for Amyotrophic Lateral Sclerosis

“…Decreasing the expression of SOD1 has also been explored as a therapeutic avenue to reduce SOD1 expression in ALS. Compounds that inhibit SOD1 transcription in model cell lines have been identified, and may serve as the basis for developing clinically viable strategies for ALS aimed at decreasing SOD1 expression (66).…”