A high-throughput screen to identify inhibitors of SOD1 transcription

Abstract

Amyotrophic lateral sclerosis (ALS) is a fatal degenerative motor neuron disease. Approximately 20 percent 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 more than 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.

Figure 1

(A) Screening of 30,000 compounds from the Chembridge library for inhibitors of the SOD1 promoter. % Inhibition of fluorescence shows the effects of compounds on the SOD1 promoter. Those demonstrating inhibition of 30% or greater were designated ‘hits’. (B) 12 point dose response curves for compounds 7687685, 7698016 and 7614572 illustrate the dose dependence of cytotoxicity (dashed line) and SOD1 inhibition (solid line) relative to DMSO treated controls. Each of these compounds shows a greater inhibition of SOD1 promoter mediated fluorescence than cytotoxicity. (C) qPCR and Western blot analyses document that 30µM 7687685 reduces levels of endogenous SOD1 mRNA and endogenous SOD1 protein in HeLa cells (n=3, p<0.05). 30 microM 7614572 and 3 microM 7698016 showed no effect on SOD1 mRNA or endogenous SOD1 protein.

Figure 2

(A) Single or multiple exposures to 30 microM 7687685 reduce the growth rate of HeLa cells. Heavy solid line: DMSO; light solid line: single treatment at start of experiment; short dashed line: once daily treatment for three days; long dashed line: treatment with siRNA directed against SOD1. In each paradigm cells were incubated in the presence of drug or siRNA for 4h, 24h, 48h or 72h. (B) Expression analysis using qPCR documents that there is off target suppression of expression of all six genes tested in HeLa cells treated with either 7687685 or SOD1 siRNA. mRNA levels for each probe are normalised to the control samples (C) Expression analysis shows off target effects of HeLa cells treated with 7687685 and SOD1 siRNA. Higher CT values reflect lower mRNA levels.

Figure 3

(A) Selected properties of compound 7687685 that predict effective penetrance of the blood-brain barrier. (B) Chemical structure of compound 7687685.