Screening for inhibitors of the SOD1 gene promoter: Pyrimethamine does not reduce SOD1 levels in cell and animal models

Abstract

Mutations in the Cu/Zn superoxide dismutase (SOD1) gene are detected in 20% of familial and 3% of sporadic amyotrophic lateral sclerosis (ALS) cases. Although mutant SOD1 is known to induce motor neuron death via multiple adverse acquired functions, its exact pathogenic mechanism is not well defined. SOD1 toxicity is dose dependent; levels of mutant SOD1 protein in transgenic mice determine disease susceptibility, onset and rate of progression. We therefore sought to identify small molecules that reduce SOD1 levels by inhibiting the SOD1 promoter. We tested pyrimethamine (previously reported to suppress SOD1 expression), several compounds currently in trials in human and murine ALS, and a set of 1040 FDA-approved compounds. In a PC12 cell-based assay, no compounds reduced SOD1 promoter activity without concomitant cytotoxicity. Additionally, pyrimethamine failed to repress levels of SOD1 protein in HeLa cells or homogenates of liver, spinal cord and brain of wild-type mice. Thirty-four compounds (including riluzole, ceftriaxone, minocyclin, PBA, lithium, acetylcysteine) in human and mouse ALS trials and an additional set of 1040 FDA-approved compounds also showed no effect on SOD1 promoter activity. This present study thus failed to identify small molecule inhibitors of SOD1 gene expression.

Introduction

Amyotrophic lateral sclerosis (ALS) is an age-dependent, neurodegenerative disease characterized pathologically by the selective loss of motor neurons in the brain and spinal cord. Symptoms of muscle weakness, cramp or spasticity begin in a single limb but become generalized, and death, due to respiratory failure, occurs within three to five years. ALS is uniformly fatal. The single FDA-approved treatment for ALS (Riluzole) increases lifespan by only 3–6 months and does not substantially alleviate symptoms [13]. The majority of ALS cases are sporadic but approximately 10% are familial. Mutations in the genes encoding FUS/TLS [24], [10], TDP-43 [23], angiogenin [8] and cytosolic Cu/Zn superoxide dismutase (SOD1) [21] have been shown to cause ALS. The most extensively studied of these genes is SOD1.

To date nearly 150 ALS-associated mutations have been reported in SOD1 [9]. Most are missense mutations, which occur throughout the protein. Through multiple mechanisms that remain fully to be defined, SOD1 mutations are pathogenic; data overwhelmingly supports the view that mutant SOD1 protein has acquired adverse cytotoxic properties. SOD1 knockout mice show no overt phenotype [20], whereas mice over-expressing mutant SOD1 develop progressive paralysis and death due to motor neuron loss [5]. Importantly, transgenic mice and rats expressing high levels of mutant SOD1 develop a disease phenotype but those expressing at a lower level do not [5], [14]. This evidence, along with the findings that siRNA directed against SOD1 prolong survival in mice [18] lead us to investigate the possibility that a reduction in SOD1 levels could attenuate ALS susceptibility and the rate of disease progression. To test this hypothesis, we developed a cell-based screen for small molecules capable of inhibiting the SOD1 promoter [3], thereby reducing levels of mutant SOD1 protein. Mutant SOD1 is thought to act in both a cell autonomous and a non-cell autonomous manner [7], [17], [12]. Reduction of levels of mutant SOD1 in motor neurons delays onset of paralysis in transgenic ALS mice [2] while diminished levels of mutant SOD1 in astrocytes and microglial cells delays microglial activation and slows disease progression after onset [29]. Thus, the potential benefits of compounds that suppress SOD1 expression may be mediated by motor neurons and surrounding non-neuronal cells. We note that there is a precedent for a beneficial influence of induced gene repression in a transgenic model of Huntington's disease [28].

For these reasons, we have developed screening assays to identify compounds that inhibit expression of the SOD1 gene. Our studies focused initially on pyrimethamine, several compounds currently in trials in human and murine ALS and a set of 1040 FDA-approved compounds. We elected to study pyrimethamine in detail because this compound has previously been reported to reduce SOD1 protein levels in lymphocytes of ALS patients by up to 60% [11]. Pyrimethamine (5-(4-chlorophenyl)-6-ethyl-2,4-pyrimidinediamine) is an anti-protozoal drug whose primary mode of action involves the preferential inhibition of protozoal dihydrofolate reductase [6]. It also induces peripheral blood lymphocyte apoptosis via activation of caspase 8- and caspase 10-dependent cascades, leading to mitochondrial depolarization [16]. How pyrimethamine might reduce activity of the SOD1 gene is not clear.