Abstract
One of the most devastating neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) or Lou Gehrig’s disease, results in paralysis and death within three to five years post diagnosis. Currently there is only one FDA-approved drug, Rilutek, which extends life by only 2-3 months, thus highlighting the need for novel drug targets and strategies. It has been hypothesized that destabilization of the Cu/Zn superoxide dismutase (SOD1) plays a role in disease progression in a variety of ALS cases; of the 153 residues in SOD1, 133 point mutations (such as G93A) have been implicated in familial (hereditary) forms of the disease. Furthermore, aberrant modification of SOD1 has also been implicated in the sporadic form of the disease. Thus, I hypothesized that stabilizing ALS-associated variants of SOD1 will ameliorate their toxic effects. Specifically I targeted two residues in SOD1, Trp32 and Cys111. Introduction of W32F or C111S mutations have been shown to ameliorate the toxic effects of ALS-associated SOD1 variants. I therefore set out to characterize compounds thought to bind either Trp32 or Cys111. In particular, microscale thermophoresis was utilized to attempt to measure the binding affinity for these compounds, which was previously unknown. Differential scanning fluorimetry was used to measure changes to SOD1 stability. In addition, we used an in-gel activity assay to assess the effect of compounds on SOD1’s native dismutase activity. The binding affinity was measured for dopamine and aniline, compounds that were previously shown to bind Trp32, which was found to be in the in low-mM (1-10 mM) range. However, dopamine was found to destabilize and reduce enzymatic activity of WT SOD1. NSC636114, a ditihione thought to bind Cys111, was found to have a binding affinity in the mid-μM range. A cyclic disulfide, NSC56224 was found to stabilize both native and demetallated SOD1.