Copper‐Zinc Superoxide Dismutase as a Convenient System for Assessment of Safety Margins In‐Vitro
Authors: Malik Ravinder , Corrales Christian , Atlasi Ryan Saleh , Valentine Joan S , Schrader Thomas , Klärner Frank‐Gerrit , Bitan Gal 
Published: November 2023

Many amyloidogenic proteins do not have known, well‐defined activities, making assessment of the safety margins of aggregation inhibitors/modulators in‐vitro difficult. In contrast, copper‐zinc superoxide dismutase (SOD1) is an antioxidant enzyme that protects cells from highly toxic reactive oxygen species formed during respiration. Mutations in SOD1 destabilize its structure leading to misfolding and formation of toxic oligomers and aggregates that kill motor neurons. These aberrant neurotoxic SOD1 assemblies are thought to cause familial amyotrophic lateral sclerosis (ALS) and possibly sporadic ALS. Therefore, inhibition and modulation of SOD1 aggregation is a promising strategy for ALS therapy development. Yet to be considered safe, effective inhibitors of SOD1 aggregation must also not disrupt the biological activity of the enzyme. Substantially lower concentrations of the inhibitors should be sufficient for inhibiting SOD1 aggregation than the concentrations that inhibit enzymatic activity. This may be particularly challenging for SOD1 mutants, which have lower enzymatic activity and higher tendency to misfold and aggregate than the wild‐type protein. Here, we demonstrate the utility of the SOD1 system for evaluating CLR01, a broad‐spectrum aggregation inhibitor, for its effects on aggregation and enzymatic activity of wild‐type and mutant SOD1. To examine inhibition of aggregation, we incubated SOD1 at a concentration of 10 μM, in the absence or presence of increasing CLR01 concentrations and measured aggregation kinetics using thioflavin T (ThT) fluorescence to obtain a dose–response analysis. Aggregation of SOD1 was attenuated in a dose‐dependent manner and completely inhibited at 10‐fold molar excess of CLR01. The effect of CLR01 on SOD1 activity was examined by an assay that utilizes a tetrazolium compound for detection of superoxide radicals generated from hypoxanthine by xanthine oxidase. SOD1 activity was measured optimally at 7.5 nM in this assay. Because the enzymatic activity and aggregation assays require three orders of magnitude difference in protein concentration, meaningful comparison can only be made between the SOD1:CLR01 concentration ratios, not the absolute concentrations themselves. The initial rates of the enzymatic reaction were plotted against CLR01 concentration to obtain a dose‐response analysis. CLR01 showed a minimal effect on the reaction rate of all the SOD1 variants tested at concentration ratios >1:100. Our study demonstrates the utility of the SOD1 system for evaluating safety margins of aggregation modulators in vitro, as opposed to more common amyloidogenic proteins, such as amyloid β‐protein, tau, and α‐synuclein, which do not have known, or easily measurable, biological activities. The data also show that CLR01 has only a minor impact on SOD1 activity at concentrations 2–3 orders of magnitude higher than those required for inhibition of aggregation, demonstrating its effectiveness and safety in this system.

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