CLR 01 inhibits SOD 1 aggregation in vitro and in vivo 1 The molecular tweezer CLR 01 inhibits aberrant superoxide dismutase 1 ( SOD 1 ) self-assembly in vitro and in the G 93 A-SOD 1 mouse model of ALS

Mutations in superoxide dismutase 1 (SOD1) cause 15%–20% of familial amyotrophic lateral sclerosis (fALS) cases. The resulting amino-acid substitutions destabilize SOD1’s protein structure, leading to its selfassembly into neurotoxic oligomers and aggregates, a process hypothesized to cause the characteristic motor-neuron degeneration in affected individuals. Currently, effective disease-modifying therapy is not available for ALS. Molecular tweezers prevent formation of toxic protein assemblies, yet their protective action has not been tested previously on SOD1 or in the context of ALS. Here, we tested the molecular tweezer CLR01—a broad-spectrum inhibitor of the self-assembly and toxicity of amyloid proteins—as a potential therapeutic agent for ALS. Using recombinant wild-type and mutant SOD1, we found that CLR01 inhibited the aggregation of all tested SOD1 forms in vitro. Next, we examined whether CLR01 could prevent the formation of misfolded SOD1 in the G93A-SOD1 mouse model of ALS and whether such inhibition would have a beneficial therapeutic effect. CLR01 treatment decreased misfolded SOD1 in the spinal cord significantly. However, these histological findings did not correlate with improvement of the disease phenotype. A small, dose-dependent decrease in disease duration was found in CLR01-treated, relative to vehicle-treated animals, yet motor function did not improve in any of the treatment groups. These results demonstrate that CLR01 can inhibit SOD1 misfolding and aggregation both in vitro and in vivo, but raise the question whether such inhibition is sufficient for achieving a therapeutic effect. Additional studies in other, less aggressive ALS models may be needed to determine the therapeutic potential of this approach. Amyotrophic lateral sclerosis (ALS) is a rapidly debilitating neuromuscular disorder characterized by loss of motor neuron function in the spinal cord, brain stem, and motor cortex. The resulting progressive muscle weakness and atrophy leads to death typically occurring 3– 5 years after onset. Approximately 90–95% of ALS cases are sporadic and the remaining cases http://www.jbc.org/cgi/doi/10.1074/jbc.RA118.005940 The latest version is at JBC Papers in Press. Published on January 2, 2019 as Manuscript RA118.005940 at U C L A -L oise D aling B om d. L b. on Jauary 3, 2019 hp://w w w .jb.org/ D ow nladed from CLR01 inhibits SOD1 aggregation in vitro and in vivo 2 are familial (fALS), caused by mutations in multiple genes (1). More than 160 mutations in the SOD1 gene, encoding the enzyme superoxide dismutase 1 (SOD1), have been described to cause amino-acid substitutions that destabilize the protein leading to its aberrant self-assembly, which causes 15–20% of fALS cases (2). SOD1 is a homodimer of a 153-amino acid polypeptide. The enzyme converts the superoxide ions produced by mitochondria into hydrogen peroxide. Some disease-associated SOD1 variants retain high activity (3) and the targeted deletion of SOD1 in transgenic mice does not induce ALS-like symptoms (4). Therefore, the mechanism by which mutant SOD1 causes ALS is believed to be not a loss of function, but rather a gain of toxic function upon misfolding and self-assembly into neurotoxic oligomers and aggregates (5,6). The latter are found in the brain and spinal cord of affected individual as intracellular inclusions. To date, two drugs have been approved for treatment of ALS—riluzole (7) and edaravone (8). These drugs slow disease progression mildly but do not alter substantially the disease course. Thus, there is an urgent need for a disease-modifying therapy that would halt the progression and ultimately cure ALS. Inhibition and modulation of aberrant protein self-assembly are promising strategies for therapy development, which have been explored quite extensively for other proteinopathies, such as Alzheimer’s disease (AD) and Parkinson’s disease (PD) (9). In contrast, to date, few studies have examined small-molecule inhibitors of SOD1 selfassembly as a potential therapy for ALS. A study using pyrimidine-2,4,6-triones showed protection from SOD1-induced cytotoxicity in cultured cells (10). Using a different mechanism, restoration of cellular protein degradation by activation of the proteasome, pyrazolones have been found to protect PC-12 cells against the toxicity of G93A-SOD1 (11). In yet a third approach, in-silico screening identified azauraciland uracil-based inhibitors, which stabilized mutant SOD1 against aggregation in human plasma (12). The therapeutic efficacy of these compounds is yet to be tested in vivo. These few examples highlight the need for further exploration of strategies directed at misfolded SOD1 selfassembly as a potential target for ALS therapy. SOD1 was the first gene linked to familial ALS (13). A transgenic mouse model of ALS, expressing human SOD1 containing a G93A substitution was first introduced in 1996 (14). Although this model is not ideal as it expresses a high copy number of the transgene and consequently displays highly aggressive disease progression, it has remained not only the most thoroughly characterized ALS animal model, but also has been regarded as the gold standard in preclinical therapeutic ALS research. This mouse model mimics motor neuron loss and progressive muscle weakness (15) similar to the clinical symptoms of human ALS. The mouse model has facilitated studying mechanistic aspects of the disease, yet the rapid course of disease may be too aggressive for many therapeutic approaches to have an effect. Previously, we reported that molecular tweezers (MTs) are broad-spectrum inhibitors of abnormal protein self-assembly and toxicity (16-18). A lead MT, called CLR01 (Figure 3A), has been found to inhibit the formation of toxic oligomers and aggregates of multiple disease-associated proteins (19), including those involved in AD (20,21) and PD (22,23). CLR01 acts as a nanochaperone by a “processspecific” mechanism, i.e., it targets the process of abnormal protein self-assembly itself rather than a particular protein. CLR01 achieves this activity by labile binding to positively charged amino-acid residues, primarily Lys and to a lower extent Arg, temporarily reversing the charge from positive to negative and disrupting hydrophobic interactions in which the butylene moiety of the Lys side chain (or the propylene chain in the Arg side chain) participate. As hydrophobic and electrostatic interactions are at U C L A -L oise D aling B om d. L b. on Jauary 3, 2019 hp://w w w .jb.org/ D ow nladed from CLR01 inhibits SOD1 aggregation in vitro and in vivo 3 important, particularly in the early stages of the aberrant self-assembly process in which oligomers and aggregation nuclei form, MTs effectively interfere with the formation of these