A copper chaperone-mimetic polytherapy for SOD1-associated amyotrophic lateral sclerosis
Preprint posted on 23 February 2021 https://www.biorxiv.org/content/10.1101/2021.02.22.432389v1
Written by: Sanne van Falier, Laurens Geene, Janny Liebregts, and Lucas Siero
Proteins need to fold correctly to exert their function. Misfolded proteins can lead to aggregate formation, which occurs in a variety of diseases. One such disease, amyotrophic lateral sclerosis (ALS), is a severe neurological disease which causes loss of voluntary muscle movement. Certain genetic ALS variations are associated with the misfolding of mutant forms of the protein superoxide dismutase (SOD1). SOD1 folding is stabilized through the insertion of both a Zn and Cu ion in the metal binding region (MBR). Mutations in this region (MBR mutants) lead to impeded ion insertion and enzymatic activity. Correct SOD1 folding also relies on the formation of a disulfide bridge between Cys57-Cys146, and aberrant folding without the disulfide bridge inhibits the dimerization of SOD1 needed for full enzymatic function. Wild-type like (WTL) mutants still show enzymatic activity, but make SOD1 more prone to aggregation.
Normally, the CCS chaperone facilitates copper insertion and disulfide bond formation, but it is unable to fully do so in mutated SOD1. In this preprint, the authors test two small molecules to recapitulate CCS function in a combination treatment: CuATSM, a drug already in clinical trials which facilitates copper ion insertion into SOD1 , and ebselen, which promotes the formation of the Cys57-Cys146 disulfide bridge . While both drugs are known to independently rescue disease related phenotypes in mice [1,2], it is yet unknown whether they can be applied in a synergistic way for ALS treatment.
To be able to study the effect of ebselen on SOD1 aggregation, the authors express EGFP-tagged SOD1 in NSC-34 motor neuron-like cells, resulting in the formation of observable inclusions. To identify cells containing inclusions, the authors optimize an analytical pipeline using CellProfiller software. With the use of machine learning, they find optimal parameters to detect inclusions at an extremely high accuracy.
Using this pipeline, they test the effect of ebselen on the inclusion formation of multiple SOD1 mutants, including WTL (A4V) and MBR (G85R) mutants. Treatment with ebselen significantly reduces the formation of inclusions, likely by facilitating the formation of the disulfide bridge. To confirm this, they test the effect of ebselen on a number of mutants including G127X, which deletes residues 127-153. This region contains Cys146 involved in the formation of the disulfide bond. Ebselen has no effect on preventing inclusion formation of SOD1 G127X, but does have a dose dependent effect on other mutants, which can form the disulfide bridge. This finding supports the notion that ebselen facilitates the formation of the disulfide bridge.
Since ebselen cannot take over the copper insertion function, a second small molecule is needed to perform this action. In previous research, CuATSM was found to be capable of delivering copper to SOD1 in cells as well as in mice . A checkerboard analysis using both molecules and WT SOD1 and WTL and MBR mutants shows that the WTL mutant responds to the polytherapy, while the MBR mutant only responds to the ebselen monotherapy. The addition of ebselen decreases the required CuATSM concentration for A4V by a factor 3, while the addition of CuATSM decreases the required amount of ebselen by a factor 10. These results indicate a synergetic drug interaction.
Native SOD1 is active as a dimer. Mutations in the SOD1 gene can negatively affect dimerization and therefore decrease enzymatic activity even further. Treatment with ebselen, CuATSM or the combination therapy rescues this phenotype by increasing dimerization, most prominently for the G93A mutant (a WTL mutation). Furthermore, all mutants show an increase in enzymatic activity after treatment with CuATSM or the combination therapy, but not with ebselen on its own. Polytherapy has the strongest effect for V148G, rescuing the enzymatic activity more than either drug separately.
Altogether, the authors conclude that CuATSM and ebselen can both be used to support correct folding of SOD1 by acting as a CCS mimetic, promoting copper binding and disulfide formation, respectively. The combination of these two drugs can function in a synergistic way for certain SOD1 mutants, which can be exploited for personalised medicine by mutation-specific treatment.
Why we chose this preprint
ALS is a terrible disease with no current treatment. It is important to find therapies that can combat specific ALS mutations, and the application of multidrug therapy can contribute to this. Combination therapy is commonly used in the treatment of cancer, but has never been exploited for ALS. The polydrug therapy tested in this preprint is especially interesting because for certain mutations it not only prevents aggregation, but also partly restores the enzymatic activity of SOD1.
We are also amazed by the accuracy of the optimized machine learning algorithm. By finding the right parameters, this algorithm can be used to screen a large number of cells for the presence of inclusions, which can be applied to find further drugs able to combat ALS and other diseases associated with inclusion formation.
Questions to the authors
Since ebselen was originally designed for hearing loss and therefore aimed at a different target protein, would it be possible to modify the drug making it even more specific for SOD1?
Previous research into ebselen showed it to be less potent at facilitating SOD1 maturation, while a much higher concentration was used (200 µM) . How do you think this is possible?
1) Roberts BR, Lim NKH, McAllum EJ, Donnelly PS, Hare DJ, Doble PA, et al. Oral Treatment with CuII(atsm) Increases Mutant SOD1 In Vivo but Protects Motor Neurons and Improves the Phenotype of a Transgenic Mouse Model of Amyotrophic Lateral Sclerosis. Journal of Neuroscience. 2014. pp. 8021–8031. doi:10.1523/jneurosci.4196-13.2014
2) Capper MJ, Wright GSA, Barbieri L, Luchinat E, Mercatelli E, McAlary L, et al. The cysteine reactive small molecule ebselen facilitates effective SOD1 maturation. Nat Commun. 2018;9: 1693.
Posted on: 9 April 2021
doi: PendingRead preprint
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