Small Molecule Keeps Mutant SOD1 Out of Trouble

Mutations in copper-zinc superoxide dismutase 1 (SOD1) derail folding of the protein, preventing it from playing nice with its siblings. This leads the mutant down a path to aggregation and destruction, but an April 27 study in Nature Communications claims to be onto a fix. Researchers led by Samar Hasnain at the University of Liverpool in England reported that the antioxidant ebselen promotes disulfide bonds in mutant SOD1, restoring its ability to fold properly and dimerize with other SOD1 proteins, steering away from aggregation. In human cells, ebselen treatment shifted levels of unfolded mutant SOD1 in favor of neatly folded proteins. Given that ebselen has already been tested for bipolar disorder and hearing loss, the researchers propose it could be quickly tested as a therapeutic to treat familial amyotrophic lateral sclerosis caused by SOD1 mutations.

  • Ebselen catalyzes disulfide formation within SOD1.
  • The chemical reversed misfolding of mutant SOD1 in cells.
  • It might be a candidate ALS drug.

“This is a novel folding and assembly corrector that could become a drug candidate,” commented Jeff Kelly of Scripps Research Institute in La Jolla, California.

Inclusions chock-full of aggregated SOD1 proteins are the pathological hallmark of dying motor neurons in people with SOD1-ALS (Jan 2009 news; Kerman et al., 2010). The disease likely begins with SOD1 misfolding, and the enzyme’s propensity to unfold and aggregate correlates with life expectancy of ALS patients and of mice that express the mutant human enzyme (Jonsson et al., 2006; Seetharaman et al., 2009; Oct 2014 news).

Disulfide bonds, as well as zinc and copper binding, are integral to SOD1 folding. First author Michael Capper and colleagues set out to restore those bonds. They screened a library of cysteine-reactive compounds for those that would oxidize cysteine residues in SOD1 and got two hits: ebselen and ebsulphur. Ebselen is an organoselenium compound with antioxidant and anti-inflammatory properties, which has been tested for treatment of multiple central nervous system disorders, including stroke, bipolar disorder, and hemorrhage (see Yamaguchi et al., 1998; Singh et al., 2013; Saito et al., 1998 ). It also bears a structural resemblance to edaravone, another antioxidant recently approved to treat ALS (Jan 2016 news; May 2017 news).

Structural analysis of the compounds bound to SOD1 revealed that ebselen and ebsulphur form covalent selenylsulfide or disulfide bonds, respectively, with cysteine-111 on SOD1. Using native mass spectrometry, the researchers found that the compounds promoted SOD1 dimerization and treating multiple dimerization-defective ALS mutants with ebselen or ebsulphur restored dimers to wild-type levels. The treatment had the strongest effect on the aggressive A4V mutant, strengthening its dimers 60-fold. Ebselen or ebsulphur did not affect SOD1’s interaction with the chaperone hCCS, which is critical for its function. Overall, ebselen outperformed ebsulphur in promoting SOD1 dimerization, so the researchers used it for future studies.

In the Dimer Groove. Ebselen (ball and stick) bound to SOD1 dimers at cysteine-111 residues at the dimer interface. Left shows ribbon model, right shows electron density map. [Courtesy of Capper et al., Nature Communications, 2018.]

All this was done in solution. Does ebselen affect SOD1 folding and function in the complex chemical milieu of the cytosol? To find out, Capper assessed the state of SOD1 in HEK293 cells using in-cell NMR. Normally, the protein starts out with free cysteines and no disulfide bonds. Zinc, then copper, bind before disulfides form. The researchers found that in cells treated with ebselen, SOD1 was in its fully oxidized state, with an intramolecular disulfide bond formed between cysteine-57 and 146. The researchers proposed that ebselen transiently interacted with both these cysteines and catalyzed the formation of their disulfide bond. This is distinct from the Cys111-mediated dimerization ebselen evoked in vitro, and the authors attributed this difference to the highly reducing environment of the cellular cytosol.

Would this Cys57-Cys146 disulfide also stabilize SOD1 mutant proteins? Yes, according to in-cell NMR experiments on cells expressing G93A or A4V SOD1 mutants. In untreated cells, the mutant proteins either existed as unfolded monomers or were barely detected due to degradation. But ebselen boosted oxidation of both mutants, and restored their mature copper- and zinc-loaded versions to the levels seen for wild-type SOD1.

Overall, it appears ebselen stabilizes SOD1 in two ways, depending on the environment. In the less-reducing environs of the test tube, ebselen formed a covalent bond with cysteine-111, which in turn promoted dimerization of the protein. In the cytosol, ebselen promoted a disulfide bond between SOD1 cysteines 57 and 146, which also stabilized the protein.

Though the researchers observed the cysteine-111 reaction only outside the cellular environment, they think it could still come into play in less strongly reducing compartments, such as the mitochondrial intermembrane space or extracellular matrix. Ebselen’s mechanism of action could also depend on dose, an idea currently being tested in SOD1-transgenic mouse models, Hasnain told Alzforum.—Jessica Shugart

Comments

  1. This is a really interesting paper reporting a dual-function SOD1 folding and assembly facilitator, ebselen. Ebselen directly reacts with either Cys 57 or Cys 146 within SOD1, forming a transient selenyl-sulfide conjugate, which spontaneously rearranges by selenyl-thiol exchange to afford the native intrasubunit disulfide bond within SOD1 that completes proper tertiary structure acquisition, releasing ebselen selenol. This properly folded monomer can then dimerize with high affinity and undergo macromolecular chaperone-mediated metallization, critical for achieving the high kinetic stability of SOD1, which prevents its aggregation that drives ALS. Two ebselens also covalently react with Cys 111 and Cys111' by transient selenyl-sulfide bond formation, further stabilizing the dimer interface via π-π interactions between two proximal ebselen-derived substructures covalently linked to Cys 111 and Cys 111'. These are transient because glutathione can attack the selenium reforming Cys 111 and 111'. One wonders whether ebselen selenol or ebselen could also non-covalently stabilize the dimer interface of SOD1, since reversible binding likely facilitates the reversible reaction with Cys 111 and Cys 111'. This paper also suggests that a two-drug strategy might be superior for treating mutant SOD-based ALS, whereby ebselen is used to facilitate intrasubunit SOD1 disulfide formation, in combination with a non-reversible electrophile that reacts selectively with Cys 111 and Cys 111' and is capable of making stabilizing π-π interactions. Alternatively, if the glutathione ebselen selenol selenyl-sulfide conjugate could re-react with Cys 111 again and again, then the stoichiometry of ebselen to SOD1 might be more manageable. To my knowledge this is a novel folding and assembly corrector, which could become a drug candidate.

    View all comments by Jeffery Kelly

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References

News Citations

  1. Bad Origami: Misfolded SOD1 in Living Color
  2. Enzyme Structure Linked to ALS Severity
  3. Does Free Radical Scavenger Edaravone Slow ALS?
  4. FDA Approves Edaravone for Treatment of ALS

Paper Citations

  1. Kerman A, Liu HN, Croul S, Bilbao J, Rogaeva E, Zinman L, Robertson J, Chakrabartty A. Amyotrophic lateral sclerosis is a non-amyloid disease in which extensive misfolding of SOD1 is unique to the familial form. Acta Neuropathol. 2010 Mar;119(3):335-44. PubMed.
  2. Jonsson PA, Graffmo KS, Andersen PM, Brännström T, Lindberg M, Oliveberg M, Marklund SL. Disulphide-reduced superoxide dismutase-1 in CNS of transgenic amyotrophic lateral sclerosis models. Brain. 2006 Feb;129(Pt 2):451-64. PubMed.
  3. Seetharaman SV, Prudencio M, Karch C, Holloway SP, Borchelt DR, Hart PJ. Immature copper-zinc superoxide dismutase and familial amyotrophic lateral sclerosis. Exp Biol Med (Maywood). 2009 Oct;234(10):1140-54. Epub 2009 Jul 13 PubMed.
  4. Yamaguchi T, Sano K, Takakura K, Saito I, Shinohara Y, Asano T, Yasuhara H. Ebselen in acute ischemic stroke: a placebo-controlled, double-blind clinical trial. Ebselen Study Group. Stroke. 1998 Jan;29(1):12-7. PubMed.
  5. Singh N, Halliday AC, Thomas JM, Kuznetsova OV, Baldwin R, Woon EC, Aley PK, Antoniadou I, Sharp T, Vasudevan SR, Churchill GC. A safe lithium mimetic for bipolar disorder. Nat Commun. 2013;4:1332. PubMed.
  6. Saito I, Asano T, Sano K, Takakura K, Abe H, Yoshimoto T, Kikuchi H, Ohta T, Ishibashi S. Neuroprotective effect of an antioxidant, ebselen, in patients with delayed neurological deficits after aneurysmal subarachnoid hemorrhage. Neurosurgery. 1998 Feb;42(2):269-77; discussion 277-8. PubMed.

Further Reading

Primary Papers

  1. Capper MJ, Wright GS, Barbieri L, Luchinat E, Mercatelli E, McAlary L, Yerbury JJ, O'Neill PM, Antonyuk SV, Banci L, Hasnain SS. The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation. Nat Commun. 2018 Apr 27;9(1):1693. PubMed.

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