The cysteine-reactive small molecule ebselen facilitates effective SOD1 maturation

Abstract

Superoxide dismutase-1 (SOD1) mutants, including those with unaltered enzymatic activity, are known to cause amyotrophic lateral sclerosis (ALS). Several destabilizing factors contribute to pathogenicity including a reduced ability to complete the normal maturation process which comprises folding, metal cofactor acquisition, intra-subunit disulphide bond formation and dimerization. Immature SOD1 forms toxic oligomers and characteristic large insoluble aggregates within motor system cells. Here we report that the cysteine-reactive molecule ebselen efficiently confers the SOD1 intra-subunit disulphide and directs correct SOD1 folding, depopulating the globally unfolded precursor associated with aggregation and toxicity. Assisted formation of the unusual SOD1 cytosolic disulphide bond could have potential therapeutic applications. In less reducing environments, ebselen forms a selenylsulphide with Cys111 and restores the monomer-dimer equilibrium of A4V SOD1 to wild-type. Ebselen is therefore a potent bifunctional pharmacological chaperone for SOD1 that combines properties of the SOD1 chaperone hCCS and the recently licenced antioxidant drug, edaravone.

Fig. 1

The structure of ebselen and ebsulphur bound to SOD1. a Cartoon representation of the SOD1 dimer with ebselen or ebsulphur molecules shown as ball and stick and bound to Cys111 residues at the dimer interface. b Electron density map (2Fo−Fc contoured at 1σ) showing the pose of two SOD1-bound ebselen molecules and proximal crystallographic waters. Electron density (maps mFo–DFc contoured at 3σ) of c ebsulphur and d ebselen showing details of binding at Cys111

Fig. 2

Conjugation of ebselen and ebsulphur to SOD1 variants increases the binding affinity of the dimer. a SOD1 variants (wild-type, A4V, H46R/H48Q, G93A and I149T) with ebselen (red), ebsulphur (blue) and without treatment (black) were buffer exchanged into 200 mM NH₄OAc and subjected to nanoESI mass spectrometry at different protein concentrations under gentle conditions optimized to maintain non-covalent interactions. Curves show the percentage of monomer signal as a function of protein concentration with determined dimer K_d values inset for each SOD1 variant. Error bars represent the standard deviation of three replicates. b The fold-increase in binding affinity of each SOD1 variant when ebselen or ebsulphur is conjugated at Cys111 where error bars represent the upper and lower K_d values calculated in a

Fig. 3

Ebselen does not prevent heterodimer formation with hCCS. Size exclusion chromatograms showing how ebselen binding to C57/146A SOD1 promotes dimerization (blue dashed line to red dashed line) but does not inhibit heterodimeric complex formation with wild-type hCCS, which exists in homodimeric state prior to complexation

Fig. 4

Ebselen efficiently oxidizes SOD1 in living cells. a Overlay of ¹H-¹⁵N NMR spectra acquired on human cells expressing [U-¹⁵N] labelled wild-type E,Zn-SOD1 in the absence (red) and in the presence (black) of ebselen in the external medium. b Overlay of ¹H-¹⁵N NMR spectra acquired on purified, disulphide-reduced [U-¹⁵N] labelled wild-type E,Zn-SOD1 before (blue) and after (magenta) treatment with 1 equivalent of ebselen. The positions of the enlarged areas (bottom panels) in the whole spectra (top panels) are indicated as dotted lines. Signals affected by the formation of the C57–C146 disulphide bond are labelled in the bottom panels

Fig. 5

Ebselen stabilizes ALS mutant SOD1 in living cells. a Overlay of ¹H-¹⁵N NMR spectra acquired on human cells expressing [U-¹⁵N] labelled SOD1 ALS mutants G93A (a) and A4V (b) in the absence (green/orange) and in the presence (black) of ebselen in the external medium. NMR signals clustered in the region around 8.3 p.p.m. (¹H) are characteristic of unstructured SOD1 species. c Western blot analysis of cell lysates expressing wild-type and mutant SOD1 in the absence and in the presence of ebselen. Original image Supplementary Fig. 11. d Expression levels of wild-type and mutant SOD1 in cell samples treated with ebselen relative to the corresponding untreated samples; error bars represent s.d. (n = 3); * indicates significant differences (ratio t-test, p < 0.05)

Fig. 6

Mechanism of SOD1 stabilization by ebselen. In the absence of ebselen, monomeric, disulphide-reduced, metal-free mutant SOD1 is highly likely to remain globally unfolded and be degraded or accumulate as small toxic oligomers or aggregates, which are characteristic of ALS neural tissues (red). However, in reaction 1, ebselen forms a selenylsulphide bond with either Cys57 or Cys146 on newly translated SOD1. 2. Selenol–disulphide exchange forms the SOD1 intra-subunit disulphide bond and frees the ebselen selenol. Reduction of hydrogen peroxide by ebselen selenol reforms the ebselen heterocycle. This recycling process indicates that relatively small amounts of ebselen could promote SOD1 maturation. 3. SOD1 disulphide formation promotes correct folding, zinc binding and dimerization. 4. Further modification of Cys111 by ebselen prevents monomerization by ALS mutations, thus maintaining stable, active, fully metalated, homodimeric SOD1