Small-molecule-mediated stabilization of familial amyotrophic lateral sclerosis-linked superoxide dismutase mutants against unfolding and aggregation

Abstract

Familial amyotrophic lateral sclerosis (FALS) is a fatal motor neuron disease that is caused by mutations in the gene encoding superoxide dismutase-type 1 (SOD1). The affected regions of the FALS brain are characterized by aggregated SOD1, and the mutations that destabilize SOD1 appear to promote its aggregation in vitro. Because dissociation of the native SOD1 dimer is required for its in vitro aggregation, we initiated an in silico screening program to find drug-like molecules that would stabilize the SOD1 dimer. A potential binding site for such molecules at the SOD1 dimer interface was identified, and its importance was validated by mutagenesis. About 1.5 million molecules from commercial databases were docked at the dimer interface. Of the 100 molecules with the highest predicted binding affinity, 15 significantly inhibited in vitro aggregation and denaturation of A4V, a FALS-linked variant of SOD1. In the presence of several of these molecules, A4V and other FALS-linked SOD1 mutants such as G93A and G85R behaved similarly to wild-type SOD1, suggesting that these compounds could be leads toward effective therapeutics against FALS.

Fig. 1.

When the cavity at the SOD1 dimer interface is partially filled by mutagenesis, the resultant protein is more stable and aggregates more slowly. (a Left) shows a surface representation of A4V mutant SOD1 dimer colored to show the two subunits. A deep cavity at the dimer interface is highlighted by the blue box. The surface was generated by using a water molecule as a probe. (a Right) A model of the SOD1 cavity in the variant A4V/V7F/V148F. Note the quartet of phenylalanine sidechains at the dimer interface. (b Left) GdnCl unfolding of A4V (black), WT (green) and the A4V/V7F/V148C triple mutant (red) plotted as fraction unfolded vs. GdnCl concentration. (b Right) Loss of SOD1 dimers over time parallels the formation of aggregates (data not shown). The rate of aggregation is inversely correlated to the stability toward denaturation: A4V (black), WT (green), and A4V/V7F/L148C (red).

Fig. 2.

The protein and small-molecule portions of the docked complexes occupy overlapping space. (a) The grid box used for our docking calculation. The green box represents the conformational search space for the ligand; the pink box represents the boundary conditions used for the docking calculation. One hundred of the 2,000 final poses obtained from docking are shown docked at the dimer interface. (b) Superposition of five SOD1 x-ray structures WT, apo-WT, S134N, H46R, and A4V (Protein Data Bank codes: 1SPD, 1HL4, 1N19, 1OEZ, and 1UXL), showing critical residues of the dimer interface cavity used in docking calculations. The mean rms deviation (Cα)is <0.6 Å, indicating that the dimer interface is rigid in nature, which suggests that docked molecules should bind to all mutants. A superposition of 20 of the top 100 docked molecules is also shown.

Fig. 3.

Fifteen compounds significantly slowed the loss A4V, G85R, and G93A SOD1 dimer under aggregating conditions. (a) EDTA-induced loss of A4V dimer in the absence (solid black line) and presence of the top 100 compounds, as predicted by the docking calculation. The average of three trials is plotted for each compound; variation was <5%. The loss of WT was very slow under these conditions (dashed red line). (b) EDTA-induced loss of G85R dimer in the absence (solid black line) and presence of the 15 best inhibitors of A4V aggregation (see a). (c) EDTA-induced loss of G93A dimer in the absence (black line) in presence of the 15 best inhibitors of A4V aggregation. (d) EDTA-induced loss of WT dimer in the absence (black line) and presence of the 15 best inhibitors of A4V aggregation. Note the difference in the scale of the y axes.

Fig. 4.

Chemical denaturation of SOD1 was inhibited by compounds that inhibited aggregation and were calculated to bind to the cavity at the interface. (a) GdnCl-induced unfolding of A4V in the presence of the 15 best aggregation inhibitors; plotted as fraction folded vs. [GdnCl]. The data has been fitted to a two-state model to allow estimation of thermodynamic parameters (Table 3). (b) Schematic and simplified representation of the reaction analyzed here (complete unfolding is a convenient experimental system but is not required for aggregation): SOD1 dimer can be stabilized relative to the unfolded state by binding to drug-like molecules. The A4V dimer (Center, red circles denote residue Val4) is much less stable than the WT dimer (Left). However, the binding of the drug molecule (purple square) to the unstable A4V dimer can decrease its free energy (ΔG of binding in green; Table 3). Binding results in depopulation of the aggregation-prone metal-free A4V monomer, decreasing the rate of aggregation.

Fig. 5.

Aggregation inhibitors have similar structural features and are predicted to bind similarly. (a) Docked poses of 6 of the 15 molecules that inhibit SOD aggregation. Four of the six molecules (panels 1, 2, 4, and 5; molecules 2, 3, 5, and 7) exhibit a very similar mode of binding. These molecules represent the major class of active molecules found in our assay and bear an aromatic ring that binds in a deep pocket. Panels 3 and 6 (triamcinolone and N6-methyladenosine, respectively) are unlike the other hits and exhibit different modes of binding, although it still binds in the same cavity. (b) An overlay of the hits obtained from screening shows a conserved aromatic moiety at the hydrophobic pocket. The remaining portion of the molecules is more variable.