Improving binding specificity of pharmacological chaperones that target mutant superoxide dismutase-1 linked to familial amyotrophic lateral sclerosis using computational methods

Abstract

We recently described a set of drug-like molecules obtained from an in silico screen that stabilize mutant superoxide dismutase-1 (SOD-1) linked to familial amyotrophic lateral sclerosis (ALS) against unfolding and aggregation but exhibited poor binding specificity toward SOD-1 in presence of blood plasma. A reasonable but not a conclusive model for the binding of these molecules was proposed on the basis of restricted docking calculations and site-directed mutagenesis of key residues at the dimer interface. A set of hydrogen bonding constraints obtained from these experiments were used to guide docking calculations with compound library around the dimer interface. A series of chemically unrelated hits were predicted, which were experimentally tested for their ability to block aggregation. At least six of the new molecules exhibited high specificity of binding toward SOD-1 in the presence of blood plasma. These molecules represent a new class of molecules for further development into clinical candidates.

Figure 1

(a) Aggregation of SOD-1^A4V monitored using size-exclusion chromatography in presence of a series of compounds having either uracil or aza-uracil like substructures. The amount of dimer observed after 48hrs of incubation is plotted after normalization of the aggregation curves. Two molecules 4, 5 bear methylation at different positions on the aza-uracil ring and are indicated by (*) on top of the bar chart. (b) Unfolding of SOD-1^A4V measured as a function of GdnCl. in presence of the small molecules tested in the left panel. The data have been fitted to a two-state model to facilitate the calculation of thermodynamics parameters shown in the table below (left). (c) Measurement of compound specificity towards a GST-SOD-1 fusion protein (green bars) in presence of buffer and blood plasma. Control experiments include binding in buffer (black bars) and GST (tag) alone (red bars).

Figure 2

(a) Docked poses for 7 of the molecules that were found to be strong inhibitors of SOD-1 aggregation described in figure 1. The dimer interface region of SOD-1 around residues 148 is shown indicating that uracil or aza-uracil substructures occupy very similar positions in all cases, and makes hydrogen bonds with Asn 53 and Val 7. The rest of the molecules, however, do not appear to have any preferred orientation in the dimer interface. (b) Effect of compounds on aggregation of SOD-1^A4V/N53A measured in a fashion similar to the one described in figure 1a. The mutant was generated with the rationale that N53→A should lead to the loss of a hydrogen bond with the small molecules. The experimental data indicates minimal or no effect on aggregation of the double mutant by the small molecules which normally stabilizes SOD-^1A4V. (c) Schematic representation of a symmetric molecule with two aza-uracil groups bound at the dimer interface of SOD-1. The schematic representation indicates the possibility of two pairs of hydrogen bonds between the protein and the small molecule. (d) Effect of various concentrations of 8 or 12 on the aggregation of SOD-1^A4V. 12 makes two pairs of hydrogen and is more effective at lower concentrations compared to 8.

Figure 3

(a,b) Glidescores obtained after docking calculations were carried out using GLIDE with a set of “false positive” molecules mixed with experimentally verified aggregation inhibitors. The top panel uses standard docking parameters of the GLIDE program. The bottom panel describes docking calculations carried out after two hydrogen bond constraints were placed on C=O of Val 7 and the side-chain of Asn53. The use of the hydrogen bonding constraints leads to substantial enrichment of the experimentally verified inhibitors over the false positives. (c) Aggregation of SOD-1^A4V monitored using size-exclusion chromatography in presence of a series of compounds that were obtained after screening of a database of 2.2 million molecules using the experimentally derived hydrogen bond constraints on Val 7 and Asn 53. The amount of dimer observed after 48hrs of incubation is plotted after normalization of the aggregation curves. (d) Binding of the new molecules obtained after docking and verified by aggregation assay as inhibitors, tested in the plasma binding assay as described above. At least six molecules show significantly improved binding specificity towards SOD-1 over blood plasma proteins. Control experiments include binding in presence of buffer and GST (tag only). The compounds show specificity towards binding to the GST-SOD-1 fusion protein but not towards GST (tag).

Figure 4

Chemical structures for the molecules that were found to have high docking scores, and satisfy the hydrogen bonding constraints imposed by the docking program.