|
Ray et al. 10.1073/pnas.0408277102. |
Fig. 6. A variant of G93A designed to fill the cavity at the interface has increased stability and aggregates more slowly. (a) voidoo-calculated cavity at the dimer interface drawn as an ezd negative density. (b) Model of the mutant G93A/V7F/V148F, showing partial capping of the dimer interface cavity with no steric clashes between the Phe sidechains. (c) GdnCl unfolding of WT superoxide dismutase 1 (SOD1), G93A, and G93A/V7F/V148F. (d) Loss of dimer over time (parallels aggregation) for WT, G93A, and G93A/V7F/V148F.
Fig. 7. Chemical structures for the 15 best inhibitors of A4V aggregation. The docking scores and estimated docking energy are given for each compound are also provided along with the chemical structures.
Fig. 8. Effects on aggregation are independent of metal binding site occupancy and are mediated by the interface cavity. (a) Loss of A4V dimer (without EDTA) in the absence (black line) and presence of the 15 best aggregation inhibitors obtained from the screen. Comparison of this data with that obtained in the presence of EDTA (Fig. 3) shows little change with respect to inhibition but a significant acceleration, probably related to demetallation. (b) Loss of Apo-A4V dimer in the absence (black line) and the presence of the top 15 A4V inhibitors. (c) Loss of A4V/V7F/V148F dimer in the absence (black line) and the presence of compounds 2, 3, 4, and 7. (d) Loss of A4V dimer in the presence of 20 randomly selected control compounds that did not dock at the SOD1 dimer interface. (e) Aggregation of a -synuclein was not effected by the 15 A4V aggregation inhibitors.
Fig. 9. The compound library was selected to enrich for drug-like properties. (a) Distribution of various physicochemical properties for the final set of compounds used for the docking screen. The plots show four of the five Lipinski rules (relaxed in this case) that are commonly used for filtering drug-like molecules from a database. (b) Flow chart showing the various steps involved in preparation of database for docking calculation.
Table 1. Vendor, database, and number of compounds used for docking
|
Company |
Library name |
Web address |
No. of compounds used |
|
Asinex |
Asinex gold and platinum collection |
www.asinex.com |
209,418 |
|
Key Organics |
Bionet |
www.keyorganics.ltd.uk |
41,000 |
|
ChemBridge |
Chembridge microformat |
www.chembridge.com |
100,000 |
|
ChemDiv |
ChemDiv |
www.chemdiv.com/main.phtml |
257,132 |
|
ChemStar |
Chemstar |
www.chemstar.ru |
49,179 |
|
Enamine |
Enamine compound collection |
www.enamine.com |
287,289 |
|
InterBioScreen |
IBS |
www.ibscreen.com |
199,363 |
|
MayBridge |
MaybBridge |
www.maybridge.com |
44,435 |
|
Laboratory for Drug Discovery in Neuroscience |
LDDN |
www.hcnr.med.harvard.edu/d_drug/ |
56,671 |
|
Molecular Diversity Preservation International |
MDPI |
www.mdpi.org |
121,244 |
|
Pharmeks |
Pharmeks Main database |
www.pharmeks.com |
105,600 |
|
Prestwick |
Prestwick Drug Like Molecule collection |
www.prestwick.com |
640 |
|
NCI/NIH Developmental Therapeutics Program |
NCI |
dtp.nci.nih.gov/index.html |
13,267 |
|
Specs and BioSpecs |
Compound collection |
www.specs.net |
240,000 |
|
Sigma |
Rare Chemical Database |
www.sigma.com |
51,294 |
|
Timtec |
Timtec |
www.timtec.net |
160,000 |
|
Tripos |
Tripos |
www.tripos.com |
4,621 |
|
Zelinsky |
www.zelinsky.com |
45,892 |
Table 2. Estimation of copper and zinc in SoD samples used in this study
|
Sample |
Protein concentration, m M* |
Copper concentration, m M |
Zinc concentration, m M |
Copper content per mole of protein, % |
Zinc per mole of protein, % |
|
WT |
178 |
157.96 |
167.67 |
88.74 |
94.1 |
|
G85R |
73 |
66.96 |
42.12 |
91.72 |
57.6 |
|
G93A |
201 |
180.78 |
191.45 |
89.9 |
95.02 |
|
A4V |
47 |
23.16 |
40.91 |
49.27 |
87.04 |
|
Apo-WT |
167 |
0.032 |
0.467 |
0.019 |
0.2 |
|
Apo-G85R |
106 |
0.063 |
0.202 |
0.059 |
0.19 |
|
Apo-G93A |
143 |
0.105 |
0.234 |
0.073 |
0.16 |
|
Apo-A4V |
86 |
0.056 |
0.134 |
0.065 |
0.15 |
*Concentration of metal and protein are estimated by using the monomeric molecular mass of superoxide dismutase.
Table 3. Thermdynamic parameters for small molecule-mediated stabilization of A4V
|
Protein/protein + compound |
Free energy ΔG, Kcal·mol–1 |
ΔΔ G for protein–drug complexes, Kcal·mol–1 |
m value,Kcal·mol–1·M–1 |
Cm, M |
|
A4V |
5.5 |
0.00 |
4.3 |
1.51 |
|
A4V+compound 1 |
8.12 |
2.62 |
4.8 |
2.12 |
|
A4V+compound 2 |
11.37 |
5.87 |
4.0 |
2.85 |
|
A4V+compound 3 |
10.91 |
5.41 |
3.8 |
2.76 |
|
A4V+compound 4 |
10.65 |
5.15 |
4.1 |
2.67 |
|
A4V+compound 5 |
7.78 |
2.28 |
3.9 |
1.99 |
|
A4V+compound 6 |
8.14 |
2.64 |
4.1 |
2.38 |
|
A4V+compound 7 |
10.21 |
4.71 |
3.9 |
2.61 |
|
A4V+compound 8 |
8.74 |
3.24 |
4.1 |
2.50 |
|
A4V+compound 9 |
7.34 |
1.84 |
3.8 |
2.00 |
|
A4V+compound 10 |
7.01 |
1.51 |
3.9 |
1.98 |
|
A4V+compound 11 |
7.78 |
2.28 |
4.1 |
2.02 |
|
A4V+compound 12 |
6.99 |
1.49 |
3.9 |
1.87 |
|
A4V+compound 13 |
7.32 |
1.82 |
3.8 |
2.12 |
|
A4V+compound 14 |
7.56 |
2.06 |
4.0 |
2.14 |
|
A4V+compound 15 |
7.65 |
2.15 |
4.2 |
2.18 |
|
WT |
12.1 |
6.60 |
4.3 |
3.21 |
m, slope of the linear extrapolation of guanidine unfolding fitted curce. Cm is the midpoint of unfolding transition.