Structure-based discovery of fiber-binding compounds that reduce the cytotoxicity of amyloid beta
- Howard Hughes Medical Institute, UCLA–DOE Institute for Genomics and Proteomics, University of California, Los Angeles, United States
Figures
Figure 1 with 3 supplements
Structure-based identification of small compound inhibitors of Aβ toxicity.
In step (A) the crystal structure (Landau et al., 2011) is determined of a complex of an amyloidogenic segment of Aβ (in this case residues 16-KLVFFA-21 of the spine of the Aβ fiber) with an …
Figure 1—figure supplement 1
Structural models of the representative BAFs and orange G docked to the side of the KLVFFA(Aβ16–21) fiber.
In step (B) (Figure 1), a large library of ∼18 thousand commercially available compounds were docked onto the Aβ16–21 fiber, and ∼400 top ranking compounds, whose binding energy and shape …
Figure 1—figure supplement 2
Structural models of the representative BAFs and orange G docked onto the full-length Aβ fiber.
In step (C) (Figure 1), the top-ranking compounds after the first docking step were further filtered by docking onto full-length Aβ fiber model (pdb entry 2LMO) (Petkova et al., 2006). The models of …
Figure 1—figure supplement 3
Alternative binding modes of BAF1 with the Aβ full-length fibers.
When identifying BAFs by two steps of computational docking (Figure 2A as well as step B and C in Figure 1), most models of the second docking step (docking to full-length Aβ fiber in step (C) …
Figure 2 with 2 supplements
Computational screening for fiber-binding compounds.
(A). Outline of our procedure for structure-based screening. We prepare two sets of compounds (shown in the upper left) for screening against both types of fibers shown in the upper right. Compound …
Figure 2—figure supplement 1
Structural comparison between docked models of BAF8 and orange G.
BAF8 has a chemical structure similar to orange G (top panels). The comparison of the shape complimentary at binding interfaces reveals that BAF8 binds more tightly to the side of fibers than orange …
Figure 2—figure supplement 2
Stereo view of the structural model of BAF8 with Aβ fiber.
A wall-eyed stereo view of BAF8 (Figure 2D) (in cyan sticks) docked to the side of an Aβ16–21 fiber (light yellow) reveals good non-polar and polar interaction across binding interfaces. The …
Figure 3 with 2 supplements
Experimental characterization of compounds that bind to amyloid fibers.
Our newly discovered BAFs diminish Aβ1–42 toxicity without significantly reducing Aβ1–42 fibrillation. (A). Eight BAFs reduce Aβ toxicity in mammalian cell lines (PC12 in orange; HeLa in green). …
Figure 3—figure supplement 1
The BAFs alone exhibit little or no toxicity on mammalian cell lines.
Incubating cells with or without BAFs for 24 hours caused little or no change for cell viability of both PC12 and HeLa. The error bars are calculated from four experiment replicates.
Figure 3—figure supplement 2
BAFs cannot reduce the cytotoxicity of amyloid fibers formed by IAPP and α-synuclein, as much as those fibers formed by Aβ.
The final concentration of IAPP is 1 µM and α-synuclein is 2 µM. The molar ratio of amyloid fibers and BAFs is 1:1. BAFs (26 and 31), which significantly reduces Aβ toxicity (Figure 3), cannot …
Figure 4
Diversified chemical structures of 8 active BAF compounds that reduce Aβ toxicity.
Orange G in an orange box is also displayed for comparison.
Figure 5 with 3 supplements
NMR evidence for binding of compounds to both Aβ16–21 and Aβ1–42 fibers.
NMR binding experiments were performed on BAF compounds and the dye orange G. By monitoring the aromatic regions of the 1H NMR spectra of BAFs 1, 8, and 31, these compounds were shown to bind to …
Figure 5—figure supplement 1
NMR peak assignment of BAF1 with Aβ16–21 fiber.
The 1D 1H NMR spectrum shows the aromatic proton regions of BAF1 upon the titration of Aβ16–21 fibers shown in Figure 5A. The insert is the chemical structure of BAF1 with the color-labeled aromatic …
Figure 5—figure supplement 2
NMR peak assignment of the control compound orange G with Aβ16–21 fiber.
The 1D 1H NMR spectrum shows the aromatic proton regions of orange G against the increasing concentrations of Aβ16–21 fibers shown in Figure 5B. The insert is the chemical structure of orange G with …
Figure 5—figure supplement 3
NMR titration of BAF8 with Aβ16–21 fibers.
To validate our computation methods, NMR titration experiments were performed. (A) One representative peak of aromatic protons of the 1D 1H NMR spectra of the compound BAF8 (at 100µM) upon Aβ16–21 …
Figure 6 with 1 supplement
Refinement of the Aβ pharmacorphore based on studies of BAF11.
(A) Atomic model of BAF11 from the initial cycle docked on the full-length Aβ fiber, viewed in perpendicular to the fiber axis (left panel) and down the fiber axis (right panel). BAF11 is shown as a …
Figure 6—figure supplement 1
Chemical structures of the lead compound BAF11 and its derivatives.
12 derivatives of the lead compound BAF11 were included to expand the set during the refinement of amyloid pharmacophore (Figure 6C). (A) Chemical structures of BAF11 derivatives. A magenta open …
Figure 7
New BAFs derived from the refined amyloid pharmacophore.
(A). Amyloid pharmacophore based on the structural overlay of active BAFs and derivatives. The overlay of the lead compounds from the initial round (BAF4, BAF8, and BAF11) elucidated the consensus …
Figure 8 with 1 supplement
Elimination of one key hydrogen bond from BAF31 causes both the loss of NMR binding to Aβ fibers and the decrease in inhibition of Aβ cyto-toxicity.
(A) Atomic model of the new inhibitor BAF31 (our most tightly binding BAF) derived from the refined pharmacophore (Figure 7, Figure 1F) in the second cycle, viewed perpendicular to the fiber axis on …
Figure 8—figure supplement 1
NMR titration of BAF31 and its derivative with the Aβ1–42 fiber.
(A). 1D 1H NMR spectrum of BAF31 (100 µM) without (in black) and with Aβ1–42 fiber (12.5 µM monomer equivalent, in a green color). The magnified peaks are shown in the right panel to highlight the …
Figure 9
Analysis of the lead compound BAF30 and its derivatives.
Structural models of BAF30 (green sticks) docked on Aβ fiber structure (in a light yellow color) are shown in (A and B). The important polar (black hydrogen bonds) interaction between BAF30 and …
Figure 10
General rule of the essential interactions between BAFs and Aβ fiber can be derived from structure-based screening of Aβ toxicity inhibitor.
The carbonyl group is used to represent the H-bond acceptor (or negative charge) of BAFs, and the naphthalene ring is used to represent the planar aromatic portion of BAFs. Based on the rounds of …
Figure 11 with 1 supplement
BAFs are designed to bind to in-register β-sheets, rather than out-of-register β-sheets.
As illustrated in (A), BAFs bind to in-register β-sheets. Our structure-based approach searches for BAFs based on in-register β-sheets in Aβ fibers. These BAFs are predicted to bind along the flat …
Figure 11—figure supplement 1
Active BAFs show no or little effects on the cyto-toxicity of pre-formed Aβ oligomers.
To assess if BAFs inhibit Aβ toxicity by directly interfering with toxic Aβ oligomers, four BAFs —1,11,26,31—, showing the inhibition to Aβ toxicity, were incubated with pre-formed Aβ oligomer and …
Figure 12
Proposed mechanism of how compound binding increases fiber stability and decreases fiber toxicity.
BAFs (green) bind to the side of amyloid fibers, stabilizing the fiber, and shifting the equilibrium from smaller and more toxic oligomers towards fibers. This shift in equilibrium reduces amyloid …
Figure 13 with 1 supplement
BAFs bind to in-register β-sheets and are compatible to both parallel and antiparallel amyloid β-sheets.
A subtlety of our procedure for compound discovery is that it involves both parallel (A) and antiparallel (B) amyloid β-sheets. In the X-ray structure of orange G bound to the segment Aβ16–21(KLVFFA)…
Figure 13—figure supplement 1
Structural models of orange G docked onto the antiparallel Aβ16–21 (A) and parallel full-length Aβ (B) fiber.
(A). The side view of orange G (in an orange color) docked on the Aβ16–21 fiber (in a grey color) with a predicted binding energy of—8 kcal/mol. (B) side view of orange G (in an orange color) docked …
Tables
Table 1
List of all tested BAF compounds
| Compound | Molecular formula | Molecular weight* | Sources/purchasing | Rescuing percentage (%) | ZINC entry |
|---|---|---|---|---|---|
| BAF1 | C20H8Br4O5 | 648 | Sigma-Aldrich | 44 ± 7 | ZINC04261875 |
| BAF2 | C19H14O5S | 354 | Sigma-Aldrich | 4 ± 3 | ZINC03860918 |
| BAF3 | C16H13NO3 | 267 | Ryan Scientific | 4 ± 5 | ZINC04289063 |
| BAF4 | C24H16N2O6 | 428 | Aldrich | 88 ± 22 | ZINC13346907 |
| BAF5 | C16H7Na3O10S3 | 524 | Sigma-Aldrich | 11 ± 7 | ZINC03594314 |
| BAF6 | C26H20N2 | 360 | Alfa-Aesar | 5 ± 7 | ZINC08078162 |
| BAF7 | C18H12N6 | 312 | Alfa-Aesar | 2 ± 2 | ZINC00039221 |
| BAF8 | C17H14N2O5S | 358 | Sigma-Aldrich | 23 ± 11 | ZINC12358966 |
| BAF9 | C19H13N3O4S | 379 | NCI plated 2007† | −3 ± 22 | ZINC03954432 |
| BAF10 | C17H13NO3 | 279 | NCI plated 2007 | 3 ± 5 | ZINC00105108 |
| BAF11 | C20H13N2O5S | 393 | NCI plated 2007 | 48 ± 12 | ZINC04521479 |
| BAF12 | C13H8Br3NO | 434 | NCI plated 2007 | 38 ± 6 | ZINC12428965 |
| BAF13 | C19H16ClNO4 | 358 | Sigma-Aldrich | 0 ± 2 | ZINC00601283 |
| BAF14 | C10H6S2O8 | 318 | Sigma-Aldrich | 3 ± 3 | ZINC01532215 |
| BAF15 | C23H28O8 | 432 | Sigma-Aldrich | 13 ± 4 | ZINC00630328 |
| BAF16 | C19H19NO5 | 341 | Sigma-Aldrich | 5 ± 8 | ZINC28616347 |
| BAF17 | C23H25N5O2 | 404 | Sigma-Aldrich | 6 ± 3 | ZINC00579168 |
| BAF18 | C24H16O2 | 336 | ChemDiv | 6 ± 2 | ZINC02168932 |
| BAF19 | C18H14N2O6 | 354 | ChemDiv | 3 ± 4 | ZINC01507439 |
| BAF20 | C25H19N5OS | 438 | ChemDiv | 8 ± 4 | ZINC15859747 |
| BAF21 | C19H14Br2O | 418 | ChemDiv | 6 ± 3 | ZINC38206526 |
| BAF22 | C21H16N2O3S2 | 408 | Life Chemicals | 3 ± 5 | ZINC04496365 |
| BAF23 | C16H11ClO5S | 351 | Enamine Ltd | 3 ± 5 | ZINC02649996 |
| BAF24 | C23H19NO3 | 357 | Sigma-Aldrich | 16 ± 5 | ZINC03953119 |
| BAF25 | C14H8Cl2N4 | 303 | Sigma-Aldrich | 4 ± 3 | ZINC00403224 |
| BAF26 | C17H10O4 | 278 | Aldrich | 46 ± 23 | ZINC05770717 |
| BAF27 | C21H16BrN3O6 | 486 | ChemBridge | 4 ± 1 | ZINC01208856 |
| BAF28 | C17H12N2O3 | 292 | ChemBridge | 2 ± 4 | ZINC00061083 |
| BAF29 | C22H10N4O2 | 362 | ChemBridge | 1 ± 5 | ZINC00639061 |
| BAF30 | C14H8O5 | 256 | Aldrich | 18 ± 13 | ZINC03870461 |
| BAF31 | C19H21NO3 | 311 | Sigma | 84 ± 12 | ZINC00011665 |
| BAF32 | C15H14O7 | 306 | Sigma-Aldrich | 15 ± 9 | ZINC03870336 |
| BAF33 | C27H33N3O8 | 528 | Sigma-Aldrich | 7 ± 2 | SIGMA-R2253§ |
| BAF34 | C30H16N4O14S4 | 785 | Aldrich | ‡ | ALDRICH-S432830§ |
| orange G | C16H12N2O7S2 | 408 | Sigma-Aldrich | −2 ± 8 | ZINC04261935 |
-
The 25 compounds (BAF1-25) are from the first round, and the nine compounds (BAF26-34) are from the second round. Another set of the 17 derivatives of the BAFs are shown in Table 3.
-
*
Molecular weight (anhydrous basis) excluding the salt and water molecules.
-
†
National Cancer Institute (NCI) free compound library (http://dtp.nci.nih.gov/).
-
‡
Toxicity results of BAF34 were not consistent among several independent replica experiments, possibly due to impurity and the high molecular weight of the compound.
-
§
ZINC entry of the compound is not applicable, and the catalog number from Sigma-Aldrich is provided.
Table 2
Detailed list of the active BAF compounds
| Compound | Molecular formula | Molecular weight* | Sources/companies | Purity | Rescuing percentage§ (%) | ZINC entry code¶ | SMILES string | |
|---|---|---|---|---|---|---|---|---|
| PC12 | Hela | |||||||
| BAF1 | C20H8Br4O5 | 647.9 | Sigma-Aldrich | ∼99% | 38 ± 11 | 44 ± 7 | ZINC04261875 | c1ccc2c(c1)C(=O)OC23c4ccc(c(c4Oc5c3ccc(c5Br)O)Br)O |
| BAF4 | C24H16N2O6 | 428.4 | Aldrich | ≥95% | 85 ± 18 | 88 ± 22 | ZINC13346907 | c1cc(c(cc1O)O)c2cc3c(cc2N)oc-4cc(=O)c(cc4n3)c5ccc(cc5O)O |
| BAF8 | C17H14N2O5S | 358.4 | Sigma-Aldrich | ≥90% | 26 ± 12 | 23 ± 11 | ZINC12358966 | Cc1ccc(c(c1)/N=N/c2c3ccccc3c(cc2O)S(=O)(=O)[O-])O |
| BAF11 | C20H13N2O5S | 393.5 | NCI plated 2007 | † | 51 ± 11 | 48 ± 12 | ZINC04521479 | c1ccc2c(c1)ccc(c2O)/N=N/c3c4ccccc4c(cc3O)S(=O)(=O)[O-] |
| BAF12 | C13H8Br3NO | 433.9 | NCI plated 2007 | † | 19 ± 6 | 38 ± 6 | ZINC12428965 | c1cc(ccc1/N=C/c2cc(cc(c2O)Br)Br)Br |
| BAF26 | C17H10O4 | 278.3 | Aldrich | ‡ | 60 ± 21 | 46 ± 23 | ZINC05770717 | c12c(cc(cc1)C(=O)C=O)Cc1c2ccc(c1)C(=O)C=O |
| BAF30 | C14H8O5 | 256.2 | Aldrich | ‡ | 37 ± 18 | 18 ± 13 | ZINC03870461 | c1cc2c(cc1O)C(=O)c3c(ccc(c3O)O)C2=O |
| BAF31 | C19H21NO3 | 311.4 | Sigma | ≥98% | 92 ± 22 | 84 ± 12 | ZINC03874841 | CCCN1CCC2=C3C1CC4=C(C3=CC(=C2)O)C(=C(C=C4)O)O |
-
BAFs 1, 4, 8, 11, 12 are from the first round. BAFs 26, 30, 31 are from the second round.
-
*
Molecular weight (anhydrous basis) excluding the salt and water molecules.
-
†
With the standard of NCI free compound library.
-
‡
Analytical data for AldrichCPR products are not available.
-
§
Rescue percentage is a scaled cell survival rate.
-
¶
Entry code for the ZINC database (http://zinc.docking.org).
Table 3
List of the representative BAFs 11, 30, 31 and their derivatives
| Compound | Molecular formula | Molecular weight | Description | Toxicity inhibition (%) | ZINC entry/catalog no. |
|---|---|---|---|---|---|
| BAF31 | C19H21NO3 | 311 | 84 ± 12 | ZINC03874841 | |
| BAF31ΔOH | C19H21NO2 | 295 | remove one hydroxyl (OH) | 15 ± 2 | ZINC03874841 |
| BAF30 | C14H8O5 | 256 | 18 ± 13 | ZINC03870461 | |
| BAF30αR | C22H20O13 | 492 | add additional R group away from binding interface | 20 ± 10 | ZINC28095922 |
| BAF30σOHAαOH | C14H8O6 | 272 | change one OH (A) position; add another OH | 9 ± 9 | ZINC03874832 |
| BAF30σOHAΔOHBαCOO | C15H8O6 | 284 | move one OH (A) position; delete an OH from loc B; add a carboxyl | 9 ± 3 | ZINC04098704 |
| BAF30σOHABαCH3 | C15H10O5 | 270 | move two OH (AB) positions; add a methyl | 6 ± 3 | ZINC03824868 |
| BAF11 | C20H13N2O5S | 393 | 48 ± 12 | ZINC04521479 | |
| BAF11ISO | C20H13N2O5S | 393 | isomer form of BAF11 | 33 ± 5 | ZINC12405071 |
| BAF11σR1 | C20H14N4O8S2 | 502 | change the aromatic group | 35 ± 9 | ZINC25558261 |
| BAF11σR2 (BAF8) | C17H14N2O5S | 358 | change the aromatic group | 22 ± 11 | ZINC12358966 |
| BAF11σR3 | C16H12N2O6S | 360 | change the aromatic group | 28 ± 4 | ZINC04900892 |
| BAF11αNO2- | C20H12N3O7S | 438 | add charged group (nitro) | 15 ± 6 | ZINC16218542 |
| BAF11ISOαCOO- | C21H12N2O7S | 436 | BAF11 isomer; add charged group (carboxyl) | 6 ± 5 | ZINC03861030 |
| BAF11ISOαSO3- | C20H11N2O11S3 | 552 | BAF11 isomer; add charged group (sulfate) | 2 ± 5 | SIGMA-33936 |
| BAF11ΔOHσR | C20H14N2O4S | 378 | remove an OH;change the position of the aromatic group | 15 ± 6 | ZINC04803992 |
| BAF11ΔOHαSO3− | C20H14N2O7S2 | 458 | remove an OH; add sulfate group | 12 ± 3 | ZINC03954029 |
| BAF11ΔOHαR | C20H18N4O5S | 426 | remove an OH; add additional group to the aromatic ring | 12 ± 6 | ZINC04416667 |
| BAF11σOHαR1 | C24H20N4O4S | 461 | swap the position of the OH and aromatics | 5 ± 5 | ZINC04804174 |
| BAF11σOHαR2 | C16H19N3O5S | 365 | swap the position of the OH and aromatics | 4 ± 6 | ZINC17378758 |
Table 4
Student’s t-test and p value analysis suggests that BAFs reduce the cytotoxicity of Aβ fibers significantly
| Average of cell viability (n = 4) | SD(σ) | Comparison to Aβ fiber alone | ||
|---|---|---|---|---|
| t value | p value | |||
| HeLa cell line | ||||
| Aβ fiber alone | 0.40 | 0.05 | / | / |
| BAF1 | 0.66 | 0.04 | 8.4 | 5E-05 |
| BAF4 | 0.93 | 0.13 | 7.4 | 1E-4 |
| BAF8 | 0.54 | 0.06 | 3.3 | 1E-2 |
| BAF11 | 0.69 | 0.07 | 6.6 | 2E-04 |
| BAF12 | 0.63 | 0.04 | 7.6 | 1E-04 |
| BAF26 | 0.68 | 0.14 | 3.8 | 5E-3 |
| BAF30 | 0.51 | 0.08 | 2.3 | 4E-2 |
| BAF31 | 0.91 | 0.07 | 11.5 | 7E-06 |
| PC12 cell line | ||||
| Aβ fiber alone | 0.37 | 0.07 | / | / |
| BAF1 | 0.61 | 0.07 | 4.9 | 1E-3 |
| BAF4 | 0.90 | 0.11 | 8.0 | 7E-05 |
| BAF8 | 0.53 | 0.07 | 3.2 | 1E-2 |
| BAF11 | 0.69 | 0.07 | 6.5 | 2E-4 |
| BAF12 | 0.49 | 0.04 | 2.9 | 2E-2 |
| BAF26 | 0.74 | 0.13 | 5.0 | 1E-3 |
| BAF30 | 0.60 | 0.11 | 3.5 | 8E-3 |
| BAF31 | 0.95 | 0.14 | 7.4 | 1E-4 |
-
The Student’s T-test and p-value are based on the comparison to Aβ fiber alone.
Table 5
Predicted binding energy and experimental measurement of the binding of two BAFs and orange G against both Aβ16–21 (KLVFFA) and full-length Aβ fibers
| Binding to KLVFFA fiber | Binding to Aβ fiber | |||
|---|---|---|---|---|
| Predicted binding energy (kcal/mol) | NMR Kd (µM) | Predicted binding energy (kcal/mol) | NMR peak reduction (%) | |
| BAF1 | −8 | 12 | −10 | 8 |
| BAF8 | −12 | 24 | −12 | 13 |
| orange G | −8 | 43 | −9 | 6 |
-
The determination of the binding parameters with KLVFFA fiber is detailed in Table 6.
Table 6
Comparison of the measured binding parameters of the representative BAFs with orange G by NMR titrations
| Compound | Predicted binding energy (kcal/mol) | fmax | Kd (µM) |
|---|---|---|---|
| BAF1 | −8 | 0.47 ± 0.04 | 12 ± 7 |
| BAF8 | −12 | 0.82 ± 0.04 | 24 ± 5 |
| Orange-G | −8 | 0.46 ± 0.06 | 43 ± 21 |
-
The second column lists the predicted binding energy for each top docked model of BAF compounds with KLVFFA fiber, and the binding energy of Orange-G with KLVFFA fiber were also calculated for comparison. Our computational method identified the BAF with better fit to the binding interface than Orange-G. We then used NMR titration to determine the binding affinity. Our previous mass spectrometric analyses of the crystal of the Orange-G with KLVFFA fibers have suggested a binding ratio of compound:fiber with the range of 1:1 to 1:10 (Landau et al., 2011). Together with our structural models and single binding site assumption, we estimated the binding ratio to be 1:3. Accordingly, calculated NMR binding parameters are listed in the table. The third column fmax is the maximum fraction of NMR signal decrease of compound upon binding saturation (‘Materials and methods’).
Table 7
BAFs reduce Aβ cyto-toxicity by targeting fibers rather than oligomers.
| Compound | Inhibition to the cyto-toxicity of Abeta oligomers (%) | Inhibition to the cyto-toxicity of Abeta fibers (%) |
|---|---|---|
| BAF1 | −4 ± 6 | 36 ± 9 |
| BAF11 | −9 ± 7 | 7 ± 7 |
| BAF26 | −6 ± 6 | 26 ± 7 |
| BAF31 | −17 ± 15 | 58 ± 7 |
-
The BAF inhibitions of toxicity from either Aβ oligomer or fibers are compared. Four BAFs, which reduce the toxicity of Aβ fibers, show no inhibitory effects to Aβ oligomer toxicity at the equal molar ratio of BAF to Aβ. The inhibition (%) are calculated using the same method defined in ‘Materials and methods’. The toxicity assay of Aβ oligomer is described in Figure 11—figure supplement 1. The toxicity assay of Aβ fiber is the same as that described in Figure 3.
Additional files
-
Supplementary file 1
Compound Library Set 1: Cambridge Structure Database (CSD) set.
- https://doi.org/10.7554/eLife.00857.037
-
Supplementary file 2
Compound Library Set 2: Flat Compound (FC) set.
- https://doi.org/10.7554/eLife.00857.038
