Borrelidin from Saltern-Derived Halophilic Nocardiopsis sp. Dissociates Amyloid- β and Tau Fibrils

MTT assay

Borrelidins (1–4) were obtained as previously reported [9]. Aβ₄₂ synthesis and MTT assay using HT22 murine hippocampal neuronal cells were performed as previously described [14–17]. Cells (2×10⁴ cells/well) were treated with 10μM Aβ₄₂ with 1–4 (20, 100μM) and incubated for 24 h at 37°C. After the incubation, Aβ-induced cytotoxicity was measured by absorbance as previously reported [16, 17].

Docking simulation

The docking of 1–4 to Aβ₄₂ were described previously [17, 18]. Briefly, the two-staged docking procedure was sequentially performed using the three-dimensional conformers of each ligand. The numbers of conformers considered for docking were 61, 42, 75, and 51 for 1–4, respectively. The low-resolution global docking was first performed by PatchDock software [19] to estimate potential binding sites in the Aβ structure (PDB ID 2LMO). Of the low-resolution docking structures, the top 500 were used for the secondary all-atom refinement by Rosetta software [20]. Docking structures were refined by the customized Rosetta ligand docking protocol, consisted of five rounds of docking/minimization, two low-repulsive followed by three normal. The docking conformation with the lowest binding energy was finally selected as the most plausible model. The binding energy was estimated by the Rosetta energy difference between ligand-bound and unbound structure.

Aβ inhibition and disaggregation

10 mM Aβ₄₂ were dissolved in DMSO and diluted with distilled water to make 25μM Aβ solutions. To measure the inhibition effect of 1–3 on Aβ aggregation, monomeric Aβ₄₂ with 1 mM of each compound was incubated for five days at 37°C. For Aβ disaggregation, 0.1 mM of Aβ₄₂ in 10% DMSO and 90% distilled water was aggregated for three days at 37°C. After the pre-incubation, 1 mM of compound was added to aggregates and incubated for additional three days at 37°C. To observe the inhibition or dissociating effect of compound, all samples were loaded into the half-area 96-well black plate. 75μL of ThT solution (5μM in 50 mM glycine buffer, pH 8.5) was added to each well [21]. Fluorescence of Aβ-bound ThT was measured at 450 nm (excitation) and 485 nm (emission) using SpectraMax M2 microplate reader (Molecular Devices).

Tau inhibition and disaggregation

Tau K18 fragment (0.5 mg/mL in PBS) was used in these assays. For tau inhibition assay, 1 mM of compound was added to tau containing 0.1 mg/mL of Heparin and 100μM of DTT, aggregation inducers. Mixed samples were incubated for five days at 37°C. For disaggregation assay, tau containing inducers was aggregated for two days at 37°C. After the pre-incubation, 1 mM of compound was added to aggregates and incubated for additional three days at 37°C. To evaluate the inhibition and disaggregation effect of compound, the fluorescence intensity was measured by ThT assay as described above.

PICUP and SDS-PAGE

To visualize inhibitory effect of compounds on Aβ aggregation, all samples were prepared under the identical conditions as used in ThT assay. For Aβ cross-linking, 10 mM Ru(Bpy) and 200 mM APS were dissolved in buffer A (0.1 M sodium phosphate, pH 7.4), and diluted with the same buffer to make 1 mM and 20 mM, respectively. Then, 1μL of Ru(Bpy) and APS were added to 10μL of each incubated sample. The mixtures were irradiated by visible light, and the reaction was quenched by adding 3μL of 5X sample buffer containing 5% reducing agent. Then, samples were boiled for 5 min at 95°C and separated by SDS-PAGE electrophoresis. After gel running, different size of Aβ peptides was visualized by silver staining according to the manufacturer’s instructions (GE Healthcare).

Statistical analysis

Graphs were obtained with GraphPad Prism 7 and statistical analysis were performed with Student’s t-tests and one-way ANOVA, followed by Bonferroni’s post hoc comparisons tests (^*p < 0.05, ^**p < 0.01, ^***p < 0.001). The error bars in all data represent the SEM.

Borrelidins bind to Aβ aggregates in docking model

As the abnormal aggregation of Aβ and tau exacerbate AD, the direct interaction between these pathological proteins and drug candidate is needed to dissociate the toxic aggregates. In this regard, we initially performed exhaustive low-resolution docking simulation to find the most plausible docking model of 1–4 to the Aβ structure. These low-resolution docking structures were then refined by Rosetta ligand optimization protocol with all-atom Rosetta energy function (REF15) [22]. The final docking model for each ligand was selected by the calculated binding energy.

Among the docking models, 1 showed the lowest binding energy (–23.04 by Rosetta Energy Unit) and 2–4 showed undistinguishable binding energies (Fig. 1B). The docking structures of 1, 2, and 4 had similar binding modes that commonly packed in the hydrophobic patch formed by the beta-strand assembly, whereas 3 was located between the beta-strand assemblies. Notably, the docking model of 1 was more sequestered inside the beta-strands than the others, which might be more effective in terms of preventing further Aβ aggregation.

The chemical structures of 1 and 2 are very similar except for the hydroxyl group (solid line circle in Fig. 1B) attached to the cyclopentane ring. In the docking model of 1, the cyclopentane ring showed tight hydrophobic contacts to the Aβ structure without the hydroxyl group attachment. However, 2 was docked more towards the exterior surface, probably due to the presence of the hydroxyl group in the cyclopentane ring. The hydroxyl group in 2 formed hydrogen bonds to the main-chain atoms of Val39.

On the other hand, the chemical structure of 4 is almost similar to 1 except for another hydroxyl group attached to the carbon chain (dotted line circle in Fig. 1B). The carbon chains in 4 faced outside, but the same part in all other ligands without the hydroxyl group was tightly packed into the hydrophobic patches in the Aβ structure. Moreover, the docking model of 3 showed a distinctive binding mode where the hydroxyl group was fully exposed to the solvent. Accordingly, the two hydroxyl groups might be responsible for the varied activities of the borrelidin derivatives on Aβ aggregation.

Borrelidins reduce Aβ-induced cytotoxicity

Since the possibility of binding between Aβ and borrelidins was confirmed through the docking model, we performed additional biochemical assays to investigate the effects of 1–4 on Aβ and tau aggregates. To evaluate their effects on Aβ-oligomer-induced neuronal cell death, we performed MTT assay using HT22, murine hippocampal cell line. Aβ were pre-aggregated at 37°C for 24 hours to form oligomers. We observed about 60% decrease in cell viability due to the toxicity induced by pre-formed aggregates. However, when the cells were treated with 1–4, a significant increase in cell viability was observed (Fig. 2A). 1 and 2 attenuated the Aβ oligomer-induced neuronal death dose dependently, and about 100% of viability as non-treated cell was shown at higher concentration (100μM) of both compounds. 4 showed the significant change in only higher concentration. These findings indicated that both concentrations (20 and 100μM) of 1–3 reduced the neurotoxicity induced by Aβ oligomers.

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Fig. 2

The effects of borrelidins, especially borrelidin (1), against Aβ and tau pathology in vitro. A) The effects of each compound on Aβ-induced cell death were measured by MTT assay in HT22 neuronal cell line. Cell viability was normalized to non-treated cells (100%). B, C) The inhibition effects of borrelidin (1) and borrelidin C, D (2, 3) were presented by ThT assay (B) and PICUP followed by SDS-PAGE gel (C). Fluorescence intensity was normalized to Aβ aggregates (100%, 5 days incubation). D) Aβ disaggregation effect of borrelidin (1) by ThT assay. Fluorescence intensity was normalized to pre-formed Aβ aggregates (100%, 3 days incubation). E, F) Tau inhibition (E) and disaggregation effects (F) of borrelidin (1) by ThT assay. Fluorescence intensity was normalized to tau aggregates (100%, 5 days incubation) and pre-formed tau aggregates (100%, 2 days incubation), respectively. Cpd, compound; aggr., aggregates. All data were represented as mean±SEM. MTT and ThT assays were analyzed by Student’s t-tests and One-way ANOVA followed by Bonferroni post-hoc comparison tests (^*p≤0.05 and ^***p≤0.001).

Borrelidins inhibit Aβ aggregation

Next, we quantified the Aβ fibril formation through ThT fluorescence intensity [23] to examine the protective mechanisms of borrelidins against neuronal death by Aβ aggregates. All compounds significantly inhibited the Aβ aggregation, especially, 1 exhibited the highest effect against Aβ aggregation, reducing ThT-positive aggregates by more than 50% (Fig. 2B). However, it is difficult to identify the effects on oligomers or protofibrils by ThT assays. Thus, all samples were visualized after covalently cross-linking of proteins to fix the original states of aggregates [24]. Through the silver stained SDS-PAGE gel, we discovered that all compounds, especially 1 and 3, remarkably decreased the formation of Aβ fibrils in size greater than 250 kDa (Fig. 2C), corresponding to ThT results (Fig. 2B). It might be because the conversion of aggregates to the monomers (4.5 kDa) by the compounds, resulting in increase of Aβ monomers when incubated with each compound.

Borrelidin dissociates pre-formed Aβ aggregates and also regulates tau pathology

Overall, 1 showed the highest possibility; interaction with Aβ aggregates, leading to the decrease of neuronal cell death via an inhibition of Aβ aggregation. However, the clearing of toxic pre-formed aggregates is considered more effective way because these accumulation starts decades in the brain before onset of symptoms. Thus, we examined whether 1, which was regarded as the most potent inhibitor, also has a clearance effect on pre-existing Aβ aggregates. We observed the levels of Aβ aggregates measured by ThT fluorescence intensity were notably reduced when incubated with 1, indicating that it could dissociate pre-existing Aβ aggregates (Fig. 2D).

As aggregation of tau proteins are also contributed to neuronal dysfunction and eventually neuronal death, we confirmed that the inhibitory effects of 1 on tau aggregation. Tau K18 fragment containing repeat domain was used in this assay. After tau incubation with 1, we found that the level of tau aggregation decreased by more than 50% in ThT assay (Fig. 2E). Moreover, ThT fluorescence intensity was also reduced when 1 was incubated with pre-aggregated tau, implying that this small molecule has inhibitory effect on tau aggregation and also can dissociate tau aggregates (Fig. 2F).