Inhibition of Tau aggregation by three Aspergillus nidulans secondary metabolites: 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde

Abstract

The aggregation of the microtubule-associated protein tau is a significant event in many neurodegenerative diseases including Alzheimer's disease. The inhibition or reversal of tau aggregation is therefore a potential therapeutic strategy for these diseases. Fungal natural products have proven to be a rich source of useful compounds having wide varieties of biological activity. We have screened Aspergillus nidulans secondary metabolites containing aromatic ring structures for their ability to inhibit tau aggregation in vitro using an arachidonic acid polymerization protocol and the previously identified aggregation inhibitor emodin as a positive control. While several compounds showed some activity, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde were potent aggregation inhibitors as determined by both a filter trap assay and electron microscopy. In this study, these three compounds were stronger inhibitors than emodin, which has been shown in a prior study to inhibit the heparin induction of tau aggregation with an IC50 of 1-5 µM. Additionally, 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde reduced, but did not block, tau stabilization of microtubules. 2,ω-Dihydroxyemodin and asperthecin have similar structures to previously identified tau aggregation inhibitors, while asperbenzaldehyde represents a new class of compounds with tau aggregation inhibitor activity. Asperbenzaldehyde can be readily modified into compounds with strong lipoxygenase inhibitor activity, suggesting that compounds derived from asperbenzaldehyde could have dual activity. Together, our data demonstrates the potential of 2,ω-dihydroxyemodin, asperthecin, and asperbenzaldehyde as lead compounds for further development as therapeutics to inhibit tau aggregation in Alzheimer's disease and neurodegenerative tauopathies.

Fig. 1

Compounds used in this study. The chemical structures are drawn for the 17 compounds used. The names of the compounds are included with their structure along with a compound number. Each compound was purified from a single HPLC peak. The purity of each compound was estimated from its ¹H NMR spectrum (see Supplemental data) and is listed in Supplemental Table S1. In F9775 B, NOESY correlations between H-13/H₃-7 and H-13/OH-8 suggested that H₃-7, OH-8, and H-13 are on the same face. The fact that the specific rotation of F9775 A and B is close to zero indicated that both compounds are racemic mixtures [48]. Stereocenters on the side chain of 3’-hydroxyversiconol have not been determined due to the free rotation of the side chain.

Fig. 2

Filter Trap Assay of Tau Filament Formation. Tau polymerization reactions were performed with 2 μM tau and 75 μM arachidonic acid either with or without 200 μM compound. The compounds used are listed on the Y-axis. The resulting amount of tau filament formation was determined by filter trap assay. The values for tau filament formation were normalized to the amount of the no compound control (dashed line). Negative values indicate that there was less detectable tau on the filter after treatment with compound than was observed with monomeric tau in the absence of arachidonic acid. Values are the average of three trials ± std. dev. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001

Fig. 3

EM Images of Polymerization Reactions. Tau polymerization reactions were performed with 2 μM tau and 75 μM arachidonic acid either with or without 200 μM compound. Aliquots of the reactions were prepared for negative stain electron microscopy. The individual images are labeled with the compound used.

Fig. 4

Filament Length Distributions. Filament lengths from electron micrographs of tau polymerization reactions were measured, placed into 50 nm bins (30–50nm, 50–100nm, 100–150nm, 150–200 nm and >200 nm) and the lengths were summed to determine the total amount of filament length in each bin. The first graph is the control reaction without compound and (1)-(17) are the graphs of the different compounds and are labeled with the compound name and number on the graph. The filament distribution for the no compound control is redrawn on each graph as a light gray line for comparison. Each point is the average distribution for images of at least 9 different fields ± std. dev. * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001

Fig. 5

IC₅₀ Determination. Polymerization reactions at 2 μM tau and 75 μM arachidonic acid were performed at several different concentrations of compound and the resulting amount of filament formation was determined by filter trap assay. The amount of polymerization was normalized to controls without compound (100%). The normalized data was plotted against the log of the inhibitor concentration and fit to a dose-response curve (solid line) to determine the IC₅₀ for A) 2,ω-dihydroxyemodin, B) asperthecin and C) asperbenzaldehyde. Data points are the average of three trials ± std. dev.

Fig. 6

Microtubule Assembly. Tubulin was incubated with either tau protein alone (●) or tau in the presence of A) 2,ω-dihydroxyemodin, B) asperthecin or C) asperbenzaldehyde at compound concentrations of 50 μM (■) or 100 μM (▲). Microtubule assembly was monitored by DAPI fluorescence (y-axis) over time (x-axis) and normalized to microtubule polymerization in the presence of paclitaxel. Every third time point is shown. Each point is the average of three independent trials ± std. dev. The data are fit to a Gompertz growth curve (solid, dashed and dotted lines for no compound, 50 μM compound and 100 μM compound respectively). * P ≤ 0.05; ** P ≤ 0.01; *** P ≤ 0.001