Asperterpenes A and B, two unprecedented meroterpenoids from Aspergillus terreus with BACE1 inhibitory activities

Abstract

Asperterpenes A (1) and B (2), two 3,5-dimethylorsellinic acid-based meroterpenoids that contain a unique β-oriented Me-21 with an unprecedented 1,2,5-trimethyl-4,9-dioxobicyclo[3.3.1]non-2-ene-3-carboxylic acid moiety, were obtained from Aspergillus terreus in very limited amounts of 3.6 mg and 1.8 mg, respectively. The absolute structure of 1 was determined using X-ray diffraction. Because of the low yield of 1, a comprehensive characterization of the BACE1 inhibitory activities of 1 was completed via molecular biological, cell and animal studies guided by in silico target confirmation (ISTC). ISTC assays suggested that compounds 1 and 2 might be BACE1 inhibitors. In cell-based tests, asperterpenes A and B, as natural products, exhibited promising inhibitory activities against BACE1, with IC₅₀ values of 78 and 59 nM, respectively. LY2811376 (the positive control), one of the most potent clinical BACE1 inhibitors, has shown an IC₅₀ value of 260 nM. In vivo, compound 1 exhibited activity similar to that of LY2811376 against Alzheimer's disease (AD) in 3xTg AD mice. Taken together, these findings demonstrate that asperterpene A, which contains a novel carbon skeleton, is the first terpenoid to exhibit effective BACE1 inhibitory activity. Moreover, 1 represents a potential lead compound and a versatile scaffold for the development of drugs for the treatment of AD.

Fig. 1. Structures of compounds 1–4 (red: new ring systems and fusion patterns of A/B rings that differ from normal analogues).

Fig. 2. X-ray crystal structure of asperterpene A (1).

Scheme 1. Plausible biosynthetic pathway of 1–3.

Fig. 3. The binding modes of compounds 1 (A) and 2 (B) modelled in silico with BACE1 were predicted in the ISTR assays (red dashed lines represent hydrogen bonds and blue dashed lines represent π–π stacking interactions).

Fig. 4. Compound 1 inhibited BACE1 activity and decreased Aβ₄₂ production. 35, 70 and 135 nM concentrations of 1 added into the medium of N2a-APP cell lines. (A) Western blot analysis of the protein levels of APP-β, APP and DM1A. (B) Quantitative analysis of the ratio of APP-β/APP in the reference groups. (C) BACE1 activity was determined using β-Secretase Activity Assay Kit. (D) Aβ₄₂ levels were quantified through ELISA. Values are shown as mean ± SD. ***p < 0.001, **p < 0.01, *p < 0.05 versus the vehicle group.

Fig. 5. Compound 1 improved learning and memory impairment in 3xTg mice. 1 (2 μg μL^–1 × 5 μL, 0.2 μg μL^–1 × 5 μL), LY2811376 (2 μg μL^–1 × 5 μL) or vehicle was infused into the cerebroventricles of 3xTg mice 48 hours before starting the task. (A) Escape latencies to find the hidden platform were recorded daily. For the memory test, the time spent in the target quadrant (B), the times that the platform was crossed (C) and the swimming tracks (D) were recorded. (F) Western blot analysis of protein levels of APP-β and APP and DM1A, and (G) the quantitative analysis of APP-β/APP. (E) BACE1 activity in vivo was determined using β-Secretase Activity Assay Kit. (H) The Aβ₄₂ levels in vivo were quantified through ELISA. Data are presented as means ± SD. ***p < 0.001, **p < 0.01, *p < 0.05 versus the vehicle control (n = 10 in each group).

Fig. 6. Compound 1 attenuated synaptic toxicity in the hippocampus of 3xTg mice. (A) Western blot analysis of the protein levels of synapsin-1, synaptophysin, psd 95 and psd 93, and their (B) quantitative analysis. (C) Representative photomicrographs of primary dendrites in the hippocampal CA3 region. (D) Quantification of dendrites and mushroom-type dendrites. Values are shown as mean ± SD ***p < 0.001, **p < 0.01, *p < 0.05 versus the vehicle group alone (n = 10 in each group).