Trehalose Conjugates of Silybin as Prodrugs for Targeting Toxic Aβ Aggregates

Abstract

Alzheimer's disease (AD) is linked to the abnormal accumulation of amyloid β peptide (Aβ) aggregates in the brain. Silybin B, a natural compound extracted from milk thistle (Silybum marianum), has been shown to significantly inhibit Aβ aggregation in vitro and to exert neuroprotective properties in vivo. However, further explorations of silybin B's clinical potential are currently limited by three main factors: (a) poor solubility, (b) instability in blood serum, and (c) only partial knowledge of silybin's mechanism of action. Here, we address these three limitations. We demonstrate that conjugation of a trehalose moiety to silybin significantly increases both water solubility and stability in blood serum without significantly compromising its antiaggregation properties. Furthermore, using a combination of biophysical techniques with different spatial resolution, that is, TEM, ThT fluorescence, CD, and NMR spectroscopy, we profile the interactions of the trehalose conjugate with both Aβ monomers and oligomers and evidence that silybin may shield the "toxic" surfaces formed by the N-terminal and central hydrophobic regions of Aβ. Finally, comparative analysis with silybin A, a less active diastereoisomer of silybin B, revealed how even subtle differences in chemical structure may entail different effects on amyloid inhibition. The resulting insight on the mechanism of action of silybins as aggregation inhibitors is anticipated to facilitate the future investigation of silybin's therapeutic potential.

Chirality Matters: Biological Activity of Optically Pure Silybin and Its Congeners

“…Unfortunately, the authors did not consider transport over the hemato-encephalitic barrier, which would be presumably impermeable for such a big molecule. The above findings prompted the group of García-Viñuales et al [102] to prepare other C-23 conjugates of silybin A and B with trehalose linked via a phosphate diester bond (Figure 8) by coupling reactions between the respective silybin-23-phosphoramidite and the appropriately protected trehalose, catalyzed by 4,5-dicyanoimidazole. Here again, a better activity in terms of inhibiting the aggregation of amyloid β peptide was found in the respective silybin B derivative.…”

“…Recent studies have shown that conjugation of a trehalose moiety to silybin A and B (Figure 8) increases water solubility without significantly affecting anti-aggregation properties [102]. An NMR study showed that silybins may act by shielding toxic Aβ40 surfaces formed by N-terminal and CHC-Aβ regions, and that the aromatic ring A of silybin is the primary site for interaction with Aβ-oligomers.…”

Natural Compounds as Inhibitors of Aβ Peptide Aggregation: Chemical Requirements and Molecular Mechanisms

“…However, Aβ40 is more abundant than Aβ42 (9:1) in the biological fluids (Qiu et al, 2015). In addition, Aβ40 was found in the amyloid deposits of patients affected by cerebral amyloid pathology, which is considered an early step in AD pathogenesis (Garcia-Vinuales et al, 2020;Greenberg et al, 2020), suggesting that both isoforms must be taken in consideration in search of neuroprotective agents.…”

“…Chemical modifications have been successfully introduced in curcumin to remove labile moieties and improve the water solubility in physiological conditions, preserving the ability to bind Aβ oligomers and plaques (Airoldi et al, 2011). The chemical conjugation of the active natural compounds with a prodrug moiety that do not inactivate its inhibitory properties and effectively improve the molecule pharmacokinetic is a promising strategy applied for quercetin (Guzzi et al, 2017) and silybin (Garcia-Vinuales et al, 2020). In particular, glycoconjugation of silybin with trehalose resulted in a significant increase of the bioavailabity, with improved solubility and half-life in blood serum.…”

“…In particular, glycoconjugation of silybin with trehalose resulted in a significant increase of the bioavailabity, with improved solubility and half-life in blood serum. NMR studies further demonstrated that the interaction of the glycoconjugated-silybin with Aβ oligomers is mainly mediated by silybin, indicating that the threalose moiety does not interfere with silybin interactions (Garcia-Vinuales et al, 2020). In this respect, NMR STD approaches provide important information to guide the effective choice of the prodrug and the linker for chemical conjugation.…”