Structure-activity relationship study of BACE1 inhibitors possessing a chelidonic or 2,6-pyridinedicarboxylic scaffold at the P(2) position.

We have previously reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. While these inhibitors exhibited potent activities in enzymatic and cellular assays (KMI-429 in particular inhibited Aß production in vivo), these inhibitors contained some natural amino acids that seemed to be required to improve enzymatic stability in vivo and permeability across the blood-brain barrier, so as to be practical drug. Recently, we synthesized non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold at the P2 position. Herein we report the SAR study of BACE1 inhibitors possessing this heterocyclic scaffold, a chelidonic or 2,6-pyridinedicarboxylic moiety.

Comparative properties of Aß1-42, Aß11-42, and [Pyr¹¹]Aß11-42 generated from O-acyl isopeptides.

The use of water-soluble O-acyl isopeptides enabled us to investigate the biochemical properties of Aß11-42 species, by preparing highly concentrated stock solutions after a pretreatment. Aß11-42 and [Pyr(11)]Aß11-42 showed comparable aggregation capability and cytotoxicity, suggesting that the pyroglutamate modification at Glu(11) does not have a crucial role in these events. However, given that Aß11-42 is converted to [Pyr(11)]Aß11-42 by a glutamyl cyclase in vivo, the potential aggregative and cytotoxic nature of [Pyr(11)]Aß11-42 that was observed in the present study provides valuable insights into the pathological functions of pyroglutamate-modified Aß species in Alzheimer's disease.

Self-assembly pathways of E22Δ-type amyloid ß peptide mutants generated from non-aggregative O-acyl isopeptide precursors.

The recently identified E22Δ-type amyloid ß peptide (Aß) mutants are reported to favor oligomerization over fibrillization and to exhibit more-potent synaptotoxicity than does wild-type (WT) Aß. Aß(E22Δ) mutants can thus be expected to serve as tools for clarifying the impact of Aß oligomers in Alzheimer's disease (or Alzheimer's-type dementia). However, the biochemical and biophysical properties of Aß(E22Δ) have not been conclusively determined. Here, we evaluated the self-assembly pathways of Aß(E22Δ) mutants generated from water-soluble, non-aggregative O-acyl isopeptide precursors. Circular dichroism spectroscopy, Western blot analysis, and thioflavin-T fluorescence intensity and cellular toxicity assays suggest that the self-assembly pathways of Aß(E22Δ) differed from those of Aß(WT). Aß1-40(E22Δ) underwent a rapid random coil→ß-sheet conformational change in its monomeric or low-molecular-weight oligomeric states, whereas Aß1-40(WT) self-assembled gradually without losing its propensity to form random coil structures. The Aß1-42(E22Δ) monomer formed ß-sheet-rich oligomers more rapidly than did Aß1-42(WT). Additionally, the Aß1-42(E22Δ) oligomers appear to differ from Aß1-42(WT) oligomers in size, shape, or both. These results should provide new insights into the functions of Aß(E22Δ) mutants.

Significance of interactions of BACE1-Arg235 with its ligands and design of BACE1 inhibitors with P2 pyridine scaffold.

Recently, we reported potent substrate-based pentapeptidic BACE1 inhibitors possessing a hydroxymethylcarbonyl isostere as a substrate transition-state mimic. Because these inhibitors contained some natural amino acids, we would need to improve their enzymatic stability in vivo and permeability across the blood-brain barrier, so that they become practically useful. Subsequently, non-peptidic and small-sized BACE1 inhibitors possessing a heterocyclic scaffold, 2,6-pyridenedicarboxylic, chelidamic or chelidonic moiety, at the P(2) position were reported. These inhibitors were designed based on the conformer of docked inhibitor in BACE1. In this study, we discuss the role and significance of interactions between Arg235 of BACE1 and its inhibitor in BACE1 inhibitory mechanism. Moreover, we designed more potent small-sized BACE1 inhibitors with a 2,6-pyridinedicarboxylic scaffold at the P(2) position, that were optimized for the interactions with Arg235 of BACE1.