Potent and Selective BACE-1 Peptide Inhibitors Lower Brain Aβ Levels Mediated by Brain Shuttle Transport

Abstract

Therapeutic approaches to fight Alzheimer's disease include anti-Amyloidβ (Aβ) antibodies and secretase inhibitors. However, the blood-brain barrier (BBB) limits the brain exposure of biologics and the chemical space for small molecules to be BBB permeable. The Brain Shuttle (BS) technology is capable of shuttling large molecules into the brain. This allows for new types of therapeutic modalities engineered for optimal efficacy on the molecular target in the brain independent of brain penetrating properties. To this end, we designed BACE1 peptide inhibitors with varying lipid modifications with single-digit picomolar cellular potency. Secondly, we generated active-exosite peptides with structurally confirmed dual binding mode and improved potency. When fused to the BS via sortase coupling, these BACE1 inhibitors significantly reduced brain Aβ levels in mice after intravenous administration. In plasma, both BS and non-BS BACE1 inhibitor peptides induced a significant time- and dose-dependent decrease of Aβ. Our results demonstrate that the BS is essential for BACE1 peptide inhibitors to be efficacious in the brain and active-exosite design of BACE1 peptide inhibitors together with lipid modification may be of therapeutic relevance.

Computer-Aided Drug Design of β-Secretase, γ-Secretase and Anti-Tau Inhibitors for the Discovery of Novel Alzheimer’s Therapeutics

“…Sequence alignment analysis showed that the amino acid sequences of human BACE1 and BACE2 gene coding regions are 45% identical and 75% homologous [22]. Numerus crystal structures of BACE1 have been resolved with and without inhibitor in its active site [23][24][25][26][27][28][29][30][31][32]. BACE1 contains an N-terminal protease domain, a connecting strand, a transmembrane region, and a cytosolic domain [23].…”

Modeling the β-secretase cleavage site and humanizing amyloid-beta precursor protein in rat and mouse to study Alzheimer’s Disease.

“…B63 antibody binds to full length APP (FL APP) and C-terminal fragments (CTF, longer exposure). 82E1 antibody speci cally detects human Aβ (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), and hence the human B-CTF, independently con rming that the rat App gene was successfully humanized. β-ACTIN was used as a loading control.…”

“…We have modelled the interaction of humanized β-CTF with BACE1, using as a template the published crystal structure of BACE1 in combination with an active site peptide inhibitor (PDB ID 5MCQ) (16). The sequence of the humanized β-CTF was aligned to the peptide in 5MCQ, aligning residue E11 from the humanized β-CTF sequence to the STA query (Threonine) of the crystallized peptide (Fig1).…”

“…Three amino acid substitutions (G676R, F681Y and R684H) in the Aβ sequence decrease BACE1 processing of rodent APP compared to human APP (1). We superposed the rodent and humanized β-CTF on a 21 amino acid peptide inhibitor at the binding site of BACE1 (Fig1), using the crystal structure PDB id:5MCQ (16). The modeled binding modes reveal two important extra interactions of the humanized β-CTF with BACE1.…”

“…Green: the three amino acids different between the human and rodent APP sequence. (B) The BACE structure is presented with its ap (16) in yellow and the 10S loop in orange; a model of rodent Aβ peptide is tted. (C) Shows the same structure but now modelled with the humanized β-CTF.…”