Design and Synthesis of a Novel Neuroprotective Brain-Targeting Amylin Receptor Antagonist
Amylin receptor antagonist AC253 is a 24-amino acid peptide that provides neuroprotection against amyloid beta (Aβ)-induced cell death and toxicity. Additionally, AC253 has been shown to improve spatial memory in mouse models of Alzheimer’s disease (AD). Screening of short overlapping fragments (12-mer) of AC253 showed that N-terminal residues were responsible for high affinity binding to the amylin receptor (AMY3). Both AC253 and R5 (a 12-mer fragment) have a short half-life in human serum and are hydrophilic peptides with a low log P (-1.2 for AC253) and are not optimized for brain uptake. From the structure-activity relationship studies, we have designed a 16-mer analog (R16) from the N-terminal region of AC253 as a novel AmyR antagonist for better activity and blood-brain permeability. The goal of this study is to design and synthesize a novel neuroprotective analog of AC253 called R16 for enhanced blood-brain barrier targeting. R16 was synthesized using solid-phase peptide synthesis, purified and characterized by MALDI-TOF mass spectrometry and reversed-phase HPLC. The proteolytic stability of R16 was evaluated in human and mouse sera to determine the half-lives and proteolytically labile sites. The half-life of R16 was found to be 8.98 hours and 0.47 hours in human and mouse serum, respectively. The N-terminal of R16 was found to be accessible to proteolytic cleavage by serum proteases. Therefore, blocking N-terminal of R16 with a blood brain targeting peptide Angiopep-2 led to the design of a proteolytic stable blood-brain barrier targeting analog of R16, namely Angiopep2- R16 conjugate. The conjugate was synthesized, purified and characterized using reversed-phase HPLC and mass spectrometry, and was found to be >95% pure. The conjugate will be further studied in the future to test its pharmacokinetics, stability, and brain activity.
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