Modifications of a Nanomolar Cyclic Peptide Antagonist for the EphA4 Receptor To Achieve High Plasma Stability

EphA4 is a receptor tyrosine kinase with a critical role in repulsive axon guidance and synaptic function. However, aberrant EphA4 activity can inhibit neural repair after injury and exacerbate neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and Alzheimer's. We previously identified the cyclic peptide APY-d2 (APYCVYRβASWSC-nh2, containing a disulfide bond) as a potent and selective EphA4 antagonist. However, APY-d2 lacks sufficient plasma stability to be useful for EphA4 inhibition in vivo through peripheral administration. Using structure-activity relationship studies, we show that protecting the peptide N-terminus from proteolytic degradation dramatically increases the persistence of the active peptide in plasma and that a positively charged peptide N-terminus is essential for high EphA4 binding affinity. Among several improved APY-d2 derivatives, the cyclic peptides APY-d3 (βAPYCVYRβASWSC-nh2) and APY-d4 (βAPYCVYRβAEWEC-nh2) combine high stability in plasma and cerebrospinal fluid with slightly enhanced potency. These properties make them valuable research tools and leads toward development of therapeutics for neurological diseases.