Biologically Active Truncated Glycopeptides Derived from Pituitary Adenylate Cyclase Activating Polypeptide

Introduction: Background and Significance of PACAP

With the massive success of Semaglutide and related GLP-1 agonists, interest in the therapeutic application of synthetic peptides has skyrocketed.(1-2) Scientists seeking to take advantage of the increased efficacy, selectivity and non-toxicity compared to “small” molecules, have begun to search for more therapeutic peptide feedstocks with refreshed vigor. Pituitary Adenylate Cyclase-Activating Polypeptide (PACAP) represents one of these promising candidates.(3) The activation of its 3 class B GPCRs, PAC1, VPAC1 and VPAC2 are associated with a myriad of effects such as neuroprotection, cardiovascular regulation, immune responses, metabolic processes, feeding and more.(4, 5) While the therapeutic promise of PACAP is substantial, it suffers from similar drawbacks as most peptide drugs, namely low cell permeability and half-life in vivo.(6, 7) However, recent work as shown glycosylation can negate these issues.(8-13) In fact, PACAP glycopeptide analogues have been shown to more effectively penetrate the Blood-Brain Barrier (BBB) and display effective neuroprotective effects in parkinsonian and ischemic stroke mouse models.(14, 15) This work will cover the design, synthesis, and biophysical properties of the next generation of PACAP glycopeptide analogues.

Design Considerations for Glycosylation. To design effective PACAP analogues, we leveraged the substantial structure-activity relationship (SAR) work available in the literature(16-19) as well as published Cryo-EM data of the native ligand bound to its receptors.(20-22) These previous works demonstrated two key findings: 1) that the first 5-10 N-terminal amino acids are essential for receptor activation and 2) C-terminal attachments can be made with limited effect on efficacy. While previous C-terminal truncations of PACAP showed limited efficacy, this is likely due to poor membrane interactions for which, the addition of the amphipathic behaviors afforded through glycosylation can be addressed.(11) As a result, we have developed PACAP glycopeptide “truncamers”, C-terminal truncations of the PACAP sequence at 10, 14, 19 and 23 amino acids, with an O-linked serine glycoside at the C-terminus. These truncations were made to coincide with one turn of the α-helix to minimize the interaction between the sugar and the extracellular domain of the receptor in its bound conformation. Met17 was also replaced with Norleucine to minimize risk of oxidation and improve shelf life.

Materials and Methods