Mimotopes of calcitonin gene-related peptide (CGRP) screened from Fv-antibody library: antagonists to CGRP receptor.

Calcitonin gene-related peptide (CGRP) is a neuropeptide synthesized in the trigeminal ganglion and it has been identified as a key mediator in the pathophysiology of migraine. The monoclonal antibodies and small-molecule antagonists for calcitonin receptor-like receptor (CLR) have been developed for therapeutics for migraine. In this study, the previously screened CGRP mimotopes sequences from Fv-antibody library were prepared as CDR3 peptides, Fv-antibodies, and heavy chains of immunoglobulin G (VH). Among these, the heavy chain-based mimotopes exhibited the highest binding affinity and antagonist activity against the CGRP receptor. To demonstrate the antagonistic activity, the binding of the CGRP mimotopes to the CGRP receptor was evaluated which prevented CGRP from interacting with the receptor, and then the influence from the inhibition of CGRP binding was demonstrated by measuring (1) intracellular cyclic adenosine monophosphate (cAMP) and (2) Ca2+ levels in the human neuroblastoma cell line SK-N-MC. Additionally, the interaction of screened CGRP mimotopes and the CLR was analyzed using a computer-aided docking simulation, and the interaction of CGRP mimotopes were analyzed to be antagonists preventing the binding of intact CGRP to CLR.