Identification of Potent, Selective, and Metabolically Stable Peptide Antagonists to the Calcitonin Gene-Related Peptide (CGRP) Receptor

Abstract

Calcitonin gene-related peptide (CGRP) is a 37-residue neuropeptide that can be converted to a CGRP(1) receptor antagonist by the truncation of its first seven residues. CGRP(8-37), 1, has a CGRP(1) receptor K(i) = 3.2 nM but is rapidly degraded in human plasma (t(1/2) = 20 min). As part of an effort to identify a prolonged in vivo circulating CGRP peptide antagonist, we found that the substitution of multiple residues in the CGRP peptide increased CGRP(1) receptor affinity >50-fold. Ac-Trp-[Arg(24),Lys(25),Asp(31),Pro(34),Phe(35)]CGRP(8-37)-NH(2), 5 (K(i) = 0.06 nM) had the highest CGRP(1) receptor affinity. Using complimentary in vitro and in vivo metabolic studies, we iteratively identified degradation sites and prepared high affinity analogues with significantly improved plasma stability. Ac-Trp-[Cit(11,18),hArg(24),Lys(25),2-Nal(27,37),Asp(31),Oic(29,34),Phe(35)]CGRP(8-37)-NH(2), 32 (K(i) = 3.3 nM), had significantly increased (>100-fold) stability over 1 or 5, with a cynomolgus monkey and human in vitro plasma half-life of 38 and 68 h, respectively.

Direct Quantitative Analysis of a 20 kDa PEGylated Human Calcitonin Gene Peptide Antagonist in Cynomolgus Monkey Serum Using In-Source CID and UPLC-MS/MS

“…In particular, peptides generally have very short circulatory half-lives due to the combined effects of a rapid renal clearance and enzymatic degradation in the blood, liver, or kidneys by endogenous proteases. To overcome these limitations, strategies such as amino acid modifications have been applied to improve peptide stability by rendering the peptides less susceptible to breakdown by endogenous proteases [3][4][5][6][7][8]. Additional improvements in peptide drugability can be obtained through PEGylation [9,10].…”

“…When the size of the attached PEG reaches 20 kDa or above, PEGylation can significantly extend in vivo circulatory half-life of the therapeutic peptides [11][12][13], as well as improve their chemical and physical stability, solubility, and potentially reduce immunogenecity [14][15][16][17]. 20 kDa PEGylated CGRP[Cit, Cit] is a human calcitonin gene peptide (CGRP) receptor antagonist developed for the treatment of migraine pain [5]. CGRP[Cit, Cit] is a peptide analog of the N-terminal truncated native human CGRP(8-37)-NH 2 (Figure 1a), which has demonstrated specific antagonist activity toward CGRP receptors [18].…”

Quantitative Structure–Activity Relationships and Docking Studies of Calcitonin Gene‐Related Peptide Antagonists

“… *Molecules were used as external test set. [5.52(IC 50 > 3E‐6)] – indicate the pIC 50 transformed from less‐accurate IC 50 values of references (7,11) (IC 50 values in the parenthesis). …”

“…Recent studies have demonstrated that it is possible to design high‐affinity CGRP antagonist analogs with significantly increased potency, selectivity, and metabolic stability as compared to the human αCGRP (8–37)‐amide antagonist peptide (7). Structure–activity studies aimed at designing novel antagonists have been challenging, however, as the structure of the receptor has only recently been reported (8).…”

Automated solid-phase peptide synthesis to obtain therapeutic peptides

“…Recently, the great methodical repertoire for extending the half-lifes of biological active peptides by covalent chemical approaches has been reviewed [8]. These methods include peptide sequence modifications by non-proteinogenic amino acids such as D- [84] or N -methylated [85–86] monomers or general truncation or mutation of biologically not relevant positions creating peptide analogs [87]. Likewise, backbone manipulation by partial or complete cyclization [88] as well as incorporation of peptide bond mimetics [89] can help to increase stability towards proteases.…”