Discovery of the Migraine Prevention Therapeutic Aimovig (Erenumab), the First FDA-Approved Antibody against a G-Protein-Coupled Receptor.

In 2018, the United States Food and Drug Administration (FDA) approved Aimovig (erenumab) for the prevention of migraine. Erenumab is the first FDA approved antibody therapeutic against a G-protein-coupled receptor, the canonical receptor of calcitonin gene related peptide (CGRP-R). A novel, epitope-focused antigen was created to reconstruct the extracellular domains of the CGRP-R in a stable conformation. Successful inoculation of XenoMouse animals and careful screening yielded multiple candidate molecules for high potency and exquisite selectivity toward the CGRP-R over related receptors. These efforts led to the discovery of erenumab which has demonstrated the desired efficacy and safety profiles in multiple clinical studies for the prevention of migraine. The innovation developed in the discovery of erenumab furthers the ability to target G-coupled protein receptors using antibody approaches.

Pharmaceutical Optimization of Peptide Toxins for Ion Channel Targets: Potent, Selective, and Long-Lived Antagonists of Kv1.3.

To realize the medicinal potential of peptide toxins, naturally occurring disulfide-rich peptides, as ion channel antagonists, more efficient pharmaceutical optimization technologies must be developed. Here, we show that the therapeutic properties of multiple cysteine toxin peptides can be rapidly and substantially improved by combining direct chemical strategies with high-throughput electrophysiology. We applied whole-molecule, brute-force, structure-activity analoging to ShK, a peptide toxin from the sea anemone Stichodactyla helianthus that inhibits the voltage-gated potassium ion channel Kv1.3, to effectively discover critical structural changes for 15× selectivity against the closely related neuronal ion channel Kv1.1. Subsequent site-specific polymer conjugation resulted in an exquisitely selective Kv1.3 antagonist (>1000× over Kv1.1) with picomolar functional activity in whole blood and a pharmacokinetic profile suitable for weekly administration in primates. The pharmacological potential of the optimized toxin peptide was demonstrated by potent and sustained inhibition of cytokine secretion from T cells, a therapeutic target for autoimmune diseases, in cynomolgus monkeys.

Peptide antagonists of the calcitonin gene-related peptide (CGRP) receptor with improved pharmacokinetics and pharmacodynamics.

Antagonism of the calcitonin gene-related peptide (CGRP) receptor may be a useful approach for migraine treatment. Selective PEGylated peptide antagonists to the CGRP receptor are described, derived from CGRP(8-37) with polymer derivatization at an engineered lysine-25 residue. Potent PEGylated peptides with improved pharmacokinetics were identified through peptide side-chain modification to mitigate metabolic liabilities. PEGylated Ac-Trp-[Cit(11,18),hArg(24),Lys(25),Asp(31),Pro(34),1-Nal(35)]CGRP(8-37)-NH2, 9, elicits a dose-dependent reduction of intradermal CGRP-induced local blood flow in rodents with an ED50 of 0.52 mg kg(-1) without any overt adverse effects.

Identification of potent, selective, and metabolically stable peptide antagonists to the calcitonin gene-related peptide (CGRP) receptor.

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.

Design and synthesis of conformationally constrained glucagon-like peptide-1 derivatives with increased plasma stability and prolonged in vivo activity.

A series of conformationally constrained derivatives of glucagon-like peptide-1 (GLP-1) were designed and evaluated. By use of [Gly (8)]GLP-1(7-37)-NH2 (2) peptide as a starting point, 17 cyclic derivatives possessing i to i + 4, i to i + 5, or i to i + 7 side chain to side chain lactam bridges from positions 18 to 30 were prepared. The effect of a helix-promoting alpha-amino-isobutyric acid (Aib) substitution at position 22 was also evaluated. The introduction of i to i + 4 glutamic acid-lysine lactam constraints in c[Glu (18)-Lys (22)][Gly (8)]GLP-1(7-37)-NH2 (6), c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-NH2 (10), and c[Glu (23)-Lys (27)][Gly (8)]GLP-1(7-37)-NH2 (11) resulted in potent functional activity and receptor affinities comparable to native GLP-1. Selected GLP-1 peptides were chemoselectively PEGylated in order to prolong their in vivo activity. PEGylated peptides [Gly (8),Aib (22)]GLP-1(7-37)-Cys ((PEG))-Ala-NH2 (23) and c[Glu (22)-Lys (26)][Gly (8)]GLP-1(7-37)-Cys ((PEG))-Ser-Gly-NH2 (24) retained picomolar functional potency and avid receptor binding properties. Importantly, PEGylated GLP-1 peptide 23 exhibited sustained in vivo efficacy with respect to blood glucose reduction and decreased body weight for several days in nonhuman primates.

Interactions between PEG and type I soluble tumor necrosis factor receptor: modulation by pH and by PEGylation at the N terminus.

The effects of polyethylene glycol (PEG) on protein structure and the molecular details that regulate its association to polypeptides are largely unknown. These issues were addressed using type I soluble tumor necrosis factor receptor (sTNF-RI) as a model system. Changes in solution viscosity established that a truncated form of sTNF-RI bound free PEG in a pH-dependent manner. Above pH 5.3, the viscosity escalated as the pH increased, while no effect occurred below pH 5.0. Conjugation of 2 kD, 5 kD, or 20 kD PEG to the N terminus attenuated the viscosity at the higher pH values. Tryptophan phosphorescence spectroscopy correlated changes in the protein structure about Trp-107, at the C terminus, with the pH-dependent and PEGylation-dependent attenuation of the viscosity. The results indicate that specific interactions between PEG and the truncated form of sTNF-RI are elicited by an increased flexibility of the truncated protein combined perhaps with removal of steric or charge barriers. Covalently bound PEG at the N terminus reduced the protein affinity for the free polymer and induced a more rigid and polar configuration around Trp-107. Deprotonation of His-105, which is perpendicular to Trp-107, was integral to the binding mechanism producing a pH-dependent switching mechanism. These findings stress the importance of surface charge and structural plasticity in determining macromolecular binding affinities and demonstrate the ability of conjugated PEG to modify the localized surface structure in proteins away from the site of conjugation.