Development of Potent, Protease-Resistant Agonists of the Parathyroid Hormone Receptor with Broad β Residue Distribution

Abstract

The parathyroid hormone receptor-1 (PTHR1) is a member of the B-family of GPCRs; these receptors are activated by long polypeptide hormones and constitute targets of drug development efforts. Parathyroid hormone (PTH; 84 residues) and PTH-related protein (PTHrP; 141 residues) are natural agonists of PTHR1, and an N-terminal fragment of PTH, PTH(1−34), is used clinically to treat osteoporosis. Conventional peptides in the 20-40-mer length range are rapidly degraded by proteases, which may limit their biomedical utility. We have used the PTHR1-ligand system to explore the impact of broadly distributed replacement of α-amino acid residues with β-amino acid residues on susceptibility to proteolysis and agonist activity. This effort led us to identify new PTHR1 agonists that contain α→β replacements throughout their sequences, manifest potent agonist activity in cellular assays, and display remarkable resistance to proteolysis. The strategy we have employed suggests a path toward identifying protease-resistant agonists of other B-family GPCRs.

Receptor selectivity from minimal backbone modification of a polypeptide agonist

“…Both enantiomers of β 2 -hNle are well tolerated in place of Met-8, but use of either β 3 -hMet or β 3 -hNle at this position causes a substantial decline in activity. The overall trend among α→β 3 replacements is consistent with a previously reported β 3 -scan of PTH(1-34)-NH 2 (46).…”

“…Only one or two β-amino acids are incorporated into the 34-residue peptides described here, and precedent suggests that this low level of modification will not have much impact on susceptibility to proteases. Since β-amino-acid residues can be used in polypeptide hormone analogs to adjust both proteolytic stability, as shown previously (46), and agonist selectivity, as shown here, it seems likely that backbone-modified analogs featuring both of these desirable attributes will ultimately be accessible.…”

“…Previous research has demonstrated that extensive backbone modification can substantially enhance the resistance of bioactive peptides to proteolysis (33,34,36,38,46). Only one or two β-amino acids are incorporated into the 34-residue peptides described here, and precedent suggests that this low level of modification will not have much impact on susceptibility to proteases.…”

Ureidopeptide GLP-1 analogues with prolonged activity in vivo via signal bias and altered receptor trafficking

“…17 We therefore hypothesize that modifying the backbone of exenatide might improve its proteolytic stability and prolong its activity in vivo. However, backbone modications at the N-terminal part of incretins and other ligands of class B GPCRs have scarcely been reported 15,[18][19][20] which reects the difficulty to mimic the complex network of interactions in the binding pocket of the receptor transmembrane domain. 21 Herein we report the utilization of a ureido residue replacement at position 2 of GLP-1 analogues to improve their pharmacodynamic properties via selective enhancement of G protein-dependent cAMP signalling and altered GLP-1R trafficking.…”

Impact of Substitution Registry on the Receptor‐Activation Profiles of Backbone‐Modified Glucagon‐like Peptide‐1 Analogues

“…Among these drugs are synthetic peptides containing unnatural components designed to optimize therapeutic performance . Unnatural modifications in clinical agents have so far been limited to side chains, but backbone‐based modifications have been evaluated in the laboratory . We have focused on agonists in which a subset of natural α‐amino acid residues is replaced by β‐amino acid residues.…”