Modifications of position 12 in a parathyroid hormone and parathyroid hormone-related protein: toward the design of highly potent antagonists

Abstract

Truncated N-terminal fragments of parathyroid hormone (PTH), [Tyr34]bovine PTH(7-34)NH2, and parathyroid hormone related protein (PTHrP), PTHrP(7-34)NH2, inhibit [Nle8,18,[125I]iodo-Tyr34]-bPTH(1-34)NH2 binding and PTH-stimulated adenylate cyclase in bone and kidney assays. However, the receptor interactions of these peptides are 2-3 orders of magnitude weaker than those of their agonist counterparts. To produce an antagonist with increased receptor-binding affinity but lacking agonist-like properties, structure-function studies were undertaken. Glycine at position 12 (present in all homologues of PTH and in PTHrP), which is predicted in both hormones to participate in a beta-turn, was examined by substituting conformational reporters, such as D- or L-Ala, Pro, and alpha-aminoisobutyric acid (Aib), in both agonist and antagonist analogues. Except for N-substituted amino acids, which substantially diminished potency, substitutions were well tolerated, indicating that this site can accept a wide latitude of modifications. To augment receptor avidity, hydrophobic residues compatible with helical secondary structure were introduced. Incorporation of the nonnatural amino acids D-Trp, D-alpha-naphthylalanine (D-alpha-Nal), or D-beta-Nal into either [Tyr34]bPTH(7-34)NH2 or [Nle8,18,Tyr34]bPTH(7-34)NH2 resulted in antagonists that were about 10-fold more active than their respective 7-34 parent compound. Similarly, [D-Trp12]PTHrP(7-34)NH2 was 6 times more potent than the unsubstituted peptide but retained partial agonistic properties, although markedly reduced, similar to PTHrP(7-34)NH2. The antagonistic potentiating effect was configurationally specific.(ABSTRACT TRUNCATED AT 250 WORDS)

PTH receptor-1 signalling—mechanistic insights and therapeutic prospects

“…Deletion of residues 1–6 from the PTH(1–34) scaffold produced analogues that no longer activate signalling via the cAMP–PKA pathway, but instead, function as competitive antagonists of PTHR1-mediated cAMP signalling. 36 Further modification of the PTH(7–34) scaffold at residue position 12 produced an analogue, DTrp 12 -PTH(7–34), which in addition to competitively antagonizing cAMP signalling at PTHR1, 37 also acts as an inverse agonist and consequently reduces basal cAMP signalling at certain constitutively active PTHR1 variants. 38 Other PTH and PTHrP ligand analogues modified at residue position 1 have been developed that preferentially activate Gα S -cAMP signalling over Gα q -PLC-β signalling.…”

“…Analogues of DTrp 12 -PTH(7–34) can inhibit PTH-induced cAMP responses in cells 37 and calcaemic responses stimulated by exogenous PTH(1–34) in rats. 97 However, trials in patients with hypercalcaemia owing to primary hyperparathyroidism revealed a complete lack of efficacy for such a DTrp 12 -PTH(7–34) analogue in reversing hypercalcaemia.…”

Backbone Modification of a Parathyroid Hormone Receptor-1 Antagonist/Inverse Agonist

“…A close analogue of 1 , containing Tyr at position 34 instead of Phe [PTH(1–34) numbering], has previously been tested in radiolabel binding assays. 12, 14 We included human PTH(1–34) (Figure 1a) as a positive control and point of comparison for peptides 1 and 2 . Bound radioactivity resulting from association of radiolabeled PTH(1–34) with PTHR1 is diminished in a dose-dependent fashion following addition of unlabeled PTH(1–34), α-peptide 1 , or α/β-peptide 2 .…”

Parathyroid Hormone-related Protein Antagonizes the Action of Parathyroid Hormone on Adult Cardiomyocytes

“…This part of the molecule has been identified as the core of a protein kinase C-activating domain of PTH and PTH-rP (7)(8)(9)(10)(11). The secondary structure of the two peptide hormones in this region is very similar (12), consisting of a helical motif. In PTH, the hydrophobic amino acids are placed on one side and the hydrophilic amino acids on the other side ( Fig.…”