Ligand-dependent effects of methionine-8 oxidation in parathyroid hormone peptide analogs

LA-PTH is a long-acting parathyroid hormone (PTH) peptide analog in
pre-clinical development for hypoparathyroidism (HP). Like native PTH,
LA-PTH contains a methionine at position 8 that is predicted to be
critical for function. We assessed the impact of methionine oxidation on
the functional properties of LA-PTH and control PTH ligands. Oxidation of
PTH(1-34) resulted in marked (~20-fold) reductions in binding affinity on
the PTH receptor-1 (PTHR1) in cell membranes, similarly diminished potency
for cAMP signaling in osteoblastic cell lines (SaOS-2 and UMR106), and
impaired efficacy for raising blood calcium in mice. Surprisingly,
oxidation of LA-PTH resulted in little or no change in these functional
responses. The signaling potency of oxidized-LA-PTH was, however, reduced
~40-fold compared to LA-PTH in cells expressing a PTHR1 construct that
lacks the N-terminal extracellular domain (ECD). Molecular modeling
revealed that while Met8 of both LA-PTH and PTH(1-34) is situated within
the orthosteric ligand-binding pocket of the receptor’s transmembrane
domain bundle (TMD), the Met8 sidechain position is shifted for the two
ligands such that upon Met8 oxidation of PTH(1-34) steric clashes occur
that are not seen with oxidized LA-PTH. The findings suggest that LA-PTH
and PTH(1-34) engage the receptor differently in the Met8-interaction
environment of the TMD bundle, and that this interaction environment can
be allosterically influenced by the ECD component of the ligand-receptor
complex. The findings should be useful for the future development of novel
PTH-based peptide therapeutics for diseases of bone and mineral ion
metabolism.