Abstract

The parathyroid hormone (PTH)-1 receptor mediates the pathophysiological effects of PTH in hyperparathyroidism and PTH-related protein (PTHrP) in humoral hypercalcemia of malignancy. A PTH1 receptor antagonist may be of therapeutic utility in these disorders. We recently identified a novel antagonist, tuberoinfundibular peptide (7-39) [TIP(7-39)], derived from the likely endogenous ligand for the PTH2 receptor TIP39. In this study its in vitro profile is evaluated and compared with that of [d-Trp¹²,Tyr³⁴]bPTH(7-34) and PTHrP(7-34), representing the two previously known structural classes of PTH1 receptor antagonists. TIP(7-39) binds with higher affinity (6.2 nM) to the PTH1 receptor than [d-Trp¹²,Tyr³⁴]bPTH(7-34) (45 nM) and PTHrP(7-34) (65 nM) and displays a 5.5-fold greater PTH1/PTH2 receptor selectivity. TIP(7-39) does not stimulate cAMP accumulation via the PTH1 receptor [in a sensitive assay that detects the activity of the weak partial agonist [Nle^8,18,Tyr³⁴]bPTH(3-34)] and does not increase intracellular calcium. Schild analysis for TIP(7-39) was consistent with purely competitive antagonism of PTH(1-34)'s stimulation of cAMP accumulation (slope = 0.99 ± 0.24). The pK_B for TIP(7-39) (7.1 ± 0.3) was higher than that for [d-Trp¹²,Tyr³⁴]bPTH(7-34) (6.5 ± 0.0) and PTHrP(7-34) (6.0 ± 0.1). Binding of¹²⁵I-TIP(7-39) to the PTH1 receptor could be measured (K_D = 1.3 ± 0.1 nM,B_max = 1.3 ± 0.1 pmol/mg), whereas binding of¹²⁵I-[Nle^8,18,d-Trp¹²,Tyr³⁴]bPTH(7-34) could not be detected. Kinetic analysis indicated that¹²⁵I-TIP(7-39) dissociates much more slowly (t_1/2 = 14 min) than [d-Trp¹²,Tyr³⁴]bPTH(7-34) (13 s) and PTHrP(7-34) (9 s). The novel antagonist TIP(7-39) therefore displays a more favorable in vitro pharmacological profile than antagonists derived from PTH and PTHrP and may be useful for demonstrating the utility of PTH1 receptor antagonism in the treatment of hypercalcemia.