Bioactivities and Secondary Structures of Constrained Analogues of Human Parathyroid Hormone:  Cyclic Lactams of the Receptor Binding Region

Abstract

In a search for analogues of human parathyroid hormone (hPTH) with improved activities and bioavailabilities, we have prepared the following three lactam analogues of hPTH-(1-31)-NH2 (1) or [Leu27]hPTH-(1-31)-NH2 (2): [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-31)-NH2 (3), [Leu27]cyclo(Lys26-Asp30)-hPTH-(1-31)-NH2 (4), and cyclo(Lys27-Asp30)-hPTH-(1-31)-NH2 (5). Analogues 1, 2, and 5 had seven or eight residues of alpha-helix, as estimated from their circular dichroism (CD) spectra, in contrast to 12 residues in cyclic analogues 3 and 4. Thus, lactams 3 and 4 stabilized a helix previously shown to exist within residues 17-29. The adenylyl cyclase activity (EC50), measured in rat osteosarcoma 17/2 cells, of 5 (40.3 +/- 2.3 nM) was half that of its linear form 1 (19.9 +/- 3.9 nM). The linear Leu27 mutant 2 was twice as active (11.5 +/- 5.2) as analogue 1, and lactam analogue 3 was 6-fold more active (3.3 +/- 0.3 nM). Lactam analogue 4 had less activity (16.9 +/- 3.3 nM) than 2, its linear form. Peptides hPTH-(1-30)-NH2 (6), [Leu27]hPTH-(1-30)-NH2 (7), and [Leu27]cyclo(Glu22-Lys26)-hPTH-(1-30)-NH2 (8) all had AC-stimulating activities similar to that of 1. When injected intravenously, with a dose of 0.8 nmol/100 g of analogue in acid saline, hypotensive effects paralleled their adenylyl cyclase activities. They behaved quite differently when applied subcutaneously. Analogues 1, 5, and 6, the weakest, showed about half the drop in blood pressure observed with 3 and 4, the most active. In contrast, the time required to reach a maximum drop in blood pressure of 4-8, after subcutaneous administration, was 2-4 times that of the other analogues. Thus, the bioavailabilities of the lactam analogues, unlike their adenylyl cyclase-stimulating activities, were highly dependent on the presence or conformation of Val31.