Analogues of parathyroid hormone modified at positions 3 and 6. Effects on receptor binding and activation of adenylyl cyclase in kidney and bone

Predictive and spectroscopic methods were used to develop a model of the structures of the 1-34 peptides of parathyroid hormone (PTH) and the PTH-related protein (PTHrP). Circular dichroism (CD) studies of bovine PTH-(1-34) and human PTHrP-(1-34)amide in the presence of trifluoroethanol suggest the presence of 24-26 alpha-helical residues. For both peptides, interactions between amino- and carboxyl-region alpha-helices are predicted to result in a hydrophobic core with externally facing hydrophilic residues that include probable determinants of receptor binding and activation. Two such residues, Ser3 and Gln6, are conserved in all known members of the PTH/PTHrP family. We have synthesized 13 novel analogues of bovine PTH-(1-34) monosubstituted at positions 3 and 6 and have determined their biological activities in renal and bone cell radioreceptor and adenylyl cyclase assays. Position 3 analogues displayed biological activity that was reduced in direct proportion to the volume of the substituent side-chain. Position 6 analogues also displayed reduced biological activity, but no simple correlation with side-chain volume or hydrophobicity was evident. The analogues fully displaced labeled PTH from binding sites in renal membranes and bone cells, but [Phe3]bPTH-(1-34), [Tyr3]bPTH-(1-34), [Phe6] bPTH-(1-34), and [Ser6]bPTH-(1-34) were only partial agonists in one or both adenylyl cyclase assays. Of these, [Phe3]bPTH-(1-34) and [Phe6]bPTH-(1-34) were tested for antagonist activity and were found to inhibit the activation of adenylyl cyclase in response to bPTH-(1-34) or hPTHrP-(1-34)amide. These results indicate that positions 3 and 6 contribute important determinants of PTH receptor binding and activation. Modification at these positions represents a novel approach to the development of antagonists of PTH action.