Firstly, parathyroid hormone (1-14) [PTH (1-14)] analogue containing various alpha-amino-iso-butyric acid residue (Aib) was synthesized by exchanging the 1st and 3rd Ala residues of alpha carbon of PTH (1-14). This analogue revealed to have the quite tight and stable alpha-helical structure using the nuclear magnetic resonance (NMR) analysis. The biological activities of these analogues were examined using a cAMP-generating assay in LLC-PK1 cell lines stably transfected with the wild-type human PTH1 receptor. Only the PTH analogue substituted with methyl moiety without acetylation showed significant cAMP generating action with 15.0 +/- 3.414 of EC50. Then, we used an ovariectomized rat model system to compare the in vivo effects of parathyroid hormone analogue with that of PTH (1-84). Daily subcutaneous administration of the unacetylated Aib1,3PTH (1-14) for 5 weeks in 30 nM/kg subcutaneously with positive control group receiving PTH (1-84) with 8 nM/ kg were performed. However, there was no significant change in spinal or femoral bone mineral density assessed by dual x-ray absorptiometry (DXA) in the Aib1,3PTH (1-14) group where definite increase of these parameters shown in the PTH (1-84) group (p < 0.001). Assessment of bone strength was evaluated with no significant differences among all groups. It was quite disappointing to see the actual discrepancies between the result of significant pharmacokinetic potency and the in vivo clinical effect of the Aib1,3PTH (1-14). However, there are several limitations to mention, such as the short duration of treatment, matter of dosage, and insufficient effect of tight alpha-helical structures with absence of C-terminus. In conclusion, our findings suggest that unacetylated Aib1,3PTH (1-14) did not exhibit any anabolic effects at the bones of ovariectomized rats.
Transfection ; Time Factors ; Structure-Activity Relationship ; Stress, Mechanical ; Spectrometry, X-Ray Emission ; Rats ; Protein Structure, Tertiary ; Protein Structure, Secondary ; Protein Conformation ; Protein Binding ; Peptides/chemistry ; Parathyroid Hormone/*analogs & derivatives/chemistry/*metabolism ; Molecular Sequence Data ; Molecular Conformation ; Models, Statistical ; Models, Molecular ; Magnetic Resonance Spectroscopy ; LLC-PK1 Cells ; Humans ; Female ; Dose-Response Relationship, Drug ; Densitometry ; Cyclic AMP/metabolism ; Cell Line ; Bone and Bones/metabolism ; Bone Density ; Biomechanics ; Animals ; Aminoisobutyric Acids/*metabolism ; Amino Acid Sequence ; Alanine/chemistry

The structure and function of short-length amino terminal PTH analogues were studied. The substitution of Leu7 with Phe in [Ala3, 10Leu7Arg11]rPTH (1-11) NH2 analogue peptides did not show any reduction in cAMP formation. Replacement of the 1st, 7th and 8th residues revealed different activities, depending upon the residue type. The substitution of Ala1 by Ser in [Ala3, 10Leu7Arg11]rPTH (1-11) NH2 caused nearly a complete loss of cAMP formation. Meanwhile, NMR analysis of [ (Ala1/ Ser1) Ala3, 10 (Leu7/Phe7) Arg11]rPTH (1-11) NH2 revealed an alpha- helical backbone structure with a flexible conformation at the carboxyl-terminus. The overall results suggest that 11-residue short oligopeptide analogues of PTH tend to form an alpha-helical structure and the different activities of those analogues could be associated with residue specificity rather than the secondary conformational structure.
Amino Acid Substitution ; Animals ; Circular Dichroism ; Cyclic AMP/metabolism ; Human ; LLC-PK1 Cells ; Nuclear Magnetic Resonance, Biomolecular ; Parathyroid Hormone/*chemistry/*metabolism ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Receptor, Parathyroid Hormone, Type 1/genetics ; Structure-Activity Relationship ; Support, Non-U.S. Gov't ; Swine

PURPOSE: Dickkopf 1 (DKK1) has been extensively investigated in mouse models of multiple myeloma, which results in osteolytic bone lesions. Elevated DKK1 levels in bone marrow plasma and serum inhibit the differentiation of osteoblast precursors. Present pharmaceutical approaches to target bone lesions are limited to antiresorptive agents. In this study, we developed a cyclized oligopeptide against DKK1-low density lipoprotein receptor-related protein (LRP) 5/6 interaction and tested the effects of the oligopeptide on tumor burden. MATERIALS AND METHODS: A cyclized oligopeptide based on DKK1-LRP5/6 interactions was synthesized chemically, and its nuclear magnetic resonance structure was assessed. Luciferase reporter assay and mRNA expressions of osteoblast markers were evaluated after oligopeptide treatment. MOPC315.BM.Luc cells were injected into the tail vein of mice, after which cyclized oligopeptide was delivered subcutaneously 6 days a week for 4 weeks. RESULTS: The cyclized oligopeptide containing NXI motif bound to the E1 domain of LRP5/6 effectively on surface plasmon resonance analysis. It abrogated the Wnt-β-catenin signaling inhibited by DKK1, but not by sclerostin, dose dependently. RT-PCR and alkaline phosphatase staining showed increased expressions of osteoblast markers according to the treatment concentrations. Bioluminescence images showed that the treatment of cyclized oligopeptide reduced tumor burden more in oligopeptide treated group than in the vehicle group. CONCLUSION: The cyclized oligopeptide reported here may be another option for the treatment of tumor burden in multiple myeloma.
Alkaline Phosphatase ; Animals ; Bone Density Conservation Agents ; Bone Marrow ; Lipoproteins ; Luciferases ; Magnetic Resonance Spectroscopy ; Mice* ; Multiple Myeloma* ; Osteoblasts ; Plasma ; RNA, Messenger ; Surface Plasmon Resonance ; Tail ; Tumor Burden* ; Veins