GnRH analogues have been extensively used in oncology to induce reversible chemical castration due to their hypophysiotropic action. In addition to that, it has recently been shown that many malignant cells, such as breast cancer cells, locally produce GnRH and express the GnRH receptor/s. In order to investigate the structure-activity relationships in both pituitary and extrapituitary biological systems, we synthesized eight new GnRH analogues with modifications in the N-terminal part and/or in position 6 and studied their pituitary binding affinity (in αT3-1 cell membranes) and effect on breast cancer (MCF-7) cell proliferation. 2-Amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid (ATPC) was incorporated instead of pGlu1-His2- and/or Gly6 was substituted by α-aminoisobutyric acid, D-Leu and D-Lys (alone or covalently linked to Gly, Ala, Sar, ATPC). Most GnRH analogues lacked the carboxy-terminal Gly10-amide of GnRH and an ethylamide residue was added to Pro9, a modification common in many potent GnRH agonists, such as leuprolide ([D-Leu6, des-Gly10]-GnRH-NHEt. Results show differential impact of these modifications on the binding affinity to the GnRH receptor in mouse pituitary cells and on the inhibition of human breast cancer cell proliferation. ATPC in the N-terminus resulted in analogues with low binding affinity but high antiproliferative effect. Substitutions in position 6 always resulted in high binding affinities. In particular, [D-Lys6(Gly), desGly10]-GnRH-NHEt and [D-Lys6(Sar), desGly10]-GnRH-NHEt have higher pituitary binding affinity than leuprolide, but only the latter had significant antiproliferative effect on both MCF-7 and MDA-MB-231 cells. These results contribute to the on-going research for more potent GnRH analogues.
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Abbreviations
- GnRH:
-
gonadotropin releasing hormone (or LHRH, luteinizing hormone releasing hormone)
- ATPC:
-
2-amino-4-pyrrolidinothieno[2,3-d]pyrimidine-6-carboxylic acid
- Aib:
-
α-aminoisobutyric acid
- DIC:
-
N,N′-diisopropylcarbodiimide
- DMF:
-
N,N′-dimethylformamide
- TFA:
-
trifluoroacetic acid
- HPLC:
-
high-performance liquid chromatography
- FBS:
-
fetal bovine serum
- IC50 :
-
concentration of tested compound required to inhibit 50% of specific binding of the radioligand during competition
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Acknowledgments
We acknowledge funding from EPAN programme of the 3rd Community Support Framework, Health, Biomedicine, Diagnostic and Therapeutic Methods’ (Ministry of Development, General Secretariat of Research and Technology, Greece). We thank Prof. Pamela Mellon for providing αT3-1 cells (School of Medicine, University of California, San Diego, CA).
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Abbreviations of common amino acids are in accordance with the recommendations of IUPAC-IUB Joint Commission on Biochemical Nomenclature: Arch. Biochem. Biophys. 206, pp.v-xxii (1988), J. Biol. Chem. 264, 668–673 (1989) or J. Peptide Sci. 9, 1–8 (2003).
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Zompra, A.A., Magafa, V., Chryssanthi, D.G. et al. Synthesis and Biological Evaluation of New GnRH Analogues on Pituitary and Breast Cancer Cells. Int J Pept Res Ther 13, 143–149 (2007). https://doi.org/10.1007/s10989-006-9057-9
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DOI: https://doi.org/10.1007/s10989-006-9057-9