ORIGINAL ARTICLE The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist Mohammed Akhter Hossain • Chrishan S. Samuel • Claudia Binder • Tim D. Hewitson • Geoffrey W. Tregear • John D. Wade • Ross A. D. Bathgate Received: 21 October 2009 / Accepted: 17 December 2009 / Published online: 31 December 2009 Ó Springer-Verlag 2009 Abstract Relaxin is a pleiotropic hormone which exerts its biological functions through its G-protein coupled receptor, RXFP1. While relaxin is well known for its reproductive and antifibrotic roles, recent studies suggest that it is produced by cancer cells and acts on RXFP1 to induce growth and metastasis. Furthermore, more recently Silvertown et al. demonstrated that lentiviral production of a human gene-2 (H2) relaxin analog reduced the growth of prostate xenograft tumors. The authors proposed that the lentivirally produced peptide was an RXFP1 antagonist; however, the processed form of the peptide produced was not demonstrated. In this study, we have chemically synthesized the H2 relaxin analog, B-R13/17K H2 relaxin, and subjected it to detailed chemical characterization by HPLC, MALDI-TOF mass spectrometry, and amino acid analysis. The biological activity of the synthetic peptide was then tested in three different cell lines. It was found to bind with 500-fold lower affinity than H2 relaxin to RXFP1 receptors over-expressed in HEK-293T cells where it acted as a partial agonist. However, in cells which natively express the RXFP1 receptor, rat renal myofibroblasts and MCF-7 cancer cells, it acted as a full antagonist. Impor- tantly, it was able to significantly inhibit cell invasion induced by H2 relaxin in MCF-7 cells consistent with the results of the lentiviral-driven expression in prostate cancer cells. The relaxin analog, B-R13/17K H2, can now be used as a tool to further understand RXFP1 function, and serve as a template for drug design for a therapeutic to treat prostate and other cancers. Keywords Relaxin Á H2 relaxin Á RXFP1 antagonist Á B-R13/17K H2 relaxin Á GPCR Introduction The relaxin family of peptides in human consists of seven members: relaxin-1 (H1 relaxin), relaxin-2 (H2 relaxin), relaxin-3 (H3 relaxin), insulin-like peptide 3 (INSL3), INSL4, INSL5, and INSL6. Mammalian non-primate spe- cies have only two relaxin peptides, relaxin-1 and relaxin-3, together with INSL3, INSL5, and INSL6 (Wilkinson et al. 2005). H2 relaxin is the equivalent of the relaxin-1 peptide in non-primate species which is referred to as ‘‘relaxin.’’ Relaxin is produced in the corpus luteum and/or placenta during pregnancy in mammals and is secreted into the blood where it has numerous essential endocrine M. A. Hossain Á C. S. Samuel Á G. W. Tregear Á J. D. Wade Á R. A. D. Bathgate (&) Florey Neuroscience Institutes, University of Melbourne, Melbourne, VIC 3010, Australia e-mail: bathgate@florey.edu.au C. S. Samuel Á G. W. Tregear Á R. A. D. Bathgate Department of Biochemistry and Molecular Biology, University of Melbourne, Melbourne, VIC 3010, Australia C. Binder Department of Hematology and Oncology, Georg-August-University, Robert-Koch Str. 40, 37075 Go ¨ttingen, Germany T. D. Hewitson Department of Nephrology, University of Melbourne, Melbourne, VIC 3010, Australia T. D. Hewitson Department of Medicine, Royal Melbourne Hospital, University of Melbourne, Melbourne, VIC 3010, Australia M. A. Hossain Á J. D. Wade School of Chemistry, University of Melbourne, Melbourne, VIC 3010, Australia 123 Amino Acids (2010) 39:409–416 DOI 10.1007/s00726-009-0454-1