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The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist

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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. Importantly, 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.

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Acknowledgments

This study was funded by a grant to JDW, GWT, and RADB from the National Health and Medical Research Council (NHMRC) of Australia (ID no. 508995). RADB, JDW, and GWT are recipients of Research Fellowship from the NHMRC, while CSS is the recipient of a National Heart Foundation of Australia/NHMRC RD Wright Fellowship. We would like to thank Sharon Layfield and Tania Ferraro for performing the binding and the cAMP assays, Matthias Schulz for technical assistance with the MCF-7 invasion assays, and Ishanee Mookerjee for assistance with the renal myofibroblast differentiation assays.

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Correspondence to Ross A. D. Bathgate.

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Hossain, M.A., Samuel, C.S., Binder, C. et al. The chemically synthesized human relaxin-2 analog, B-R13/17K H2, is an RXFP1 antagonist. Amino Acids 39, 409–416 (2010). https://doi.org/10.1007/s00726-009-0454-1

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