Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

High-throughput screening campaign identifies a small molecule agonist of the relaxin family peptide receptor 4


Relaxin/insulin-like family peptide receptor 4 (RXFP4) is a class A G protein-coupled receptor (GPCR), and insulin-like peptide 5 (INSL5) is its endogenous ligand. Although the precise physiological role of INSL5/RXFP4 remains elusive, a number of studies have suggested it to be a potential therapeutic target for obesity and other metabolic disorders. Since selective agonists of RXFP4 are scarcely available and peptidic analogs of INSL5 are hard to make, we conducted a high-throughput screening campaign against 52,000 synthetic and natural compounds targeting RXFP4. Of the 109 initial hits discovered, only 3 compounds were confirmed in secondary screening, with JK0621-D008 displaying the best agonism at human RXFP4. Its S-configuration stereoisomer (JK1) was subsequently isolated and validated by a series of bioassays, demonstrating a consistent agonistic effect in cells overexpressing RXFP4. This scaffold may provide a valuable tool to further explore the biological functions of RXFP4.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6


  1. 1.

    Liu C, Kuei C, Sutton S, Chen J, Bonaventure P, Wu J, et al. INSL5 is a high affinity specific agonist for GPCR142 (GPR100). J Biol Chem. 2005;280:292–300.

    CAS  Article  Google Scholar 

  2. 2.

    Fredriksson R, Lagerstrom MC, Lundin LG, Schioth HB. The G-protein-coupled receptors in the human genome form five main families. Phylogenetic analysis, paralogon groups, and fingerprints. Mol Pharmacol. 2003;63:1256–72.

    CAS  Article  Google Scholar 

  3. 3.

    Boels K, Schaller HC. Identification and characterisation of GPR100 as a novel human G-protein-coupled bradykinin receptor. Br J Pharmacol. 2003;140:932–8.

    CAS  Article  Google Scholar 

  4. 4.

    Kong RC, Shilling PJ, Lobb DK, Gooley PR, Bathgate RA. Membrane receptors: structure and function of the relaxin family peptide receptors. Mol Cell Endocrinol. 2010;320:1–15.

    CAS  Article  Google Scholar 

  5. 5.

    Akhter Hossain M, Bathgate RA, Kong CK, Shabanpoor F, Zhang S, Haugaard-Jonsson LM, et al. Synthesis, conformation, and activity of human insulin-like peptide 5 (INSL5). ChemBioChem. 2008;9:1816–22.

    Article  Google Scholar 

  6. 6.

    Conklin D, Lofton-Day CE, Haldeman BA, Ching A, Whitmore TE, Lok S, et al. Identification of INSL5, a new member of the insulin superfamily. Genomics. 1999;60:50–6.

    CAS  Article  Google Scholar 

  7. 7.

    Ang SY, Hutchinson DS, Patil N, Evans BA, Bathgate RAD, Halls ML, et al. Signal transduction pathways activated by insulin-like peptide 5 at the relaxin family peptide RXFP4 receptor. Br J Pharmacol. 2017;174:1077–89.

    CAS  Article  Google Scholar 

  8. 8.

    Luo X, Li T, Zhu Y, Dai Y, Zhao J, Guo ZY, et al. The insulinotrophic effect of insulin-like peptide 5 in vitro and in vivo. Biochem J. 2015;466:467–73.

    CAS  Article  Google Scholar 

  9. 9.

    Aparicio S, Dixon J, Hendrick A. Use of GPR100 receptor in diabetes and obesity regulation. US Patent Appl 20080269118.

  10. 10.

    Burnicka-Turek O, Mohamed BA, Shirneshan K, Thanasupawat T, Hombach-Klonisch S, Klonisch T, et al. INSL5-deficient mice display an alteration in glucose homeostasis and an impaired fertility. Endocrinology. 2012;153:4655–65.

    CAS  Article  Google Scholar 

  11. 11.

    Grosse J, Heffron H, Burling K, Akhter Hossain M, Habib AM, Rogers GJ, et al. Insulin-like peptide 5 is an orexigenic gastrointestinal hormone. Proc Natl Acad Sci U S A. 2014;111:11133–8.

    CAS  Article  Google Scholar 

  12. 12.

    DeChristopher B, Park SH, Vong L, Bamford D, Cho HH, Duvadie R, et al. Discovery of a small molecule RXFP3/4 agonist that increases food intake in rats upon acute central administration. Bioorg Med Chem Lett. 2019;29:991–4.

    CAS  Article  Google Scholar 

  13. 13.

    Luo X, Bathgate RA, Zhang WJ, Liu YL, Shao XX, Wade JD, et al. Design and recombinant expression of insulin-like peptide 5 precursors and the preparation of mature human INSL5. Amino Acids. 2010;39:1343–52.

    CAS  Article  Google Scholar 

  14. 14.

    Patil NA, Hughes RA, Rosengren KJ, Kocan M, Ang SY, Tailhades J, et al. Engineering of a novel simplified human insulin-like peptide 5 agonist. J Med Chem. 2016;59:2118–25.

    CAS  Article  Google Scholar 

  15. 15.

    Belgi A, Hossain MA, Shabanpoor F, Chan L, Zhang S, Bathgate RA, et al. Structure and function relationship of murine insulin-like peptide 5 (INSL5): free C-terminus is essential for RXFP4 receptor binding and activation. Biochemistry. 2011;50:8352–61.

    CAS  Article  Google Scholar 

  16. 16.

    Van der Westhuizen ET, Sexton PM, Bathgate RA, Summers RJ. Responses of GPCR135 to human gene 3 (H3) relaxin in CHO-K1 cells determined by microphysiometry. Ann N Y Acad Sci. 2005;1041:332–7.

    Article  Google Scholar 

  17. 17.

    Zhang JH, Chung TD, Oldenburg KR. A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen. 1999;4:67–73.

    CAS  Article  Google Scholar 

  18. 18.

    Bathgate RA, Ivell R, Sanborn BM, Sherwood OD, Summers RJ. International union of pharmacology LVII: recommendations for the nomenclature of receptors for relaxin family peptides. Pharmacol Rev. 2006;58:7–31.

    CAS  Article  Google Scholar 

  19. 19.

    Faure M, Voyno-Yasenetskaya TA, Bourne HR. cAMP and beta gamma subunits of heterotrimeric G proteins stimulate the mitogen-activated protein kinase pathway in COS-7 cells. J Biol Chem. 1994;269:7851–4.

    CAS  PubMed  Google Scholar 

  20. 20.

    Crespo P, Xu N, Simonds WF, Gutkind JS. Ras-dependent activation of MAP kinase pathway mediated by G-protein beta gamma subunits. Nature. 1994;369:418–20.

    CAS  Article  Google Scholar 

  21. 21.

    Lopez-Ilasaca M, Crespo P, Pellici PG, Gutkind JS, Wetzker R. Linkage of G protein-coupled receptors to the MAPK signaling pathway through PI3-kinase gamma. Science. 1997;275:394–7.

    CAS  Article  Google Scholar 

  22. 22.

    Bathgate RAD, Halls ML, van der Westhuizen ET, Callander GE, Kocan M, Summers RJ, et al. Relaxin family peptides and their receptors. Physiol Rev. 2013;93:405–80.

    CAS  Article  Google Scholar 

  23. 23.

    Hu MJ, Shao XX, Wang JH, Wei D, Guo YQ, Liu YL, et al. Mechanism for insulin-like peptide 5 distinguishing the homologous relaxin family peptide receptor 3 and 4. Sci Rep. 2016;6:29648.

    Article  Google Scholar 

  24. 24.

    Alvarez-Jaimes L, Sutton SW, Nepomuceno D, Motley ST, Cik M, et al. In vitro pharmacological characterization of RXFP3 allosterism: an example of probe dependency. PLoS ONE. 2012;7:e30792.

    CAS  Article  Google Scholar 

  25. 25.

    Chen CZ, Southall N, Xiao J, Marugan JJ, Ferrer M, Hu X, et al. Identification of small-molecule agonists of human relaxin family receptor 1 (RXFP1) by using a homogenous cell-based cAMP assay. J Biomol Screen. 2013;18:670–7.

    CAS  Article  Google Scholar 

  26. 26.

    Xiao J, Huang Z, Chen CZ, Agoulnik IU, Southall N, Hu X, et al. Identification and optimization of small-molecule agonists of the human relaxin hormone receptor RXFP1. Nat Commun. 2013;4:1953.

    Article  Google Scholar 

Download references


We are indebted to Ji Wu and Qiang Shen for technical assistance. This work was partially supported by grants from the National Natural Science Foundation of China 81872915 (MWW), 81573479 (DHY), and 81773792 (DHY); the National Science & Technology Major Project “Key New Drug Creation and Manufacturing Program” of China (2018ZX09735-001 to MWW, 2018ZX09711002-002-005 to DHY and 2018ZX09711002-002-011 to QL); the National Key R&D Program of China 2018YFA0507000 (MWW); and the Novo Nordisk-CAS Research Fund (NNCAS-2017-1-CC to DHY). Research at the Florey was supported by the Victorian Government’s Operational Infrastructure Support Program. We are grateful to Tania Ferraro and Sharon Layfield for assistance with assays at the Florey. RADB is an NHMRC Senior Research Fellow. The funders had no role in the study design, data collection and analysis, decision to publish, or paper preparation.

Author information




MWW and RADB designed the research. GYL, XQC, ATD, and JL performed the research. YZ established the hRXFP4-Gα16-CHO cell line. LL and QL synthesized JK1. GYL, LL, QL, DHY, RADB, and MWW analyzed the data. GYL, QL, MWW, and RADB drafted the paper, and GYL, QL, DHY, RADB, and MWW edited and revised the paper.

Corresponding authors

Correspondence to De-hua Yang or Ross A. D. Bathgate or Ming-wei Wang.

Ethics declarations

Competing interests

The authors declare no competing interests.

Supplementary information

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Lin, Gy., Lin, L., Cai, Xq. et al. High-throughput screening campaign identifies a small molecule agonist of the relaxin family peptide receptor 4. Acta Pharmacol Sin 41, 1328–1336 (2020).

Download citation


  • RXFP4
  • INSL5
  • high-throughput screening
  • agonist
  • JK1

Further reading


Quick links