The peptide hormone relaxin (RLN) and its G protein-coupled receptor RXFP1 are expressed in several types of cancer cells, including prostate, endometrial, thyroid and others. It was shown that the overexpression of relaxin is often associated with advanced metastatic disease. Stimulation of RLN/RXFP1 signaling increases cell proliferation, invasion, migration, adhesion, and decreases cell apoptosis in vitro and in viv. RLN activates a set of signaling pathways and genes previously shown to be involved in tumorigenesis. More importantly, a suppression of RLN or RXFP1 by siRNA knockdown or through peptide antagonist expression in prostate cancer cells has an inhibitory effect on tumor growth and metastasis. This establishes the RLN signaling pathway as a promising novel anticancer target. To date no small molecule or non-peptide antagonists of the relaxin receptor are known. The current application is designed to fill this gap through high throughput screening (HTS) of a >400,000 small molecule compound library at NIH NCGC. An easily detectable and reliable indication of RXFP1 activation is an increase of cAMP production. Using HEK293T cells stably transfected with RXFP1 we have optimized a time-resolved fluorescence resonance energy transfer cAMP assay for quantitative HTS of RXFP1 antagonists in a 1536-well format. This assay will be applied for the RXFP1 antagonist screening campaign. After the primary screen the active compounds will be selected in a series of secondary assays designed to identify specific relaxin receptor antagonists. These include a counterscreen against cells transfected with related GPCR, cells stimulated with non-specific activator of cAMP, and a conformation screen using an orthogonal detection method. Tertiary cell-based assays will be used to select antagonists affecting RLN-induced responses in prostate cancer cells. Finally, we will analyze the antagonist specificity, efficacy, potency, mode of action, and their effects on prostate cancer cells. In collaboration with NCGC we have generated, developed, and obtained all reagents necessary for the proposed assays and tested all experimental procedures. The discovery of relaxin receptor antagonists will provide a basis for their testing as anticancer agents.

Public Health Relevance

We have shown that the inhibition of relaxin hormone signaling suppresses prostate cancer progression. We will perform a high throughput screening of a large library of small molecules to isolate chemical compounds that disrupt relaxin signaling and can be potentially used as the anti-cancer drugs.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01CA177711-02
Application #
8735900
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Forry, Suzanne L
Project Start
2013-09-17
Project End
2015-08-31
Budget Start
2014-09-01
Budget End
2015-08-31
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Florida International University
Department
Genetics
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33199
Agoulnik, Alexander I; Agoulnik, Irina U; Hu, Xin et al. (2017) Synthetic non-peptide low molecular weight agonists of the relaxin receptor 1. Br J Pharmacol 174:977-989
Kaftanovskaya, Elena M; Soula, Mariluz; Myhr, Courtney et al. (2017) Human Relaxin Receptor Is Fully Functional in Humanized Mice and Is Activated by Small Molecule Agonist ML290. J Endocr Soc 1:712-725
Kocan, Martina; Sarwar, Mohsin; Ang, Sheng Y et al. (2017) ML290 is a biased allosteric agonist at the relaxin receptor RXFP1. Sci Rep 7:2968
Hu, Xin; Myhr, Courtney; Huang, Zaohua et al. (2016) Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists. Biochemistry 55:1772-83
Agoulnik, Irina U; Agoulnik, Alexander I (2015) Long-Lasting Consequences of Testosterone Exposure. Endocrinology 156:3488-9
Kaftanovskaya, Elena M; Lopez, Carolina; Ferguson, Lydia et al. (2015) Genetic ablation of androgen receptor signaling in fetal Leydig cell lineage affects Leydig cell functions in adult testis. FASEB J 29:2327-37
Huang, Zaohua; Myhr, Courtney; Bathgate, Ross A D et al. (2015) Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290. Front Endocrinol (Lausanne) 6:128