Design and development of analogues of dimers of insulin‐like peptide 3 B‐chain as high‐affinity antagonists of the RXFP2 receptor

Abstract

Insulin-like peptide 3 (INSL3) is one of 10 members of the human relaxin-insulin superfamily of peptides. It is a peptide hormone that is expressed by fetal and postnatal testicular Leydig cells and postnatal ovarian thecal cells. It mediates testicular descent during fetal life and suppresses sperm apoptosis in adult males, whereas, in females, it causes oocyte maturation. INSL3 has also been shown to promote thyroid tumor growth and angiogenesis in human. These actions of INSL3 are mediated through its G protein-coupled receptor, RXFP2. INSL3, a two-chained peptide, binds to its receptor primarily via its B-chain, whereas elements of the A-chain are essential for receptor activation. In an attempt to design a high-affinity antagonist with potential clinical application as an anticancer agent as well as a contraceptive, we have previously prepared a synthetic parallel dimer of INSL3 B-chain and demonstrated that it binds to RXFP2 with high affinity. In this work, we undertook full pharmacological characterization of this peptide and show that it can antaogonize INSL3-mediated cAMP signaling through RXFP2. Further refinement by truncation of 18 residues yielded a minimized analogue that retained full binding affinity and INSL3 antagonism. It is an attractive lead peptide for in vivo evaluation as an inhibitor of male and female fertility and of INSL3-mediated carcinogenesis.

Protective Role of Testicular Hormone INSL3 From Atrophy and Weakness in Skeletal Muscle

“…To this aim, we evaluated downstream Akt/mTOR/S6-phosphorylation at different times after stimulation with 10 nM INSL3 (Figures 2A,B ). In order to address the specific involvement of RXFP2 in INSL3-mediated effects, we performed the same experiment in presence of the INSL3-β chain dimer (β-chain, 20 nM), the high affinity antagonist of RXFP2 receptor ( 19 ). INSL3 treatment caused a time-dependent increase of both phospho-Akt, phospho-mTOR and phospho-S6 levels.…”

“…In differentiation experiments, culture medium containing INSL3, and β-chain when necessary, was renewed every other day. INSL3-β chain is an antagonist of RXFP2 receptor ( 19 ) and it was provided by Prof. Ross Bathgate (Florey Institute of Neuroscience and Mental Health, The University of Melbourne, Victoria, Australia).…”

Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor

“…To simply assess whether the sensor was able to respond to rapid changes in cAMP we utilized a specific receptor antagonist. As there is no relevant RXFP1 antagonist we utilized a well‐characterized RXFP2 antagonist INSL3 B‐chain dimer . Blockade of RXFP2 by the antagonist 10 minutes before adding the agonist INSL3 abolished the ability of INSL3 to increase cAMP activity (Figure F).…”

“…As there is no relevant RXFP1 antagonist we utilized a well-characterized RXFP2 antagonist INSL3 B-chain dimer. 22 Blockade of RXFP2 by the antagonist 10 minutes before adding the agonist INSL3 abolished the ability of INSL3 to increase cAMP activity ( Figure 2F). Importantly, addition of antagonist 10 minutes after addition of INSL3 resulted in real-time reversal of the BRET signal highlighting the CAMYEL sensor is able to respond to dynamic changes in cAMP activity.…”

“…DMEM, DMEM/F12, RPMI 1640, trypsin/EDTA, L‐glutamine, penicillin/streptomycin, DPBS, Gateway enzymes, Lipofectamine 2000 (Invitrogen); FBS (Scientifix); BSA, forskolin, HEPES, PTX (Sigma‐Aldrich); Viafect (Promega); coelenterazine h (Nanolight); CulturPlate96 (PerkinElmer); RXFP1 agonists: H2 relaxin and the small molecule ML290; RXFP2 agonists: INSL3 and H2 relaxin; antagonist: INSL3 B‐chain dimer; RXFP3 agonists: analogue 2 (H3 relaxin analogue), peptide 5 (single‐chain H3 relaxin analogue), R3/I5; antagonist: R3 B1‐22R; RXFP4 agonists: INSL5 and analogue 13 (INSL5 analogue) …”