Engineering of chimeric peptides as antagonists for the G protein-coupled receptor, RXFP4

Abstract

Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. However, INSL5 with two chains and three disulfide bridges is an extremely difficult peptide to assemble by chemical or recombinant means. In a recent study, we were able to engineer a simplified INSL5 analogue 13 which is a relaxin family peptide receptor 4 (RXFP4)-specific agonist. To date, however, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature. The focus of this study was to utilize the non-specific RXFP3/RXFP4 antagonist ΔR3/I5 as a template to rationally design an RXFP4 specific antagonist. Unexpectedly, we demonstrated that ΔR3/I5 exhibited partial agonism at RXFP4 when expressed in CHO cells which is associated with only partial antagonism of INSL5 analogue activation. In an attempt to improve RXFP4 specificity and antagonist activity we designed and chemically synthesized a series of analogues of ΔR3/I5. While all the chimeric analogues still demonstrated partial agonism at RXFP4, one peptide (Analogue 17) exhibited significantly improved RXFP4 specificity. Importantly, analogue 17 has a simplified structure which is more amenable to chemical synthesis. Therefore, analogue 17 is an ideal template for further development into a specific high affinity RXFP4 antagonist which will be an important tool to probe the physiological role of RXFP4/INSL5 axis.

A Novel Antagonist Peptide Reveals a Physiological Role of Insulin-Like Peptide 5 in Control of Colorectal Function

“…INSL5-A13AGP also demonstrated antagonism but with a lower potency than INSL5-A13NR, whereas INSL5-A13HR had no antagonist activity. Consistent with the small agonistic effect of INSL5-A13NR, the peptide was not able to completely block the cAMP inhibitory effects of an agonist in a similar manner to the antagonism we have recently demonstrated for the nonspecific RXFP3/4 antagonist, ΔR3/I5 . To further demonstrate the RXFP4 antagonist activity of INSL5-A13NR, we developed a sensitive NanoBRET based β-Arrestin2 recruitment assay.…”

“…Because INSL5-A13NR only demonstrates partial antagonism in this cAMP activity assay, similar to the RXFP3/4 antagonist ΔR3/I5, we also assessed the ability of INSL5-A13NR to antagonize agonist-induced β-Arrestin recruitment. It has been previously demonstrated that agonist activation of RXFP4 leads to the recruitment of both β-Arrestin1 and β-Arrestin2 leading to receptor internalization .…”

“…One of these chimeric peptides, INSL5-A17, acted as a partial agonist at RXFP4, showing very weak antagonism. 14 It also showed activity on the related RXFP3 receptor. In this study, we have taken a novel approach to create a high affinity RXFP4-specific antagonist.…”

“…Recently, we followed a chimeric peptide approach and generated a series of chimeric peptides consisting of the INSL5 A-chain and the relaxin-3 B-chain. One of these chimeric peptides, INSL5-A17, acted as a partial agonist at RXFP4, showing very weak antagonism . It also showed activity on the related RXFP3 receptor.…”

Human Insulin-like Peptide 5 (INSL5). Identification of a Simplified Version of Two-Chain Analog A13

“…A13 and its two truncated analogs (A13: B3− 24, A13: B5−24) showed random coil structures in 10 mM phosphate buffer (PB) (Figure 3A). As previous study of INSL5-related peptides revealed that the α-helical structure could be induced by trifluoroethanol (TFE), 22 thus, we further studied the CD spectroscopy of A13: B3−24 and A13: B5−24 in phosphate buffer containing 20% TFE. TFE is known to improve hydrogen bonding of peptides and proteins and thus stabilizes the α-helical structure of peptide.…”