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.
Highlights
Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes
All the analogues were made with free C-termini as we have previously shown that INSL5 and H3 relaxin analogues with amidated C-termini were less active compared with native INSL5 or H3 relaxin with free C-termini[9]
The antagonism of RXFP3 and RXFP4 was compared, and the results showed that ΔR3/I5 dose-dependently shifted the agonism curves to the right confirming ΔR3/I5 to be an antagonist for both RXFP3 and RXFP416
Summary
Insulin-like peptide 5 (INSL5) is a very important pharma target for treating human conditions such as anorexia and diabetes. Studies on the biology of INSL5 have been limited by the lack of sufficient quantities of the native peptide and the absence of small molecule RXFP4-specific agonists Synthesis of both human INSL5 and mouse INSL5 has proven to be extraordinarily challenging by either chemical[8,9] or recombinant means[10]. We recently undertook structure-activity relationship (SAR) studies on INSL5 and utilized the knowledge to engineer a simplified, potent RXFP4 agonist peptide, analogue 13 (Table 1)[11]. To date, no RXFP4-specific antagonist (peptide or small molecule) has been reported in the literature and such a compound would be enormously valuable to understand the physiology of INSL5 and validate RXFP4 as potential therapeutic target for the treatment of human conditions such as obesity and diabetes.
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