Abstract:The relaxin family peptides play a variety of biological functions by activating four G protein-coupled receptors, RXFP1–4. Among them, insulin-like peptide 5 (INSL5) and relaxin-3 share the highest sequence homology, but they have distinct receptor preference: INSL5 can activate RXFP4 only, while relaxin-3 can activate RXFP3, RXFP4, and RXFP1. Previous studies suggest that the A-chain is responsible for their different selectivity for RXFP1. However, the mechanism by which INSL5 distinguishes the homologous R… Show more
“…R B12 in the B-chain of ΔR3/I5 was previously reported to be one of the residues important for binding to both RXFP3 and RXFP4 16 . The corresponding residue L B9 in the B-chain of INSL5, on the other hand, was reported to be one of the most critical residues by which INSL5 exhibits selectivity for RXFP4 over RXFP3 27 . Therefore, in the first attempt, we replaced the R B12 residues in the B-chain of ΔR3/I5 with corresponding L B9 residue of INSL5 B-chain in order to achieve selectivity for RXFP4 over RXFP3 (Table 1).…”
Section: Resultsmentioning
confidence: 99%
“…3D) compared with ΔR3/I5. The fact that the agonist peptide R3/I5 with the same mutation (R B12 /L B9 ) was shown to exhibit ~1600 less RXFP3 potency without losing much RXFP4 potency 27 suggest that the binding mode of the agonist (R3/I5) peptide for RXFP3 and RXFP4 might be different to that of the antagonist peptide (ΔR3/I5).Figure 3Competition binding curves for ΔR3/I5 and novel chimeric analogues 14–20 , ( A – C ) in comparison to Eu-B1-22R in RXFP3-expressing cells and ( D – F ) in comparison to Eu-INSL5 in RXFP4-expressing cells. The data are the result of n = 3–4 independent experiments and are expressed as mean ± SEM.…”
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.
“…R B12 in the B-chain of ΔR3/I5 was previously reported to be one of the residues important for binding to both RXFP3 and RXFP4 16 . The corresponding residue L B9 in the B-chain of INSL5, on the other hand, was reported to be one of the most critical residues by which INSL5 exhibits selectivity for RXFP4 over RXFP3 27 . Therefore, in the first attempt, we replaced the R B12 residues in the B-chain of ΔR3/I5 with corresponding L B9 residue of INSL5 B-chain in order to achieve selectivity for RXFP4 over RXFP3 (Table 1).…”
Section: Resultsmentioning
confidence: 99%
“…3D) compared with ΔR3/I5. The fact that the agonist peptide R3/I5 with the same mutation (R B12 /L B9 ) was shown to exhibit ~1600 less RXFP3 potency without losing much RXFP4 potency 27 suggest that the binding mode of the agonist (R3/I5) peptide for RXFP3 and RXFP4 might be different to that of the antagonist peptide (ΔR3/I5).Figure 3Competition binding curves for ΔR3/I5 and novel chimeric analogues 14–20 , ( A – C ) in comparison to Eu-B1-22R in RXFP3-expressing cells and ( D – F ) in comparison to Eu-INSL5 in RXFP4-expressing cells. The data are the result of n = 3–4 independent experiments and are expressed as mean ± SEM.…”
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.
“…While H3 relaxin cross‐reacts with other receptors including RXFP4 receptors, INSL5 is very selective (RXFP4) and does not interact with RXFP3 receptors. The mechanism by which INSL5 distinguishes RXFP4 and RXFP3 receptors has recently been identified (Hu et al, ). The authors have identified four determinants (E B2 , L B9 , Y B17 and a rigid B‐chain C‐terminus) on INSL5 that are responsible for its inactivity at RXFP3 receptors (Hu et al, ).…”
The human relaxin peptide family consists of seven cystine-rich peptides, four of which are known to signal through relaxin family peptide receptors, RXFP1-4. As these peptides play a vital role physiologically and in various diseases, they are of considerable importance for drug discovery and development. Detailed structure-activity relationship (SAR) studies towards understanding the role of important residues in each of these peptides have been reported over the years and utilized for the design of antagonists and minimized agonist variants. This review summarizes the current knowledge of the SAR of human relaxin 2 (H2 relaxin), human relaxin 3 (H3 relaxin), human insulin-like peptide 3 (INSL3) and human insulin-like peptide 5 (INSL5).
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“…Among them, RXFP4 and its endogenous ligand INSL5 have been linked to insulin secretion and appetite [ 8 , 11 ], thereby making them a potential therapeutic target for obesity and other metabolic disorders. Given the complexity of the interaction between RXFP4 and INSL5 [ 23 ], it is not surprising that the number of ligands selective for the receptor is rather limited. It is also well recognized that the synthesis of INSL5 and its peptide analogs is technically challenging [ 13 , 14 ], and nonpeptidic agonists are thus in high demand.…”
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.
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