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Chimeric peptides derived from Relaxin 2, Relaxin 3, and INSL5 as receptor agonists for RXFPs

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Engineering of a Novel Simplified Human Insulin-Like Peptide 5 Agonist.

Insulin-like peptide 5 (INSL5) has recently been discovered as only the second orexigenic gut hormone after ghrelin. As we have previously reported,INSL5 is extremely difficult to assemble and oxidize into its two-chain three-disulfide structure. The focus of this study was to generate structure-activity relationships (SARs) of INSL5 and use it to develop a potent and simpler INSL5 mimetic with RXFP4 agonist activity. A series of human and mouse INSL5 (hINSL5/mINSL5) analogues were designed and chemically synthesized, resulting in a chimeric INSL5 analogue exhibiting more than 10-fold higher potency (0.35 nM) at human RXFP4 compared with native hINSL5 (4.57 nM). The SAR study also identified a key residue (K(A15)) in the A-chain of mINSL5 that contributes to improved RXFP4 affinity and potency of mINSL5 compared with hINSL5. This knowledge ultimately led us to engineer a minimized hINSL5 mimetic agonist that retains native hINSL5-like RXFP4 affinity and potency at human RXFP4. This minimized analogue was synthesized in 17.5-fold higher yield and in less time compared with hINSL5.

Patil NA, Hughes RA, Rosengren KJ et al., J Med Chem. 2016 Mar 10;59(5):2118-25. doi: 10.1021/acs.jmedchem.5b01786. Epub 2016 Feb 12.

Insulin-like peptide 5 is a microbially regulated peptide that promotes hepatic glucose production

Objective: Insulin-like peptide 5 (INSL5) is a recently identified gut hormone that is produced predominantly by L-cells in the colon, but its function is unclear. We have previously shown that colonic expression of the gene for the L-cell hormone GLP-1 is high in mice that lack a microbiota and thus have energy-deprived colonocytes. Our aim was to investigate if energy deficiency also affected colonic Insl5 expression and to identify a potential role of INSL5.
Methods: We analyzed colonic Insl5 expression in germ-free (GF), conventionally raised (CONV-R), conventionalized (CONV-D) and antibiotic-treated mice, and also assessed the effect of dietary changes on colonic Insl5 expression. In addition, we characterized the metabolic phenotype of Insl5-/- mice.
Results: We showed that colonic Insl5expression was higher in GF and antibiotic-treated mice than in CONV-R mice, whereasInsl5 expression in the brain was higher in CONV-R versus GF mice. We also observed that colonic Insl5 expression was suppressed by increasing the energy supply in GF mice by colonization or high-fat feeding. We did not observe any differences in food intake, gut transit or oral glucose tolerance betweenInsl5-/- and wild-type mice. However, we showed impaired intraperitoneal glucose tolerance in Insl5-/- mice. We also observed improved insulin tolerance and reduced hepatic glucose production in Insl5-/- mice.
Conclusions: We have shown that colonic Insl5expression is regulated by the gut microbiota and energy availability. We propose that INSL5 is a hormone that could play a role in promoting hepatic glucose production during periods of energy deprivation.

**Remark : The authors used the Phoenix Pharmaceutical’s antibody (#G-035-40) in an immunohistochemical analysis to probe for INSL5 expression in mouse colon (Supplementary Figure 1A, and 1B). They found that INSL5 expression was co-localized with GLP-1 and PYY in colonic L-cells of the wild type mice. However, similar anti-INSL5 staining was not present in INSL5 gene knock-out mice.

Ying Shiuan Lee, Filipe De Vadder, Valentina Tremaroli et al., Molecular Metabolism , April 2016 ( 5); No. 4 :1-8. DOI: http://dx.doi.org/10.1016/j.molmet.2016.01.007

The novel nonapeptide acein targets angiotensin converting enzyme in the brain and induces dopamine release.

BACKGROUND AND PURPOSE: Using an in-house bioinformatics programme, we identified and synthesized a novel nonapeptide, H-Pro-Pro-Thr-Thr-Thr-Lys-Phe-Ala-Ala-OH. Here, we have studied its biological activity, in vitro and in vivo, and have identified its target in the brain.
EXPERIMENTAL APPROACH: The affinity of the peptide was characterized using purified whole brain and striatal membranes from guinea pigs and rats . Its effect on behaviour in rats following intra-striatal injection of the peptide was investigated. A photoaffinity UV cross-linking approach combined with subsequent affinity purification of the ligand covalently bound to its receptor allowed identification of its target.
KEY RESULTS: The peptide bound with high affinity to a single class of binding sites, specifically localized in the striatum and substantia nigra of brains from guinea pigs and rats. When injected within the striatum of rats, the peptide stimulated in vitro and in vivo dopamine release and induced dopamine-like motor effects. We purified the target of the peptide, a ~151 kDa protein that was identified by MS/MS as angiotensin converting enzyme (ACE I). Therefore, we decided to name the peptide acein.
CONCLUSION AND IMPLICATIONS: The synthetic nonapeptide acein interacted with high affinity with brain membrane-bound ACE. This interaction occurs at a different site from the active site involved in the well-known peptidase activity, without modifying the peptidase activity. Acein, in vitro and in vivo, significantly increased stimulated release of dopamine from the brain. These results suggest a more important role for brain ACE than initially suspected.

Neasta J, Valmalle C, Coyne AC et al., Br J Pharmacol. 2016 Apr;173(8):1314-28. doi: 10.1111/bph.13424. Epub 2016 Mar 8.

H2 relaxin is a biased ligand relative to H3 relaxin at the relaxin family peptide receptor 3 (RXFP3).

Relaxin family peptide 3 receptors (RXFP3) are activated by H3-relaxin to inhibit forskolin-stimulated cAMP accumulation and stimulate extracellular signal-regulated kinase (ERK) 1/2 phosphorylation. In this study, we sought to identify novel signaling pathways coupled to RXFP3 and to investigate whether other members of the relaxin peptide family activated these pathways. Two patterns of signaling were observed in RXFP3-expressing Chinese hamster ovary (CHO)-K1 and human embryonic kidney (HEK)-293 cells (CHO-RXFP3 and HEK-RXFP3) and murine septal neuron SN56 cell lines: 1) strong inhibition of forskolin-stimulated cAMP accumulation, ERK1/2 activation and nuclear factor (NF)-kappaB reporter gene activation in cells stimulated with H3 relaxin, with weaker activity observed for H2 relaxin, porcine relaxin, or insulin-like peptide (INSL) 3 and 2) strong stimulation of activator protein (AP)-1 reporter genes by H2 relaxin, with weaker activation observed with H3 or porcine relaxin. Two distinct ligand binding sites were identified on RXFP3-expressing cells using two different radioligands. (125)I-INSL5 A-chain/relaxin-3 B-chain chimera bound with high affinity to the RXFP3-expressing cells with competition by H3 relaxin or a H3 relaxin B-chain dimeric peptide, consistent with previous reports. Binding studies with (125)I-H2 relaxin revealed a distinct binding site with potent competition observed with H2 relaxin, H3 relaxin, or INSL3 and weaker competition with porcine relaxin. Thus H3 relaxin potently activates all signaling pathways coupled to RXFP3, whereas H2 relaxin is an AP-1-biased ligand relative to H3 relaxin.

van der Westhuizen ET, Christopoulos A, Sexton PM et al., Mol Pharmacol. 2010 May;77(5):759-72. doi: 10.1124/mol.109.061432. Epub 2010 Feb 16.

Recent progress in relaxin-3-related research.

Relaxin-3 (R3) is the latest member of the insulin (INSL) superfamily, which is composed of peptides with diverse sequences held together by characteristic disulfide links connecting A and B peptide-Chains. R3 has nearly exclusive expression in the brainstem and was demonstrated to be an additional ligand for LGR7. We recently identified R3 as a ligand for two orphan G-protein coupled receptors, GPCR135 and GPCR142. The predominant brain expression for both R3 and GPCR135, coupled with their high affinity interaction, strongly suggests that R3 is the endogenous ligand for GPCR135. Both R3 and GPCR135 from different species are highly conserved from genetic sequences to in vitro pharmacology. By contrast, GPCR142 is a pseudogene in the rat, and the mouse gene is less conserved with human GPCR142, suggesting that GPCR142 may have a diminished role as a receptor for R3 in the rodent. In addition, the tissue expression pattern of GPCR142, which is primarily in peripheral tissue, is drastically different from that of R3, suggesting that GPCR142 may have an endogenous ligand other than R3. Sequence analysis amongINSL/relaxin family members shows that INSL5 is the closest member of R3. We were able to demonstrate that INSL-5 is an agonist for GPCR142 but not for GPCR135. We also showed that the mRNA expression pattern of INSL-5 overlaps with that of GPCR142. By substituting the A-Chain of R3 with the A-Chain of INSL-5, we devised a chimeric peptide (R3/I5) that is about 1000-fold selective for GPCR135 and GPCR142 over LGR7. Autoradiographic distribution of GPCR135 binding sites using [125I]R3/I5 in rat brain shows that GPCR135 receptor is most prominent in areas known for the processing of sensory signals.

Liu C, Bonaventure P, Sutton SW et al., Ann N Y Acad Sci. 2005 May;1041:47-60.

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036-21;036-22;036-23;036-24;036-25;036-26;035-71;035-80


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