General method for selective labelling of double‐chain cysteine‐rich peptides with a lanthanide chelate via solid‐phase synthesis

Shabanpoor, Fazel; Separovic, Frances; Wade, John D.

doi:10.1002/psc.1307

Cited by 13 publications

(6 citation statements)

References 20 publications

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“…The receptor binding affinity of the peptides was measured using CHO-K1 cells stably expressing RXFP3 and RXFP4. The competition binding assays were carried out as previously described [24] , [27] . Briefly, a single concentration of Eu-labelled I5/R3 (0.5 nM) or Eu-labelled mouse INSL5 (2.5 nM) were used in presence of increasing concentration of peptides.…”

Section: Methodsmentioning

confidence: 99%

C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

et al. 2013

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Human relaxin-3 is a neuropeptide that is structurally similar to human insulin with two chains (A and B) connected by three disulfide bonds. It is expressed primarily in the brain and has modulatory roles in stress and anxiety, feeding and metabolism, and arousal and behavioural activation. Structure-activity relationship studies have shown that relaxin-3 interacts with its cognate receptor RXFP3 primarily through its B-chain and that its A-chain does not have any functional role. In this study, we have investigated the effect of modification of the B-chain C-terminus on the binding and activity of the peptide. We have chemically synthesised and characterized H3 relaxin as C-termini acid (both A and B chains having free C-termini; native form) and amide forms (both chains’ C-termini were amidated). We have confirmed that the acid form of the peptide is more potent than its amide form at both RXFP3 and RXFP4 receptors. We further investigated the effects of amidation at the C-terminus of individual chains. We report here for the first time that amidation at the C-terminus of the B-chain of H3 relaxin leads to significant drop in the binding and activity of the peptide at RXFP3/RXFP4 receptors. However, modification of the A-chain C-terminus does not have any effect on the activity. We have confirmed using circular dichroism spectroscopy that there is no secondary structural change between the acid and amide form of the peptide, and it is likely that it is the local C-terminal carboxyl group orientation that is crucial for interacting with the receptors.

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Section: Methodsmentioning

confidence: 99%

C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

et al. 2013

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“…Binding assays were conducted as described. 45 Briefly, the competition binding assay was done using a single concentration of Eu-labeled INSL5A/H3 relaxin B (0.5 nM), 46 Eulabeled H2 relaxin (1 nM), and Eu-labeled mouse INSL5 (2.5 nM) in the presence of an increasing concentration of H3 relaxin analogues in comparison to the native receptor ligands. Each concentration point was performed in triplicate, and the data were expressed as the mean ± SEM of three independent experiments.…”

Section: Journal Of Medicinal Chemistrymentioning

confidence: 99%

Minimization of Human Relaxin-3 Leading to High-Affinity Analogues with Increased Selectivity for Relaxin-Family Peptide 3 Receptor (RXFP3) over RXFP1

Shabanpoor¹,

Hossain²,

Ryan³

et al. 2012

J. Med. Chem.

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164

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Relaxin-3 is a neuropeptide that is implicated in the regulation of stress responses and memory. The elucidation of its precise physiological role(s) has, however, been hampered by cross-activation of the relaxin-2 receptor, RXFP1, in the brain. The current study undertook to develop analogues of human relaxin-3 (H3 relaxin) that can selectively bind and activate its receptor, RXFP3. We developed a high-affinity selective agonist (analogue 2) by removal of the intra-A chain disulfide bond and deletion of 10 residues from the N terminus of the A chain. Further truncation of this analogue from the C terminus of the B chain to Cys(B22) and addition of an Arg(B23) led to a high-affinity, RXFP3-selective, competitive antagonist (analogue 3). Central administration of analogue 2 in rats increased food intake, which was blocked by prior coadministration of analogue 3. These novel RXFP3-selective peptides represent valuable pharmacological tools to study the physiological roles of H3 relaxin/RXFP3 systems in the brain and important leads for the development of novel compounds for the treatment of affective and cognitive disorders.

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“…5 CHO-K1 cells stably expressing RXFP3 were plated into a 96 well plate (Viewplate; opaque white wall and clear bottom, PerkinElmer, Glen Waverly, Australia) at a density of 5 × 10 4 cells/well and grown over night to reach ~90% confluence before experimentation. Binding assays were conducted as described (Shabanpoor et al 2011)Briefly, the competition binding assay was conducted using a single concentration of Eu-labelled INSL5A/H3 relaxin B (Eu-R3/I5) (0.5 nM) in the presence of increasing concentrations of rat/mouse relaxin-3.…”

Section: Whole Cell Rxfp3 Binding Assaymentioning

confidence: 99%

Chemical synthesis and orexigenic activity of rat/mouse relaxin-3

et al. 2013

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The insulin-like peptide, relaxin-3 was first identified just a decade ago via a genomic database search and is now recognized to be a key neuropeptide with several roles including the regulation of arousal, stress responses and neuroendocrine homeostasis. It also has significant potential as a drug to treat stress and obesity. Its actions are mediated via its cognate G protein-coupled receptor, RXFP3, which is found in abundant numbers in the brain. However, much remains to be determined with respect to the mechanism of neurological action of this peptide. Consequently, the chemical synthesis of the rat and mouse (which share identical primary structures) two-chain, three disulfide peptide was undertaken and the resulting peptide subjected to detailed in vitro and in vivo assay. Use of efficient solid-phase synthesis methods provided the two regioselectively S-protected A- and B-chains which were readily combined via sequential disulfide bond formation. The synthetic rat/mouse relaxin-3 was obtained in high purity and good overall yield. It demonstrated potent orexigenic activity in rats in that central intracerebroventricular infusion led to significantly increased food intake and water drinking.

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General method for selective labelling of double‐chain cysteine‐rich peptides with a lanthanide chelate via solid‐phase synthesis

Cited by 13 publications

References 20 publications

C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

C-Terminus of the B-Chain of Relaxin-3 Is Important for Receptor Activity

Minimization of Human Relaxin-3 Leading to High-Affinity Analogues with Increased Selectivity for Relaxin-Family Peptide 3 Receptor (RXFP3) over RXFP1

Chemical synthesis and orexigenic activity of rat/mouse relaxin-3

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