A single-chain derivative of the relaxin hormone is a functionally selective agonist of the G protein-coupled receptor, RXFP1

Hossain, Mohammed Akhter; Kočan, Martina; Yao, Song T.; Royce, Simon G.; Nair, Vinojini B.; Siwek, Christopher; Patil, Nitin A.; Harrison, Ian; Rosengren, K. Johan; Selemidis, Stavros; Summers, Roger J.; Wade, John D.; Bathgate, Ross A. D.; Samuel, Chrishan S.

doi:10.1039/c5sc04754d

Cited by 78 publications

(122 citation statements)

References 73 publications

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“…Unlike ML290, B7-33 competes with relaxin binding at the orthosteric site and promotes cAMP accumulation following stimulation of rat and mouse RXFP1 as well as human RXFP1 albeit with 3–5 orders of magnitude lower potency than H2 relaxin 44 . Like ML290, B7-33 has little if any activity at RXFP2 44 .…”

Section: Discussionmentioning

confidence: 99%

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1

Kočan

Sarwar

Ang

et al. 2017

Sci Rep

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Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gαs and GαoB but weak coupling to Gαi3. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.

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Section: Discussionmentioning

confidence: 99%

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1

Kočan

Sarwar

Ang

et al. 2017

Sci Rep

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“…The β-chain is mediated through pERK1/2 and the cGMP pathway. 16 RXFP1 activation by the β-chain increases phosphorylation of extracellular signal-regulated protein kinase 1 and 2 (pERK1/2) that enhances neuronal nitric oxide synthase (NOS) activity and cyclic guanosine monophosphate (cGMP) generation. 14 Activation of this pathway disrupts profibrotic TGF-β/Smad2 phosphorylation (pSmad2) signaling 17 to inhibit collagen synthesis, promote expression of MMPs, 18 and decrease expression of TIMPs.…”

Section: Relaxin Hormonementioning

confidence: 99%

“…The cAMP pathway is also involved in enhancement of proangiogenic signaling. The β‐chain is mediated through pERK1/2 and the cGMP pathway …”

Section: Introductionmentioning

confidence: 99%

Relaxin‐2 therapy reverses radiation‐induced fibrosis and restores bladder function in mice

Ikeda

Zabbarova

Birder

et al. 2018

Neurourology and Urodynamics

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Aim To determine the efficacy of human relaxin-2 (hRLX2) in reversing radiation-induced bladder fibrosis and lower urinary tract dysfunction (LUTD). Radiation cystitis is a consequence of radiotherapy for pelvic malignancies. Acutely, irradiation leads to reactive oxygen/nitrogen species in urothelial cells, apoptosis, barrier disruption, and inflammation. Chronically, this results in collagen deposition, bladder fibrosis, and attenuated storage and voiding functions. In severe cases, cystectomies are performed as current therapies do not reverse fibrosis. Methods We developed a mouse model for selective bladder irradiation (10 Gray; 1 Gy = 100 rads) resulting in chronic fibrosis within 6 weeks, with decreased bladder compliance, contractility, and overflow incontinence. Seven weeks post-irradiation, female C57Bl/6 mice were continuously infused with hRLX2 (400 μg/kg/day/14 days) or vehicle (saline) via subcutaneous osmotic pumps. Mice were evaluated in vivo using urine spot analysis, cystometrograms and external urethral sphincter electromyograms; and in vitro using length-tension measurements, Western blots, histology, and immunohistochemistry. Results hRLX2 reversed fibrosis, decreased collagen content, improved bladder wall architecture, and increased bladder compliance, detrusor smooth muscle Cav1.2 expression and detrusor contractility in mice with chronic radiation cystitis. hRLX2 treatment outcomes were likely caused by the activation of RXFP1/2 receptors which are expressed on the detrusor. Conclusion hRLX2 may be a new therapeutic option for rescuing bladders with chronic radiation cystitis.

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“…Our newly‐developed THP‐1 CAMYEL cell line will be a valuable tool for drug discovery targeting RXFP1, as it is sensitive and able to detect concentration‐dependent increases in cAMP activity over time in the absence of phosphodiesterase inhibitors. Importantly, the assay showed a similar potency of H2 relaxin to that observed using traditional cAMP assays such as enzyme immunoassay and HTRF cAMP assays . The THP‐1 CAMYEL assay also streamlines the process of screening ligands, as these suspension cells can be plated out on the day of the assay, and no extra steps for lysis or addition of detection reagents are required.…”

Section: Discussionmentioning

confidence: 65%

“…In particular, RXFP1 is a promising therapeutic target for cardiovascular diseases and fibrosis. Novel ligands acting at RXFP1, including peptide analogues (e.g., ) and small molecules, have been screened in THP‐1 cells due to the robust cAMP activation by relaxin in these cells. Our newly‐developed THP‐1 CAMYEL cell line will be a valuable tool for drug discovery targeting RXFP1, as it is sensitive and able to detect concentration‐dependent increases in cAMP activity over time in the absence of phosphodiesterase inhibitors.…”

Section: Discussionmentioning

confidence: 99%

Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor

Valkovic

Leckey

Whitehead

et al. 2018

Pharmacology Res & Perspec

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Relaxin family peptide (RXFPs) 1‐4 receptors modulate the activity of cyclic adenosine monophosphate (cAMP) to produce a range of physiological functions. RXFP1 and RXFP2 increase cAMP via Gαs, whereas RXFP3 and RXFP4 inhibit cAMP via Gαi/o. RXFP1 also shows a delayed increase in cAMP downstream of Gαi3. In this study we have assessed whether the bioluminescence resonance energy transfer (BRET)‐based biosensor CAMYEL (cAMP sensor using YFP‐Epac‐Rluc), which allows real‐time measurement of cAMP activity in live cells, will aid in understanding ligand‐ and cell‐specific RXFP signaling. CAMYEL detected concentration‐dependent changes in cAMP activity at RXFP1‐4 in recombinant cell lines, using a variety of ligands with potencies comparable to those seen in conventional cAMP assays. We used RXFP2 and RXFP3 antagonists to demonstrate that CAMYEL detects dynamic changes in cAMP by reversing cAMP activation or inhibition respectively, with real‐time addition of antagonist after agonist stimulation. To demonstrate the utility of CAMYEL to detect cAMP activation in native cells expressing low levels of RXFP receptor, we cloned CAMYEL into a lentiviral vector and transduced THP‐1 cells, which express low levels of RXFP1. THP‐1 CAMYEL cells demonstrated robust cAMP activation in response to relaxin. However, the CAMYEL assay was unable to detect the Gαi3‐mediated phase of RXFP1 cAMP activation in PTX‐treated THP‐1 cells or HEK293A cells with knockout of Gαs. Our data demonstrate that cytoplasmically‐expressed CAMYEL efficiently detects real‐time cAMP activation by Gαs or inhibition by Gαi/o but may not detect cAMP generated in specific intracellular compartments such as that generated by Gαi3 upon RXFP1 activation.

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A single-chain derivative of the relaxin hormone is a functionally selective agonist of the G protein-coupled receptor, RXFP1

Abstract: A single-chain derivative of the relaxin hormone ameliorates fibrosis without side-effects.

Cited by 78 publications

References 73 publications

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1

Relaxin‐2 therapy reverses radiation‐induced fibrosis and restores bladder function in mice

Real‐time examination of cAMP activity at relaxin family peptide receptors using a BRET‐based biosensor

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