Noncovalent Peptide Stapling Using Alpha-Methyl-l-Phenylalanine for α-Helical Peptidomimetics.

Peptides and peptidomimetics are attractive drug candidates because of their high target specificity and low-toxicity profiles. Developing peptidomimetics using hydrocarbon (HC)-stapling or other stapling strategies has gained momentum because of their high stability and resistance to proteases; however, they have limitations. Here, we take advantage of the α-methyl group and an aromatic phenyl ring in a unique unnatural amino acid, α-methyl-l-phenylalanine (αF), and propose a novel, noncovalent stapling strategy to stabilize peptides. We utilized this strategy to create an α-helical B-chain mimetic of a complex insulin-like peptide, human relaxin-3 (H3 relaxin). Our comprehensive data set (in vitro, ex vivo, and in vivo) confirmed that the new high-yielding B-chain mimetic, H3B10-27(13/17αF), is remarkably stable in serum and fully mimics the biological function of H3 relaxin. H3B10-27(13/17αF) is an excellent scaffold for further development as a drug lead and an important tool to decipher the physiological functions of the neuropeptide G protein-coupled receptor, RXFP3.

A Real-Time, Plate-Based BRET Assay for Detection of cGMP in Primary Cells.

Cyclic guanosine monophosphate (cGMP) is a second messenger involved in the regulation of numerous physiological processes. The modulation of cGMP is important in many diseases, but reliably assaying cGMP in live cells in a plate-based format with temporal resolution is challenging. The Förster/fluorescence resonance energy transfer (FRET)-based biosensor cGES-DE5 has a high temporal resolution and high selectivity for cGMP over cAMP, so we converted it to use bioluminescence resonance energy transfer (BRET), which is more compatible with plate-based assays. This BRET variant, called CYGYEL (cyclic GMP sensor using YFP-PDE5-Rluc8), was cloned into a lentiviral vector for use across different mammalian cell types. CYGYEL was characterised in HEK293T cells using the nitric oxide donor diethylamine NONOate (DEA), where it was shown to be dynamic, reversible, and able to detect cGMP with or without the use of phosphodiesterase inhibitors. In human primary vascular endothelial and smooth muscle cells, CYGYEL successfully detected cGMP mediated through either soluble or particulate guanylate cyclase using DEA or C-type natriuretic peptide, respectively. Notably, CYGYEL detected differences in kinetics and strength of signal both between ligands and between cell types. CYGYEL remained selective for cGMP over cAMP, but this selectivity was reduced compared to cGES-DE5. CYGYEL streamlines the process of cGMP detection in plate-based assays and can be used to detect cGMP activity across a range of cell types.

Understanding relaxin signalling at the cellular level.

The peptide hormone relaxin mediates many biological actions including anti-fibrotic, vasodilatory, angiogenic, anti-inflammatory, anti-apoptotic, and organ protective effects across a range of tissues. At the cellular level, relaxin binds to the G protein-coupled receptor relaxin family peptide receptor 1 (RXFP1) to activate a variety of downstream signal transduction pathways. This signalling cascade is complex and also varies in diverse cellular backgrounds. Moreover, RXFP1 signalling shows crosstalk with other receptors to mediate some of its physiological functions. This review summarises known signalling pathways induced by acute versus chronic treatment with relaxin across a range of cell types, it describes RXFP1 crosstalk with other receptors, signalling pathways activated by other ligands targeting RXFP1, and it also outlines physiological relevance of RXFP1 signalling outputs. Comprehensive understanding of the mechanism of relaxin actions in fibrosis, vasodilation, as well as organ protection, will further support relaxin's clinical potential.

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

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.