Apelin is the endogenous ligand for the G protein-coupled receptor APJ and exerts a key role in regulating cardiovascular functions. We report herein a novel series of macrocyclic analogues of apelin-13 in which the N- and C-terminal residues as well as the macrocycle composition were chemically modified to modulate structure–activity relationships on the APJ receptor. To this end, the binding affinity and the ability to engage G protein-dependent and G protein-independent signalling pathways of the resulting analogues were assessed. In this series, the position and the nature of the C-terminal aromatic residue is a determinant for APJ interaction and β-arrestin recruitment, as previously demonstrated for linear apelin-13 derivatives. We finally discovered compounds 1, 4, 11 and 15, four potent G protein-biased apelin receptor agonists exhibiting affinity in the nanomolar range for APJ. These macrocyclic compounds represent very useful pharmacological tools to explore the therapeutic potential of the apelinergic system.

Human influenza viruses cause acute respiratory symptoms that can lead to death. Due to the emergence of antiviral drug‐resistant strains, there is an urgent requirement for novel antiviral agents and innovative therapeutic strategies. Using the peptidomimetic ketobenzothiazole protease inhibitor RQAR‐Kbt (IN‐1, aka N‐0100) as a starting point, we report how substituting P2 and P4 positions with natural and unnatural amino acids can modulate the inhibition potency toward matriptase, a prototypical type II transmembrane serine protease (TTSP) that acts as a priming protease for influenza viruses. We also introduced modifications of the peptidomimetics N‐terminal groups, leading to significant improvements (from μM to nM, 60 times more potent than IN‐1) in their ability to inhibit the replication of influenza H1N1 virus in the Calu‐3 cell line derived from human lungs. The selectivity towards other proteases has been evaluated and explained using molecular modeling with a crystal structure recently obtained by our group. By targeting host cell TTSPs as a therapeutic approach, it may be possible to overcome the high mutational rate of influenza viruses and consequently prevent potential drug resistance.

To probe ligand-receptor binding at the atomic-level, a frequent approach involves multidimensional nuclear magnetic resonance (NMR) spectroscopy experiments relying on ¹³C- and/or ¹⁵N-enrichment alongside ¹H. Alternatively, the lack of fluorine in biomolecules may be exploited through specific incorporation of ¹⁹F nuclei into a sample. The ¹⁹F nucleus is highly sensitive to environmental changes and allows for one-dimensional NMR spectroscopic study, with perturbation to chemical shift and spin dynamics diagnostic of structural change, ligand binding, and modified conformational sampling. This was applied to the apelinergic system, which comprises a rhodopsin-like G protein-coupled receptor (the apelin receptor (AR)/APJ) and two families of cognate ligands, the apelin and apela (ELABELA/toddler) peptides. Specifically, AR fragments consisting of either the N-terminal tail and first transmembrane (TM) α-helix (AR55) or the first three transmembrane α-helices (TM1-3) were prepared with biosynthetic fluorotryptophan incorporation. Interactions of each AR fragment with a high-affinity, 2,4,5-trifluorophenylalanine labeled apelin analogue were compared by ¹⁹F NMR. Distinct ranges of ¹⁹F chemical shifts for ligand and receptor provide unambiguous tracking of both species, with distinct binding behaviour observed for each AR fragment implying that AR55 is not sufficient to recapitulate the physiological binding event. Site-specific perturbation was also apparent for the apelin analogue as a function of substitution site, indicating an orientational binding preference. As a whole, this strategy of distinctive ¹⁹F labelling for ligand and receptor provides a relatively fast (i.e., employing 1D NMR experiments) and highly sensitive method to simultaneously and definitively track binding in both species.