Chemoselective Acylation of Hydrazinopeptides to Access Fluorescent Probes for Time-Resolved FRET Assays on GPCRs.

Fluorescence techniques represent a powerful tool to investigate dynamic and functional architecture of GPCRs. Thus, fluorescent GPCR ligands have found various applications in cellular imaging, in the development of binding assays as replacements for radioligands in the study of ligand-receptor but also in receptor-receptor interactions at the cell surface or in native tissues. To extend the applicability of these techniques, the design and the synthesis of fluorescent probes are critical steps. As there are numerous peptide receptors in the GPCR family, fluorescent peptide-based probes are of importance. Herein, we present a convenient method to facilitate the solution-phase fluorescent labeling of peptides which is based on the chemoselective acylation of α-hydrazinopeptides. This approach combines the advantages to use commercially available amine-reactive dyes and very mild conditions that are fully compatible with the chemical sensitivity of the dyes. It gives a rapid access to fluorescent peptidic probes compatible with the time-resolved fluorescence resonance energy transfer (TR-FRET) techniques.

A Time-Resolved FRET Cell-Based Binding Assay for the Apelin Receptor.

Analogues of apelin-13 carrying diverse spacers and an ad hoc DY647-derived fluorophore were designed and synthesized by chemoselective acylation of α-hydrazinopeptides. The resulting probes retain very high affinity and efficacy for both the wild-type and SNAP-tagged apelin receptor (ApelinR). They give a time-resolved FRET (TR-FRET) signal with rare-earth lanthanides used as donor fluorophores grafted onto the SNAP-tagged receptor. This specific signal allowed the validation of a binding assay with a high signal-to-noise ratio. In such an assay, the most potent sub-nanomolar fluorescent probe was found to be competitively displaced by the endogenous apelin peptides with binding constants similar to those obtained in a classical radioligand assay. We have thus validated the first TR-FRET cell-based binding assay for ApelinR with potential high-throughput screening applications.

Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

Apelin, a (neuro)vasoactive peptide, plays a prominent role in controlling cardiovascular functions and water balance. Because the in vivo apelin half-life is in the minute range, we aimed to identify metabolically stable apelin-17 (K17F) analogs. We generated P92 by classic chemical substitutions and LIT01-196 by original addition of a fluorocarbon chain to the N terminus of K17F. Both analogs were much more stable in plasma (half-life >24 h for LIT01-196) than K17F (4.6 min). Analogs displayed a subnanomolar affinity for the apelin receptor and behaved as full agonists with regard to cAMP production, ERK phosphorylation, and apelin receptor internalization. Ex vivo, these compounds induced vasorelaxation of rat aortas and glomerular arterioles, respectively, precontracted with norepinephrine and angiotensin II, and increased cardiac contractility. In vivo, after intracerebroventricular administration in water-deprived mice, P92 and LIT01-196 were 6 and 160 times, respectively, more efficient at inhibiting systemic vasopressin release than K17F. Administered intravenously (nmol/kg range) in normotensive rats, these analogs potently increased urine output and induced a profound and sustained decrease in arterial blood pressure. In summary, these new compounds, which favor diuresis and improve cardiac contractility while reducing vascular resistances, represent promising candidates for the treatment of heart failure and water retention/hyponatremic disorders.-Gerbier, R., Alvear-Perez, R., Margathe, J.-F., Flahault, A., Couvineau, P., Gao, J., De Mota, N., Dabire, H., Li, B., Ceraudo, E., Hus-Citharel, A., Esteoulle, L., Bisoo, C., Hibert, M., Berdeaux, A., Iturrioz, X., Bonnet, D., Llorens-Cortes, C. Development of original metabolically stable apelin-17 analogs with diuretic and cardiovascular effects.

Convenient Access to Fluorescent Probes by Chemoselective Acylation of Hydrazinopeptides: Application to the Synthesis of the First Far-Red Ligand for Apelin Receptor Imaging.

Herein, we develop a convenient method to facilitate the solution-phase fluorescent labelling of peptides based on the chemoselective acylation of α-hydrazinopeptides. This approach combines the advantages of using commercially available amine-reactive dyes and very mild conditions, which are fully compatible with the chemical sensitivity of the dyes. The usefulness of this approach was demonstrated by the labelling of apelin-13 peptide. Various fluorescent probes were readily synthesized, enabling the rapid optimization of their affinities for the apelin receptor. Thus, the first far-red fluorescent ligand with sub-nanomolar affinity for the apelin receptor was characterized and shown to track the receptor efficiently in living cells by fluorescence confocal microscopy.

Selective nonpeptidic fluorescent ligands for oxytocin receptor: design, synthesis, and application to time-resolved FRET binding assay.

The design and the synthesis of the first high-affinity fluorescent ligands for oxytocin receptor (OTR) are described. These compounds enabled the development of a TR-FRET based assay for OTR, readily amenable to high throughput screening. The validation of the assay was achieved by competition experiments with both peptide and nonpeptide OTR ligands as competitors. These probes represent the first selective fluorescent ligands for the oxytocin G protein-coupled receptor.

Time-resolved FRET binding assay to investigate hetero-oligomer binding properties: proof of concept with dopamine D1/D3 heterodimer.

G protein-coupled receptors (GPCRs) have been described to form hetero-oligomers. The importance of these complexes in physiology and pathology is considered crucial, and heterodimers represent promising new targets to discover innovative therapeutics. However, there is a lack of binding assays to allow the evaluation of ligand affinity for GPCR hetero-oligomers. Using dopamine receptors and more specifically the D1 and D3 receptors as GPCR models, we developed a new time-resolved FRET (TR-FRET) based assay to determine ligand affinity for the D1/D3 heteromer. Based on the high-resolution structure of the dopamine D3 receptor (D3R), six fluorescent probes derived from a known D3R partial agonist (BP 897) were designed, synthesized and evaluated as high affinity and selective ligands for the D3/D2 receptors, and for other dopamine receptor subtypes. The highest affinity ligand 21 was then employed in the development of the D1/D3 heteromer assay. The TR-FRET was monitored between a fluorescent tag donor carried by the D1 receptor (D1R) and a fluorescent acceptor D3R ligand 21. The newly reported assay, easy to implement on other G protein-coupled receptors, constitutes an attractive strategy to screen for heteromer ligands.

Diastereoselective synthesis of novel aza-diketopiperazines via a domino cyclohydrocarbonylation/addition process.

Herein, we report an unprecedented, short and diastereo-selective synthesis of newly reported aza-diketopiperazine (aza-DKP) scaffolds starting from amino acids. The strategy is based on a Rh(I)-catalyzed hydroformylative cyclohydrocarbonylation of allyl-substituted aza-DKP, followed by a diastereoselective functionalization of the platform. This methodology allows the synthesis of novel bicyclic and tricyclic aza-DKP scaffolds incorporating six- or seven-membered rings, with potential applications in medicinal chemistry.

Structure-activity relationship studies toward the discovery of selective apelin receptor agonists.

Apelin is the endogenous ligand for the previously orphaned G protein-coupled receptor APJ. Apelin and its receptor are widely distributed in the brain, heart, and vasculature, and are emerging as an important regulator of body fluid homeostasis and cardiovascular functions. To further progress in the pharmacology and the physiological role of the apelin receptor, the development of small, bioavailable agonists and antagonists of the apelin receptor, is crucial. In this context, E339-3D6 (1) was described as the first nonpeptidic apelin receptor agonist. We show here that 1 is actually a mixture of polymethylated species, and we describe an alternative and versatile solid-phase approach that allows access to highly pure 27, the major component of 1. This approach was also applied to prepare a series of derivatives in order to identify the crucial structural determinants required for the ligand to maintain its affinity for the apelin receptor as well as its capacity to promote apelin receptor signaling and internalization. The study of the structure-activity relationships led to the identification of ligands 19, 21, and 38, which display an increased affinity compared to that of 27. The latter and 19 behave as full agonists with regard to cAMP production and apelin receptor internalization, whereas 21 is a biased agonist toward cAMP production. Interestingly, the three ligands display a much higher stability in mouse plasma (T1/2 > 10 h) than the endogenous apelin-17 peptide 2 (T1/2 < 4 min).

Combinatorial aid for underprivileged scaffolds: solution and solid-phase strategies for a rapid and efficient access to novel aza-diketopiperazines (Aza-DKP).

An efficient solution-phase synthesis of aza-diketopiperazines (aza-DKP, triazinediones) is reported. A structurally diverse collection of c-[aza-alkylGly-Pro] derivatives and yet unreported 2,4,5-trisubstituted-1,2,4-triazine-3,6-diones has been synthesized starting from Fmoc-l-Pro-OH and various Fmoc-l-amino acids. To extend the practical value of this class of dipeptidomimetics, a general solid-phase synthesis approach amenable to library production was developed on both Wang-PS and HMBA-PS resins. The final acidic treatment of the resins in TFA/water mixture at room temperature enabled the rapid and quantitative cyclization/release highly pure triazinediones. The conformational preferences and the spatial organization of the three substituents of a representative 2,4,5-trisubstituted-1,2,4-triazine-3,6-dione were investigated by X-ray diffraction and (1)H NMR spectroscopy.

Solid-phase, multicomponent reactions of methyleneaziridines: synthesis of 1,3-disubstituted propanones.

[reaction: see text] Polymer-supported methyleneaziridines undergo ring opening by Grignard reagents under copper catalysis to yield metalloenamines which are alkylated in situ to yield ketimines. Filtration and washing of these Merrifield resin-bound intermediates prior to hydrolysis provides the corresponding 1,3-disubstituted propanones in a high state of purity without recourse to column chromatography.