Localized apelin-17 analogue-bicelle interactions as a facilitator of membrane-catalyzed receptor recognition and binding.

The apelinergic system encompasses two peptide ligand families, apelin and apela, along with the apelin receptor (AR or APJ), a class A G-protein-coupled receptor. This system has diverse physiological effects, including modulating heart contraction, vasodilation/constriction, glucose regulation, and vascular development, with involvement in a variety of pathological conditions. Apelin peptides have been previously shown to interact with and become structured upon binding to anionic micelles, consistent with a membrane-catalyzed mechanism of ligand-receptor binding. To overcome the challenges of observing nuclear magnetic resonance (NMR) spectroscopy signals of a dilute peptide in biological environments, 19F NMR spectroscopy, including diffusion ordered spectroscopy (DOSY) and saturation transfer difference (STD) experiments, was used herein to explore the membrane-interactive behaviour of apelin. NMR-optimized apelin-17 analogues with 4-trifluoromethyl-phenylalanine at various positions were designed and tested for bioactivity through ERK activation in stably-AR transfected HEK 293 T cells. Far-UV circular dichroism (CD) spectropolarimetry and 19F NMR spectroscopy were used to compare the membrane interactions of these analogues with unlabelled apelin-17 in both zwitterionic/neutral and net-negative bicelle conditions. Each analogue binds to bicelles with relatively weak affinity (i.e., in fast exchange on the NMR timescale), with preferential interactions observed at the cationic residue-rich N-terminal and mid-length regions of the peptide leaving the C-terminal end unencumbered for receptor recognition, enabling a membrane-anchored fly-casting mechanism of peptide search for the receptor. In all, this study provides further insight into the membrane-interactive behaviour of an important bioactive peptide, demonstrating interactions and biophysical behaviour that cannot be neglected in therapeutic design.

Optimization of Ketobenzothiazole-Based Type II Transmembrane Serine Protease Inhibitors to Block H1N1 Influenza Virus Replication.

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.

From garden to lab: C-3 chemical modifications of tomatidine unveil broad-spectrum ATP synthase inhibitors to combat bacterial resistance.

Antibiotic resistance is escalating alarmingly worldwide. Bacterial resistance mechanisms are surfacing and proliferating across the globe, jeopardizing our capacity to manage prevalent infectious illnesses. Without drastic measures, we risk entering a post-antibiotic era, where even trivial infections and injuries can cause death again. In this context, we have developed a new class of antibiotics based on tomatidine (TO), a natural product derived from tomato plants, with a novel mode of action by targeting bacterial ATP synthases. The first generation of compounds proved highly specific for small-colony variants (SCVs) of Staphylococcus aureus. However, optimization of this scaffold through extensive structure-activity relationship studies has enabled us to broaden its effectiveness to include both Gram-positive and Gram-negative bacteria. Notably, the results showed that specific C3-modification of TO could improve ATP synthase inhibition and also bypass the outer membrane barrier of Gram-negative bacteria to gain substantial growth inhibition including against multi-resistant strains.

Signaling Modulation via Minimal C-Terminal Modifications of Apelin-13.

Apelin is an endogenous peptide that is involved in many diseases such as cardiovascular diseases, obesity, and cancer, which has made it an attractive target for drug discovery. Herein, we explore the penultimate and final sequence positions of [Pyr1]-apelin-13 (Ape13) via C-terminal N α-alkylated amide bonds and the introduction of positive charges, potentially targeting the allosteric sodium pocket, by assessing the binding affinity and signaling profiles at the apelin receptor (APJ). Synthetic analogues modified within this segment of Ape13 showed high affinity (K i 0.12-0.17 nM vs Ape13 K i 0.7 nM), potent Gαi1 activation (EC50 Gαi1 0.4-0.9 nM vs Ape13 EC50 1.1 nM), partial agonist behavior disfavoring ß-arrestin 2 recruitment for positively charged ligands (e.g., 49 (SBL-AP-058), EC50 ß-arr2 275 nM, E max 54%) and high plasma stability for N-alkyl ligands (t 1/2 > 7 h vs Ape13 t 1/2 0.5 h). Combining the benefits of the N α-alkylated amide bond with the guanidino substitution in a constrained ligand led to 63 (SBL-AP-049), which displayed increased plasma stability (t 1/2 5.3 h) and strong reduction of ß-arrestin 2 signaling with partial maximal efficacy (EC50 ß-arr 864 nM, E max 48%), significantly reducing the hypotensive effect in vivo.

Comparative Analysis of Cyclization Techniques in Stapled Peptides: Structural Insights into Protein-Protein Interactions in a SARS-CoV-2 Spike RBD/hACE2 Model System.

Medicinal chemistry is constantly searching for new approaches to develop more effective and targeted therapeutic molecules. The design of peptidomimetics is a promising emerging strategy that is aimed at developing peptides that mimic or modulate the biological activity of proteins. Among these, stapled peptides stand out for their unique ability to stabilize highly frequent helical motifs, but they have failed to be systematically reported. Here, we exploit chemically diverse helix-inducing i, i + 4 constraints-lactam, hydrocarbon, triazole, double triazole and thioether-on two distinct short sequences derived from the N-terminal peptidase domain of hACE2 upon structural characterization and in silico alanine scan. Our overall objective was to provide a sequence-independent comparison of α-helix-inducing staples using circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. We identified a 9-mer lactam stapled peptide derived from the hACE2 sequence (His34-Gln42) capable of reaching its maximal helicity of 55% with antiviral activity in bioreporter- and pseudovirus-based inhibition assays. To the best of our knowledge, this study is the first comprehensive investigation comparing several cyclization methods with the goal of generating stapled peptides and correlating their secondary structures with PPI inhibitions using a highly topical model system (i.e., the interaction of SARS-CoV-2 Spike RBD with hACE2).

Size-Reduced Macrocyclic Analogues of [Pyr1]-apelin-13 Showing Negative Gα12 Bias Still Produce Prolonged Cardiac Effects.

We previously reported a series of macrocyclic analogues of [Pyr1]-apelin-13 (Ape13) with increased plasma stability and potent APJ agonist properties. Based on the most promising compound in this series, we synthesized and then evaluated novel macrocyclic compounds of Ape13 to identify agonists with specific pharmacological profiles. These efforts led to the development of analogues 39 and 40, which possess reduced molecular weight (MW 1020 Da vs Ape13, 1534 Da). Interestingly, compound 39 (Ki 0.6 nM), which does not activate the Gα12 signaling pathway while maintaining potency and efficacy similar to Ape13 to activate Gαi1 (EC50 0.8 nM) and ß-arrestin2 recruitment (EC50 31 nM), still exerts cardiac actions. In addition, analogue 40 (Ki 5.6 nM), exhibiting a favorable Gα12-biased signaling and an increased in vivo half-life (t1/2 3.7 h vs <1 min of Ape13), produces a sustained cardiac response up to 6 h after a single subcutaneous bolus injection.

Constraining the Side Chain of C-Terminal Amino Acids in Apelin-13 Greatly Increases Affinity, Modulates Signaling, and Improves the Pharmacokinetic Profile.

Side-chain-constrained amino acids are useful tools to modulate the biological properties of peptides. In this study, we applied side-chain constraints to apelin-13 (Ape13) by substituting the Pro12 and Phe13 positions, affecting the binding affinity and signaling profile on the apelin receptor (APJ). The residues 1Nal, Trp, and Aia were found to be beneficial substitutions for Pro12, and the resulting analogues displayed high affinity for APJ (Ki 0.08-0.18 nM vs Ape13 Ki 0.7 nM). Besides, constrained (d-Tic) or α,α-disubstituted residues (Dbzg; d-α-Me-Tyr(OBn)) were favorable for the Phe13 position. Compounds 47 (Pro12-Phe13 replaced by Aia-Phe, Ki 0.08 nM) and 53 (Pro12-Phe13 replaced by 1Nal-Dbzg, Ki 0.08 nM) are the most potent Ape13 analogues activating the Gα12 pathways (53, EC50 Gα12 2.8 nM vs Ape13, EC50 43 nM) known to date, displaying high affinity, resistance to ACE2 cleavage as well as improved pharmacokinetics in vitro (t1/2 5.8-7.3 h in rat plasma) and in vivo.

Gαi-biased apelin analog protects against isoproterenol-induced myocardial dysfunction in rats.

Apelin receptor (APJ) activation by apelin-13 (APLN-13) engages both Gαi proteins and ß-arrestins, stimulating distinct intracellular pathways and triggering physiological responses like enhanced cardiac contractility. Substituting the C-terminal phenylalanine of APLN-13 with α-methyl-l-phenylalanine [(l-α-Me)Phe] or p-benzoyl-l-phenylalanine (Bpa) generates biased analogs inducing APJ functional selectivity toward Gαi proteins. Using these original analogs, we proposed to investigate how the canonical Gαi signaling of APJ regulates the cardiac function and to assess their therapeutic impact in a rat model of isoproterenol-induced myocardial dysfunction. In vivo and ex vivo infusions of either Bpa or (l-α-Me)Phe analogs failed to enhance rats' left ventricular (LV) contractility compared with APLN-13. Inhibition of Gαi with pertussis toxin injection optimized the cardiotropic effect of APLN-13 and revealed the inotropic impact of Bpa. Moreover, both APLN-13 and Bpa efficiently limited the forskolin-induced and PKA-dependent phosphorylation of phospholamban at the Ser16 in neonatal rat ventricular myocytes. However, only Bpa significantly reduced the inotropic effect of forskolin infusion in isolated-perfused heart, highlighting its efficient bias toward Gαi. Compared with APLN-13, Bpa also markedly improved isoproterenol-induced myocardial systolic and diastolic dysfunctions. Bpa prevented cardiac weight increase, normalized both ANP and BNP mRNA expressions, and decreased LV fibrosis in isoproterenol-treated rats. Our results show that APJ-driven Gαi/adenylyl cyclase signaling is functional in cardiomyocytes and acts as negative feedback of the APLN-APJ-dependent inotropic response. Biased APJ signaling toward Gαi over the ß-arrestin pathway offers a promising strategy in the treatment of cardiovascular diseases related to myocardial hypertrophy and high catecholamine levels.NEW & NOTEWORTHY By using more potent Gαi-biased APJ agonists that strongly inhibit cAMP production, these data point to the negative inotropic effect of APJ-mediated Gαi signaling in the heart and highlight the potential protective impact of APJ-dependent Gαi signaling in cardiovascular diseases associated with left ventricular hypertrophy.

Structure-Activity Relationship and Bioactivity of Short Analogues of ELABELA as Agonists of the Apelin Receptor.

ELABELA (ELA) is the second endogenous ligand of the apelin receptor (APJ). Although apelin-13 and ELA both target APJ, there is limited information on structure-activity relationship (SAR) of ELA. In the present work, we identified the shortest bioactive C-terminal fragment ELA23-32, which possesses high affinity for APJ (Ki 4.6 nM) and produces cardiorenal effects in vivo similar to those of ELA. SAR studies on conserved residues (Leu25, His26, Val29, Pro30, Phe31, Pro32) show that ELA and apelin-13 may interact differently with APJ. His26 and Val29 emerge as important for ELA binding. Docking and binding experiments suggest that Phe31 of ELA may bind to a tight groove distinct from that of Phe13 of Ape13, while the Phe13 pocket may be occupied by Pro32 of ELA. Further characterization of signaling profiles on the Gαi1, Gα12, and ß-arrestin2 pathways reveals the importance of aromatic residue at the Phe31 or Pro32 position for receptor activation.

Inhibitors of type II transmembrane serine proteases in the treatment of diseases of the respiratory tract - A review of patent literature.

INTRODUCTION: Type II transmembrane serine proteases (TTSPs) of the human respiratory tract generate high interest owing to their ability, among other roles, to cleave surface proteins of respiratory viruses. This step is critical in the viral invasion of coronaviruses, including SARS-CoV-2 responsible for COVID-19, but also influenza viruses and reoviruses. Accordingly, these cell surface enzymes constitute appealing therapeutic targets to develop host-based therapeutics against respiratory viral diseases. Additionally, their deregulated levels or activity has been described in non-viral diseases such as fibrosis, cancer, and osteoarthritis, making them potential targets in these indications. AREAS COVERED: Areas covered: This review includes WIPO-listed patents reporting small molecules and peptide-based inhibitors of type II transmembrane serine proteases of the respiratory tract. EXPERT OPINION: Expert opinion: Several TTSPs of the respiratory tract represent attractive pharmacological targets in the treatment of respiratory infectious diseases (notably COVID-19 and influenza), but also against idiopathic pulmonary fibrosis and lung cancer. The current emphasis is primarily on TMPRSS2, matriptase, and hepsin, yet other TTSPs await validation. Compounds listed herein are predominantly peptidomimetic inhibitors, some with covalent reversible mechanisms of action and high potencies. Their selectivity profile, however, are often only partially characterized. Preclinical data are promising and warrant further advancement in the above diseases.

Data set describing the in vitro biological activity of JMV2009, a novel silylated neurotensin(8-13) analog.

Neurotensin (NT) is a tridecapeptide displaying interesting antinociceptive properties through its action on its receptors, NTS1 and NTS2. Neurotensin-like compounds have been shown to exert better antinociceptive properties than morphine at equimolar doses. In this article, we characterized the molecular effects of a novel neurotensin (8-13) (NT(8-13)) analog containing an unnatural amino acid. This compound, named JMV2009, displays a Silaproline in position 10 in replacement of a proline in the native NT(8-13). We first examined the binding affinities of this novel NT(8-13) derivative at both NTS1 and NTS2 receptor sites by performing competitive displacement of iodinated NT on purified cell membranes. Then, we evaluated the ability of JMV2009 to activate NTS1-related G proteins as well as to promote the recruitment of ß-arrestins 1 and 2 by using BRET-based cellular assays in live cells. We next assessed its ability to induce p42/p44 MAPK phosphorylation and NT receptors internalization using western blot and cell-surface ELISA, respectively. Finally, we determined the in vitro plasma stability of this NT derivative. This article is associated with the original article "Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog" published in European Journal of Pharmacology[1]. The reader is directed to the associated article for results interpretation, comments, and discussion.

Pain relief devoid of opioid side effects following central action of a silylated neurotensin analog.

Neurotensin (NT) exerts naloxone-insensitive antinociceptive action through its binding to both NTS1 and NTS2 receptors and NT analogs provide stronger pain relief than morphine on a molecular basis. Here, we examined the analgesic/adverse effect profile of a new NT(8-13) derivative denoted JMV2009, in which the Pro10 residue was substituted by a silicon-containing unnatural amino acid silaproline. We first report the synthesis and in vitro characterization (receptor-binding affinity, functional activity and stability) of JMV2009. We next examined its analgesic activity in a battery of acute, tonic and chronic pain models. We finally evaluated its ability to induce adverse effects associated with chronic opioid use, such as constipation and analgesic tolerance or related to NTS1 activation, like hypothermia. In in vitro assays, JMV2009 exhibited high binding affinity for both NTS1 and NTS2, improved proteolytic resistance as well as agonistic activities similar to NT, inducing sustained activation of p42/p44 MAPK and receptor internalization. Intrathecal injection of JMV2009 produced dose-dependent antinociceptive responses in the tail-flick test and almost completely abolished the nociceptive-related behaviors induced by chemical somatic and visceral noxious stimuli. Likewise, increasing doses of JMV2009 significantly reduced tactile allodynia and weight bearing deficits in nerve-injured rats. Importantly, repeated agonist treatment did not result in the development of analgesic tolerance. Furthermore, JMV2009 did not cause constipation and was ineffective in inducing hypothermia. These findings suggest that NT drugs can act as an effective opioid-free medication for the management of pain or can serve as adjuvant analgesics to reduce the opioid adverse effects.

Simultaneous Ligand and Receptor Tracking through NMR Spectroscopy Enabled by Distinct 19F Labels.

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.

The hypotensive effect of activated apelin receptor is correlated with ß-arrestin recruitment.

The apelinergic system is an important player in the regulation of both vascular tone and cardiovascular function, making this physiological system an attractive target for drug development for hypertension, heart failure and ischemic heart disease. Indeed, apelin exerts a positive inotropic effect in humans whilst reducing peripheral vascular resistance. In this study, we investigated the signaling pathways through which apelin exerts its hypotensive action. We synthesized a series of apelin-13 analogs whereby the C-terminal Phe13 residue was replaced by natural or unnatural amino acids. In HEK293 cells expressing APJ, we evaluated the relative efficacy of these compounds to activate Gαi1 and GαoA G-proteins, recruit ß-arrestins 1 and 2 (ßarrs), and inhibit cAMP production. Calculating the transduction ratio for each pathway allowed us to identify several analogs with distinct signaling profiles. Furthermore, we found that these analogs delivered i.v. to Sprague-Dawley rats exerted a wide range of hypotensive responses. Indeed, two compounds lost their ability to lower blood pressure, while other analogs significantly reduced blood pressure as apelin-13. Interestingly, analogs that did not lower blood pressure were less effective at recruiting ßarrs. Finally, using Spearman correlations, we established that the hypotensive response was significantly correlated with ßarr recruitment but not with G protein-dependent signaling. In conclusion, our results demonstrated that the ßarr recruitment potency is involved in the hypotensive efficacy of activated APJ.

A Systematic Exploration of Macrocyclization in Apelin-13: Impact on Binding, Signaling, Stability, and Cardiovascular Effects.

The apelin receptor generates increasing interest as a potential target across several cardiovascular indications. However, the short half-life of its cognate ligands, the apelin peptides, is a limiting factor for pharmacological use. In this study, we systematically explored each position of apelin-13 to find the best position to cyclize the peptide, with the goal to improve its stability while optimizing its binding affinity and signaling profile. Macrocyclic analogues showed a remarkably higher stability in rat plasma (half-life >3 h versus 24 min for Pyr-apelin-13), accompanied by improved affinity (analogue 15, Ki 0.15 nM and t1/2 6.8 h). Several compounds displayed higher inotropic effects ex vivo in the Langendorff isolated heart model in rats (analogues 13 and 15, maximum response at 0.003 nM versus 0.03 nM of apelin-13). In conclusion, this study provides stable and active compounds to better characterize the pharmacology of the apelinergic system.

ELABELA Improves Cardio-Renal Outcome in Fatal Experimental Septic Shock.

OBJECTIVES: Apelin-13 was recently proposed as an alternative to the recommended ß-adrenergic drugs for supporting endotoxin-induced myocardial dysfunction. Since Apelin-13 signals through its receptor (Apelin peptide jejunum) to exert singular inotropic/vasotropic actions and to optimize body fluid balance, this candidate pathway might benefit septic shock management. Whether the newly discovered ELABELA (ELA), a second endogenous ligand of the Apelin peptide jejunum receptor highly expressed in the kidney, further improves cardio-renal impairment remains unknown. DESIGN, SETTING, AND SUBJECTS: Interventional study in a rat model of septic shock (128 adult males) to assess the effects of ELA and Apelin-13 on vascular and cardio-renal function. Experiments were performed in a tertiary care University-based research institute. INTERVENTIONS: Polymicrobial sepsis-induced cardiac dysfunction was produced by cecal ligation puncture to assess hemodynamic efficacy, cardioprotection, and biomechanics under acute or continuous infusions of the apelinergic agonists ELA or Apelin-13 (39 and 15 µg/kg/hr, respectively) versus normal saline. MEASUREMENTS AND MAIN RESULTS: Apelinergic agonists improved 72-hour survival after sepsis induction, with ELA providing the best clinical outcome after 24 hours. Apelinergic agonist infusion counteracted cecal ligation puncture-induced myocardial dysfunction by improving left ventricular pressure-volume relationship. ELA-treated cecal ligation puncture rats were the only group to 1) display a significant improvement in left ventricular filling as shown by increased E-wave velocity and left ventricular end-diastolic volume, 2) exhibit a higher plasma volume, and 3) limit kidney injury and free-water clearance. These beneficial renal effects were superior to Apelin-13, likely because full-length ELA enabled a distinctive regulation of pituitary vasopressin release. CONCLUSIONS: Activation of the apelinergic system by exogenous ELA or Apelin-13 infusion improves cardiovascular function and survival after cecal ligation puncture-induced sepsis. However, ELA proved better than Apelin-13 by improving fluid homeostasis, cardiovascular hemodynamics recovery, and limiting kidney dysfunction in a vasopressinergic-dependent manner.

The signaling signature of the neurotensin type 1 receptor with endogenous ligands.

The human neurotensin 1 receptor (hNTS1) is a G protein-coupled receptor involved in many physiological functions, including analgesia, hypothermia, and hypotension. To gain a better understanding of which signaling pathways or combination of pathways are linked to NTS1 activation and function, we investigated the ability of activated hNTS1, which was stably expressed by CHO-K1 cells, to directly engage G proteins, activate second messenger cascades and recruit ß-arrestins. Using BRET-based biosensors, we found that neurotensin (NT), NT(8-13) and neuromedin N (NN) activated the Gαq-, Gαi1-, GαoA-, and Gα13-protein signaling pathways as well as the recruitment of ß-arrestins 1 and 2. Using pharmacological inhibitors, we further demonstrated that all three ligands stimulated the production of inositol phosphate and modulation of cAMP accumulation along with ERK1/2 activation. Interestingly, despite the functional coupling to Gαi1 and GαoA, NT was found to produce higher levels of cAMP in the presence of pertussis toxin, supporting that hNTS1 activation leads to cAMP accumulation in a Gαs-dependent manner. Additionally, we demonstrated that the full activation of ERK1/2 required signaling through both a PTX-sensitive Gi/o-c-Src signaling pathway and PLCß-DAG-PKC-Raf-1-dependent pathway downstream of Gq. Finally, the whole-cell integrated signatures monitored by the cell-based surface plasmon resonance and changes in the electrical impedance of a confluent cell monolayer led to identical phenotypic responses between the three ligands. The characterization of the hNTS1-mediated cellular signaling network will be helpful to accelerate the validation of potential NTS1 biased ligands with an improved therapeutic/adverse effect profile.

Structure-activity relationship of novel macrocyclic biased apelin receptor agonists.

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 ß-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.

Identification of 2-({[1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]carbonyl}amino)tricyclo[3.3.1.13,7]decane-2-carboxylic Acid (NTRC-844) as a Selective Antagonist for the Rat Neurotensin Receptor Type 2.

Neurotensin receptor type 2 (NTS2) compounds display analgesic activity in animal pain models. We have identified the first high-affinity NTS2-selective antagonist (8) that is active in vivo. This study also revealed that the NTS2 FLIPR assay designation for a compound, agonist, partial agonist, and so forth, did not correlate with its in vivo activity as observed in the thermal tail-flick acute model of pain. This suggests that calcium mobilization is not the signaling pathway involved in NTS2-mediated analgesia as assessed by the thermal tail-flick model. Finally, we found a significant bias between rat and human for compound 9 in the NTS2 binding assay.

Intracuff 160 mg alkalinized lidocaine reduces cough upon emergence from N2O-free general anesthesia: a randomized controlled trial.

PURPOSE: Chemical and mechanical irritation of the tracheal mucosa influences the incidence of cough at emergence from general anesthesia, potentially leading to significant postoperative complications. This study evaluates the benefits of endotracheal tube (ETT) intracuff alkalinized lidocaine during N2O-free general anesthesia by 1) assessing the in vitro effect of alkalinization on lidocaine diffusion kinetics across the cuff's membrane and 2) evaluating, in a randomized controlled clinical trial, the impact of 160 mg of intracuff alkalinized lidocaine on cough upon emergence from anesthesia for surgery lasting > 120 min. METHODS: In the in vitro study, diffusion kinetics of various intracuff alkalinized lidocaine amounts (40, 80, and 160 mg) were compared to their non-alkalinized lidocaine controls. In the clinical trial, 80 adult patients (American Society of Anesthesiologists physical status I-III) undergoing urological or gynecological surgery expected to last > 120 min and scheduled for N2O-free general anesthesia were enrolled. The ETT cuffs (high-volume, low-pressure) were filled with either 160 mg of alkalinized lidocaine or a comparable volume of 0.9% saline. The primary outcome was the incidence of cough upon emergence from anesthesia. Sore throat, hoarseness, and postoperative nausea and vomiting were evaluated as secondary outcomes. RESULTS: Our in vitro study confirmed that alkalinization increases lidocaine diffusion across the membrane of ETT cuffs and suggested that the lidocaine diffusion rate is associated with the initial intracuff lidocaine quantity. Our clinical trial demonstrated that, compared with the saline group, 160 mg of intracuff alkalinized lidocaine reduced the incidence of cough upon emergence from N2O-free general anesthesia (76% vs 34%, respectively; difference 42%; 95% confidence interval, 21% to 62%; P < 0.001) while having no clinical impact on secondary outcomes. CONCLUSIONS: The use of 160 mg of intracuff alkalinized lidocaine is associated with a decreased incidence of cough upon emergence from N2O-free general anesthesia > 120 min. This trial was registered at www.clinicaltrials.gov (NCT01774292).