Alexandre Murza

Elucidation of the Structure–Activity Relationships of Apelin: Influence of Unnatural Amino Acids on Binding, Signaling, and Plasma Stability

Apelin is the endogenous ligand of the APJ receptor, a member of the G‐protein‐coupled receptor family. The apelin–APJ complex has been detected in many tissues and is emerging as a promising target for several pathophysiological conditions. There is currently little information on the structure–activity relationship (SAR) of the apelin hormone. In an effort to better delineate SAR, we synthesized analogues of apelin‐13 modified at selected positions with unnatural amino acids, with a particular emphasis on the C‐terminal portion. Analogues were then tested in binding and functional assays by evaluating Gi/o‐mediated decreases in cAMP levels and by assessing β‐arrestin2 recruitment to the APJ receptor. The plasma stability of new compounds was also assessed. Several analogues were found to possess increased binding and higher stability than the parent peptide.

Conjugation of a brain-penetrant peptide with neurotensin provides antinociceptive properties

Neurotensin (NT) has emerged as an important modulator of nociceptive transmission and exerts its biological effects through interactions with 2 distinct GPCRs, NTS1 and NTS2. NT provides strong analgesia when administered directly into the brain; however, the blood-brain barrier (BBB) is a major obstacle for effective delivery of potential analgesics to the brain. To overcome this challenge, we synthesized chemical conjugates that are transported across the BBB via receptor-mediated transcytosis using the brain-penetrant peptide Angiopep-2 (An2), which targets LDL receptor-related protein-1 (LRP1). Using in situ brain perfusion in mice, we found that the compound ANG2002, a conjugate of An2 and NT, was transported at least 10 times more efficiently across the BBB than native NT. In vitro, ANG2002 bound NTS1 and NTS2 receptors and maintained NT-associated biological activity. In rats, i.v. ANG2002 induced a dose-dependent analgesia in the formalin model of persistent pain. At a dose of 0.05 mg/kg, ANG2002 effectively reversed pain behaviors induced by the development of neuropathic and bone cancer pain in animal models. The analgesic properties of ANG2002 demonstrated in this study suggest that this compound is effective for clinical management of persistent and chronic pain and establish the benefits of this technology for the development of neurotherapeutics.

Discovery and Structure–Activity Relationship of a Bioactive Fragment of ELABELA that Modulates Vascular and Cardiac Functions

ELABELA (ELA) was recently discovered as a novel endogenous ligand of the apelin receptor (APJ), a G protein-coupled receptor. ELA signaling was demonstrated to be crucial for normal heart and vasculature development during embryogenesis. We delineate here ELAs structure-activity relationships and report the identification of analogue 3 (ELA(19-32)), a fragment of ELA that binds to APJ, activates the Gαi1 and β-arrestin-2 signaling pathways, and induces receptor internalization similarly to its parent endogenous peptide. An alanine scan performed on 3 revealed that the C-terminal residues are critical for binding to APJ and signaling. Finally, using isolated-perfused hearts and in vivo hemodynamic and echocardiographic measurements, we demonstrate that ELA and 3 both reduce arterial pressure and exert positive inotropic effects on the heart. Altogether, these results present ELA and 3 as potential therapeutic options in managing cardiovascular diseases.

C-Terminal modifications of apelin-13 significantly change ligand binding, receptor signaling, and hypotensive action.

Apelin is the endogenous ligand of the APJ receptor, a member of the G protein-coupled receptor family. This system plays an important role in the regulation of blood pressure and cardiovascular functions. To better understand the role of its C-terminal Phe(13) residue on ligand binding, receptor signaling, and hypotension, we report a series of modified analogues in which Phe(13) was substituted by unnatural amino acids. These modifications delivered new compounds exhibiting higher affinity and potency to inhibit cAMP accumulation compared to apelin-13. In particular, analogues Bpa(13) or (α-Me)Phe(13) were 30-fold more potent to inhibit cAMP accumulation than apelin-13. Tyr(OBn)(13) substitution led to a 60-fold improvement in binding affinity and induced stronger and more sustained drop in blood pressure compared to apelin-13. Our study identified new potent analogues of apelin-13, which represent valuable probes to better understand its structure-function relationship.

Stability and degradation patterns of chemically modified analogs of apelin-13 in plasma and cerebrospinal fluid

Apelin is the endogenous ligand of APJ, which belongs to the superfamily of G protein‐coupled receptors. In recent years, the apelin/APJ system has been detected in many tissues and emerges as a promising target for the treatment of various pathophysiological conditions. Pyr1‐apelin‐13 is the major isoform of apelin in human plasma; however its stability and proteolytic degradation pattern remain poorly understood. The aim of the present study was first to identify the cleavage sites of Pyr1‐apelin‐13 in mouse, rat and human plasma and rat cerebrospinal fluid, then to determine its stability to proteolytic degradation following intravenous administration in rats. Secondly, key residues were substituted by natural and unnatural amino acids in order to examine the impact on in vitro stability and degradation pattern. The kinetics of degradation revealed that the Leu5‐Ser6 peptide bond of Pyr1‐apelin‐13 is the first cleavage observed in plasma, independently of the species. Replacement of Phe13 by unnatural amino acids showed a 10‐fold increase in plasma stability although the hydrolysis of Pro12‐Phe13 bond, previously described as a site of cleavage by ACE‐2, was not observed. In vivo, this Pro12‐Phe13 bond was cleaved yet appears as a minor product compared to hydrolysis of the Pro10‐Met11 bond. This study pinpoints the most critical amino acids targeted by proteases and will be instrumental for the design of Pyr1‐apelin‐13 analogs possessing increased stability.

ELABELA Improves Cardio-Renal Outcome in Fatal Experimental Septic Shock

Objectives: Apelin-13 was recently proposed as an alternative to the recommended &bgr;-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

&NA; 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 &bgr;‐arrestins. Using BRET‐based biosensors, we found that neurotensin (NT), NT(8‐13) and neuromedin N (NN) activated the G&agr;q‐, G&agr;i1‐, G&agr;oA‐, and G&agr;13‐protein signaling pathways as well as the recruitment of &bgr;‐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 G&agr;i1 and G&agr;oA, 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 G&agr;s‐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 PLC&bgr;‐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.

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.

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

Graphical abstract Figure. No caption available. ABSTRACT 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 G&agr;i1 and G&agr;oA G‐proteins, recruit &bgr;‐arrestins 1 and 2 (&bgr;arrs), 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 &bgr;arrs. Finally, using Spearman correlations, we established that the hypotensive response was significantly correlated with &bgr;arr recruitment but not with G protein‐dependent signaling. In conclusion, our results demonstrated that the &bgr;arr 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.