Design, Synthesis, and In Vitro Characterization of Proteolytically-Stable Opioid-Neurotensin Hybrid Peptidomimetics.

Linking an opioid to a nonopioid pharmacophore represents a promising approach for reducing opioid-induced side effects during pain management. Herein, we describe the optimization of the previously reported opioid-neurotensin hybrids (OPNT-hybrids), SBL-OPNT-05 & -10, containing the µ-/δ-opioid agonist H-Dmt-d-Arg-Aba-ß-Ala-NH2 and NT(8-13) analogs optimized for NTS2 affinity. In the present work, the constrained dipeptide Aba-ß-Ala was modified to investigate the optimal linker length between the two pharmacophores, as well as the effect of expanding the aromatic moiety within constrained dipeptide analogs, via the inclusion of a naphthyl moiety. Additionally, the N-terminal Arg residue of the NT(8-13) pharmacophore was substituted with ß3 hArg. For all analogs, affinity was determined at the MOP, DOP, NTS1, and NTS2 receptors. Several of the hybrid ligands showed a subnanomolar affinity for MOP, improved binding for DOP compared to SBL-OPNT-05 & -10, as well as an excellent NTS2-affinity with high selectivity over NTS1. Subsequently, the Gαi1 and ß-arrestin-2 pathways were evaluated for all hybrids, along with their stability in rat plasma. Upon MOP activation, SBL-OPNT-13 and -18 were the least effective at recruiting ß-arrestin-2 (E max = 17 and 12%, respectively), while both compounds were also found to be partial agonists at the Gαi1 pathway, despite improved potency compared to DAMGO. Importantly, these analogs also showed a half-life in rat plasma in excess of 48 h, making them valuable tools for future in vivo investigations.

Multitarget ligands that comprise opioid/nonopioid pharmacophores for pain management: Current state of the science.

Chronic pain, which affects more than one-third of the world's population, represents one of the greatest medical challenges of the 21st century, yet its effective management remains sub-optimal. The 'gold standard' for the treatment of moderate to severe pain consists of opioid ligands, such as morphine and fentanyl, that target the µ-opioid receptor (MOP). Paradoxically, these opioids also cause serious side effects, including constipation, respiratory depression, tolerance, and addiction. In addition, the development of opioid-use disorders, such as opioid diversion, misuse, and abuse, has led to the current opioid crisis, with dramatic increases in addiction, overdoses, and ultimately deaths. As pain is a complex, multidimensional experience involving a variety of pathways and mediators, dual or multitarget ligands that can bind to more than one receptor and exert complementary analgesic effects, represent a promising avenue for pain relief. Indeed, unlike monomodal therapeutic approaches, the modulation of several endogenous nociceptive systems can often result in an additive or even synergistic effect, thereby improving the analgesic-to-side-effect ratio. Here, we provide a comprehensive overview of research efforts towards the development of dual- or multi-targeting opioid/nonopioid hybrid ligands for effective and safer pain management. We reflect on the underpinning discovery rationale by discussing the design, medicinal chemistry, and in vivo pharmacological effects of multitarget antinociceptive compounds.

Plasma endocannabinoids are independently associated with the metabolic function of white adipose tissue.

CONTEXT: Little is known about the link between the endocannabinoid system and the in vivo metabolic function of white adipose tissue (WAT). OBJECTIVE: We aimed to evaluate whether endocannabinoids (EC) are linked to postprandial fatty acid metabolism and WAT metabolic function. DESIGN: Men and women, with (IGT, n=20) or without impaired glucose tolerance (NGT, n=20) underwent meal testing with oral and intravenous stable isotope palmitate tracers and positron emission tomography with intravenous [11C]-palmitate and oral [18F]-fluoro-thia-heptadecanoic acid to determine systemic and organ-specific dietary fatty acid (DFA) and non-esterified fatty acid (NEFA) metabolism and partitioning. We determined fasting and postprandial plasma levels of EC by UHPLC-MS/MS. RESULTS: All EC of the 2-monoacylglycerol (2-MAG) family displayed a progressive postprandial increase up to 360 min after meal intake that was more pronounced in women with IGT. N-acylethanolamine (NAE) levels decreased between fasting and 180 min, followed by a return to pre-prandial values at 360 min and were also increased in women with IGT. Postprandial area under the curve (AUC) of palmitate appearance rate was significantly and independently associated with postprandial AUC of anandamide (AEA; P=0.0003) and total energy expenditure (P=0.0009). DFA storage in abdominal subcutaneous adipose tissue was positively predicted by fasting 2-arachidonoylglycerol (2-AG; P<0.04). CONCLUSION: EC levels of the NAE family independently follow plasma NEFA metabolism, whereas 2-MAG closely follow the spillover of triglyceride-rich lipoprotein intravascular lipolytic products. Whether these associations are causal requires further investigation.

Development of Macrocyclic Neurotensin Receptor Type 2 (NTS2) Opioid-Free Analgesics.

The opioid crisis has highlighted the urgent need to develop non-opioid alternatives for managing pain, with an effective, safe, and non-addictive pharmacotherapeutic profile. Using an extensive structure-activity relationship approach, here we have identified a new series of highly selective neurotensin receptor type 2 (NTS2) macrocyclic compounds that exert potent, opioid-independent analgesia in various experimental pain models. To our knowledge, the constrained macrocycle in which the Ile12 residue of NT(7-12) was substituted by cyclopentylalanine, Pro7 and Pro10 were replaced by allyl-glycine followed by side-chain to side-chain cyclization is the most selective analog targeting NTS2 identified to date (Ki 2.9 nM), showing 30,000-fold selectivity over NTS1. Of particular importance, this macrocyclic analog is also able to potentiate the analgesic effects of morphine in a dose- and time-dependent manner. Exerting complementary analgesic actions via distinct mechanisms of nociceptive transmission, NTS2-selective macrocycles can therefore be exploited as opioid-free analgesics or as opioid-sparing therapeutics, offering superior pain relief with reduced adverse effects to pain patients.

Rational In Silico Design of Selective TMPRSS6 Peptidomimetic Inhibitors via Exploitation of the S2 Subpocket.

TMPRSS6 is a potential therapeutic target for the treatment of iron overload due to its role in regulating levels of hepcidin. Although potent TMPRSS6 inhibitors have been previously developed, their lack of specificity requires optimization to avoid potential side effects before pursuing preclinical development with in vivo models. Here, using computer-aided drug design based on a TMPRSS6 homology model, we reveal that the S2 position of TMPRSS6 offers a potential avenue to achieve selectivity against other members of the TTSP family. Accordingly, we synthesized novel peptidomimetic molecules containing lipophilic amino acids at the P2 position to exploit this unexplored pocket. This enabled us to identify TMPRSS6-selective small molecules with low nanomolar affinity. Finally, pharmacokinetic parameters were determined, and a compound was found to be potent in cellulo toward its primary target while retaining TTSP-subtype selectivity and showing no signs of alteration in in vitro TEER experiments.

Discovery of a CCR2-targeting pepducin therapy for chronic pain.

Targeting the CCL2/CCR2 chemokine axis has been shown to be effective at relieving pain in rodent models of inflammatory and neuropathic pain, therefore representing a promising avenue for the development of non-opioid analgesics. However, clinical trials targeting this receptor for inflammatory conditions and painful neuropathies have failed to meet expectations and have all been discontinued due to lack of efficacy. To overcome the poor selectivity of CCR2 chemokine receptor antagonists, we generated and characterized the function of intracellular cell-penetrating allosteric modulators targeting CCR2, namely pepducins. In vivo, chronic intrathecal administration of the CCR2-selective pepducin PP101 was effective in alleviating neuropathic and bone cancer pain. In the setting of bone metastases, we found that T cells infiltrate dorsal root ganglia (DRG) and induce long-lasting pain hypersensitivity. By acting on CCR2-expressing DRG neurons, PP101 attenuated the altered phenotype of sensory neurons as well as the neuroinflammatory milieu of DRGs, and reduced bone cancer pain by blocking CD4+ and CD8+ T cell infiltration. Notably, PP101 demonstrated its efficacy in targeting the neuropathic component of bone cancer pain, as evidenced by its anti-nociceptive effects in a model of chronic constriction injury of the sciatic nerve. Importantly, PP101-induced reduction of CCR2 signaling in DRGs did not result in deleterious tumor progression or adverse behavioral effects. Thus, targeting neuroimmune crosstalk through allosteric inhibition of CCR2 could represent an effective and safe avenue for the management of chronic pain.

Dopamine-sensitive neurons in the mesencephalic locomotor region control locomotion initiation, stop, and turns.

The locomotor role of dopaminergic neurons is traditionally attributed to their ascending projections to the basal ganglia, which project to the mesencephalic locomotor region (MLR). In addition, descending dopaminergic projections to the MLR are present from basal vertebrates to mammals. However, the neurons targeted in the MLR and their behavioral role are unknown in mammals. Here, we identify genetically defined MLR cells that express D1 or D2 receptors and control different motor behaviors in mice. In the cuneiform nucleus, D1-expressing neurons promote locomotion, while D2-expressing neurons stop locomotion. In the pedunculopontine nucleus, D1-expressing neurons promote locomotion, while D2-expressing neurons evoke ipsilateral turns. Using RNAscope, we show that MLR dopamine-sensitive neurons comprise a combination of glutamatergic, GABAergic, and cholinergic neurons, suggesting that different neurotransmitter-based cell types work together to control distinct behavioral modules. Altogether, our study uncovers behaviorally relevant cell types in the mammalian MLR based on the expression of dopaminergic receptors.

Apelin stimulation of the vascular skeletal muscle stem cell niche enhances endogenous repair in dystrophic mice.

Impaired skeletal muscle stem cell (MuSC) function has long been suspected to contribute to the pathogenesis of muscular dystrophy (MD). Here, we showed that defects in the endothelial cell (EC) compartment of the vascular stem cell niche in mouse models of Duchenne MD, laminin α2-related MD, and collagen VI-related myopathy were associated with inefficient mobilization of MuSCs after tissue damage. Using chemoinformatic analysis, we identified the 13-amino acid form of the peptide hormone apelin (AP-13) as a candidate for systemic stimulation of skeletal muscle ECs. Systemic administration of AP-13 using osmotic pumps generated a pro-proliferative EC-rich niche that supported MuSC function through angiocrine factors and markedly improved tissue regeneration and muscle strength in all three dystrophic mouse models. Moreover, EC-specific knockout of the apelin receptor led to regenerative defects that phenocopied key pathological features of MD, including vascular defects, fibrosis, muscle fiber necrosis, impaired MuSC function, and reduced force generation. Together, these studies provide in vivo proof of concept that enhancing endogenous skeletal muscle repair by targeting the vascular niche is a viable therapeutic avenue for MD and characterized AP-13 as a candidate for further study for the systemic treatment of MuSC dysfunction.

Ligand-dependent intracellular trafficking of the G protein-coupled P2Y6 receptor.

Endosomal trafficking is intricately linked to G protein-coupled receptors (GPCR) fate and signaling. Extracellular uridine diphosphate (UDP) acts as a signaling molecule by selectively activating the GPCR P2Y6. Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y6 receptors in response to its endogenous agonist UDP and synthetic selective agonist 5-iodo-UDP (MRS2693). Confocal microscopy and cell surface ELISA revealed delayed internalization kinetics in response to MRS2693 vs. UDP stimulation in AD293 and HCT116 cells expressing human P2Y6. Interestingly, UDP induced clathrin-dependent P2Y6 internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y6 was associated with Rab4, 5, and 7 positive vesicles independent of the agonist. We have measured a higher frequency of receptor expression co-occurrence with Rab11-vesicles, the trans-Golgi network, and lysosomes in response to MRS2693. Interestingly, a higher agonist concentration reversed the delayed P2Y6 internalization and recycling kinetics in the presence of MRS2693 stimulation without changing its caveolin-dependent internalization. This work showed a ligand-dependent effect affecting the P2Y6 receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y6 signaling.

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.

Modulating the activity of human nociceptors with a SCN10A promoter-specific viral vector tool.

Despite the high prevalence of chronic pain as a disease in our society, there is a lack of effective treatment options for patients living with this condition. Gene therapies using recombinant AAVs are a direct method to selectively express genes of interest in target cells with the potential of, in the case of nociceptors, reducing neuronal firing in pain conditions. We designed a recombinant AAV vector expressing cargos whose expression was driven by a portion of the SCN10A (NaV1.8) promoter, which is predominantly active in nociceptors. We validated its specificity for nociceptors in mouse and human dorsal root ganglia and showed that it can drive the expression of functional proteins. Our viral vector and promoter package drove the expression of both excitatory or inhibitory DREADDs in primary human DRG cultures and in whole cell electrophysiology experiments, increased or decreased neuronal firing, respectively. Taken together, we present a novel viral tool that drives expression of cargo specifically in human nociceptors. This will allow for future specific studies of human nociceptor properties as well as pave the way for potential future gene therapies for chronic pain.

Opening the amino acid toolbox for peptide-based NTS2-selective ligands as promising lead compounds for pain management.

Chronic pain is one of the most critical health issues worldwide. Despite considerable efforts to find therapeutic alternatives, opioid drugs remain the gold standard for pain management. The administration of µ-opioid receptor (MOR) agonists is associated with detrimental and limiting adverse effects. Overall, these adverse effects strongly overshadow the effectiveness of opioid therapy. In this context, the development of neurotensin (NT) ligands has shown to be a promising approach for the management of chronic and acute pain. NT exerts its opioid-independent analgesic effects through the binding of two G protein-coupled receptors (GPCRs), NTS1 and NTS2. In the last decades, modified NT analogues have been proven to provide potent analgesia in vivo. However, selective NTS1 and nonselective NTS1/NTS2 ligands cause antinociception associated with hypothermia and hypotension, whereas selective NTS2 ligands induce analgesia without altering the body temperature and blood pressure. In light of this, various structure-activity relationship (SAR) studies provided findings addressing the binding affinity of ligands towards NTS2. Herein, we comprehensively review peptide-based NTS2-selective ligands as a robust alternative for future pain management. Particular emphasis is placed on SAR studies governing the desired selectivity and associated in vivo results.

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).

Structural insight into apelin receptor-G protein stoichiometry.

The technique of cryogenic-electron microscopy (cryo-EM) has revolutionized the field of membrane protein structure and function with a focus on the dominantly observed molecular species. This report describes the structural characterization of a fully active human apelin receptor (APJR) complexed with heterotrimeric G protein observed in both 2:1 and 1:1 stoichiometric ratios. We use cryo-EM single-particle analysis to determine the structural details of both species from the same sample preparation. Protein preparations, in the presence of the endogenous peptide ligand ELA or a synthetic small molecule, both demonstrate these mixed stoichiometric states. Structural differences in G protein engagement between dimeric and monomeric APJR suggest a role for the stoichiometry of G protein-coupled receptor- (GPCR-)G protein coupling on downstream signaling and receptor pharmacology. Furthermore, a small, hydrophobic dimer interface provides a starting framework for additional class A GPCR dimerization studies. Together, these findings uncover a mechanism of versatile regulation through oligomerization by which GPCRs can modulate their signaling.

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.

Apelin-13 in septic shock: effective in supporting hemodynamics in sheep but compromised by enzymatic breakdown in patients.

Sepsis is a prevalent life-threatening condition related to a systemic infection, and with unresolved issues including refractory septic shock and organ failures. Endogenously released catecholamines are often inefficient to maintain blood pressure, and low reactivity to exogenous catecholamines with risk of sympathetic overstimulation is well documented in septic shock. In this context, apelinergics are efficient and safe inotrope and vasoregulator in rodents. However, their utility in a larger animal model as well as the limitations with regards to the enzymatic breakdown during sepsis, need to be investigated. The therapeutic potential and degradation of apelinergics in sepsis were tested experimentally and in a cohort of patients. (1) 36 sheep with or without fecal peritonitis-induced septic shock (a large animal experimental design aimed to mimic the human septic shock paradigm) were evaluated for hemodynamic and renal responsiveness to incremental doses of two dominant apelinergics: apelin-13 (APLN-13) or Elabela (ELA), and (2) 52 subjects (33 patients with sepsis/septic shock and 19 healthy volunteers) were investigated for early levels of endogenous apelinergics in the blood, the related enzymatic degradation profile, and data regarding sepsis outcome. APLN-13 was the only one apelinergic which efficiently improved hemodynamics in both healthy and septic sheep. Endogenous apelinergic levels early rose, and specific enzymatic breakdown activities potentially threatened endogenous apelin system reactivity and negatively impacted the outcome in human sepsis. Short-term exogenous APLN-13 infusion is helpful in stabilizing cardiorenal functions in ovine septic shock; however, this ability might be impaired by specific enzymatic systems triggered during the early time course of human sepsis. Strategies to improve resistance of APLN-13 to degradation and/or to overcome sepsis-induced enzymatic breakdown environment should guide future works.

Optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus controls locomotion in a mouse model of Parkinson's disease.

In Parkinson's disease (PD), the loss of midbrain dopaminergic cells results in severe locomotor deficits, such as gait freezing and akinesia. Growing evidence indicates that these deficits can be attributed to the decreased activity in the mesencephalic locomotor region (MLR), a brainstem region controlling locomotion. Clinicians are exploring the deep brain stimulation of the MLR as a treatment option to improve locomotor function. The results are variable, from modest to promising. However, within the MLR, clinicians have targeted the pedunculopontine nucleus exclusively, while leaving the cuneiform nucleus unexplored. To our knowledge, the effects of cuneiform nucleus stimulation have never been determined in parkinsonian conditions in any animal model. Here, we addressed this issue in a mouse model of PD, based on the bilateral striatal injection of 6-hydroxydopamine, which damaged the nigrostriatal pathway and decreased locomotor activity. We show that selective optogenetic stimulation of glutamatergic neurons in the cuneiform nucleus in mice expressing channelrhodopsin in a Cre-dependent manner in Vglut2-positive neurons (Vglut2-ChR2-EYFP mice) increased the number of locomotor initiations, increased the time spent in locomotion, and controlled locomotor speed. Using deep learning-based movement analysis, we found that the limb kinematics of optogenetic-evoked locomotion in pathological conditions were largely similar to those recorded in intact animals. Our work identifies the glutamatergic neurons of the cuneiform nucleus as a potentially clinically relevant target to improve locomotor activity in parkinsonian conditions. Our study should open avenues to develop the targeted stimulation of these neurons using deep brain stimulation, pharmacotherapy, or optogenetics.

The neuronal potassium current IA is a potential target for pain during chronic inflammation.

Voltage-gated ion channels play a key role in the action potential (AP) initiation and its propagation in sensory neurons. Modulation of their activity during chronic inflammation creates a persistent pain state. In this study, we sought to determine how peripheral inflammation caused by complete Freund's adjuvant (CFA) alters the fast sodium (INa ), L-type calcium (ICaL ), and potassium (IK ) currents in primary afferent fibers to increase nociception. In our model, intraplantar administration of CFA induced mechanical allodynia and thermal hyperalgesia at day 14 post-injection. Using whole-cell patch-clamp recording in dissociated small (C), medium (Aδ), and large-sized (Aß) rat dorsal root ganglion (DRG) neurons, we found that CFA prolonged the AP duration and increased the amplitude of the tetrodotoxin-resistant (TTX-r) INa in Aß fibers. In addition, CFA accelerated the recovery of INa from inactivation in C and Aδ nociceptive fibers but enhanced the late sodium current (INaL ) only in Aδ and Aß neurons. Inflammation similarly reduced the amplitude of ICaL in each neuronal cell type. Fourteen days after injection, CFA reduced both components of IK (IKdr and IA ) in Aδ fibers. We also found that IA was significantly larger in C and Aδ neurons in normal conditions and during chronic inflammation. Our data, therefore, suggest that targeting the transient potassium current IA represents an efficient way to shift the balance toward antinociception during inflammation, since its activation will selectively decrease the AP duration in nociceptive fibers. Altogether, our data indicate that complex interactions between IK , INa , and ICaL reduce pain threshold by concomitantly enhancing the activity of nociceptive neurons and reducing the inhibitory action of Aß fibers during chronic inflammation.

Elabela Protects Spontaneously Hypertensive Rats From Hypertension and Cardiorenal Dysfunctions Exacerbated by Dietary High-Salt Intake.

Objectives: Arterial hypertension, when exacerbated by excessive dietary salt intake, worsens the morbidity and mortality rates associated with cardiovascular and renal diseases. Stimulation of the apelinergic system appears to protect against several circulatory system diseases, but it remains unknown if such beneficial effects are conserved in severe hypertension. Therefore, we aimed at determining whether continuous infusion of apelinergic ligands (i.e., Apelin-13 and Elabela) exerted cardiorenal protective effects in spontaneously hypertensive (SHR) rats receiving high-salt diet. Methods: A combination of echocardiography, binding assay, histology, and biochemical approaches were used to investigate the cardiovascular and renal effects of Apelin-13 or Elabela infusion over 6 weeks in SHR fed with normal-salt or high-salt chow. Results: High-salt intake upregulated the cardiac and renal expression of APJ receptor in SHR. Importantly, Elabela was more effective than Apelin-13 in reducing high blood pressure, cardiovascular and renal dysfunctions, fibrosis and hypertrophy in high-salt fed SHR. Unlike Apelin-13, the beneficial effects of Elabela were associated with a counter-regulatory role of the ACE/ACE2/neprilysin axis of the renin-angiotensin-aldosterone system (RAAS) in heart and kidneys of salt-loaded SHR. Interestingly, Elabela also displayed higher affinity for APJ in the presence of high salt concentration and better resistance to RAAS enzymes known to cleave Apelin-13. Conclusion: These findings highlight the protective action of the apelinergic system against salt-induced severe hypertension and cardiorenal failure. As compared with Apelin-13, Elabela displays superior pharmacodynamic and pharmacokinetic properties that warrant further investigation of its therapeutic use in cardiovascular and kidney diseases.

Pharmacodynamic and pharmacokinetic profiles of a neurotensin receptor type 2 (NTS2) analgesic macrocyclic analog.

The current opioid crisis highlights the urgent need to develop safe and effective pain medications. Thus, neurotensin (NT) compounds represent a promising approach, as the antinociceptive effects of NT are mediated by activation of the two G protein-coupled receptor subtypes (i.e., NTS1 and NTS2) and produce potent opioid-independent analgesia. Here, we describe the synthesis and pharmacodynamic and pharmacokinetic properties of the first constrained NTS2 macrocyclic NT(8-13) analog. The Tyr11 residue of NT(8-13) was replaced with a Trp residue to achieve NTS2 selectivity, and a rationally designed side-chain to side-chain macrocyclization reaction was applied between Lys8 and Trp11 to constrain the peptide in an active binding conformation and limit its recognition by proteolytic enzymes. The resulting macrocyclic peptide, CR-01-64, exhibited high-affinity for NTS2 (Ki 7.0 nM), with a more than 125-fold selectivity over NTS1, as well as an improved plasma stability profile (t1/2 > 24 h) compared with NT (t1/2 ~ 2 min). Following intrathecal administration, CR-01-64 exerted dose-dependent and long-lasting analgesic effects in acute (ED50 = 4.6 µg/kg) and tonic (ED50 = 7.1 µg/kg) pain models as well as strong mechanical anti-allodynic effects in the CFA-induced chronic inflammatory pain model. Of particular importance, this constrained NTS2 analog exerted potent nonopioid antinociceptive effects and potentiated opioid-induced analgesia when combined with morphine. At high doses, CR-01-64 did not cause hypothermia or ileum relaxation, although it did induce mild and short-term hypotension, all of which are physiological effects associated with NTS1 activation. Overall, these results demonstrate the strong therapeutic potential of NTS2-selective analogs for the management of pain.