Search

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 Ile 12 residue of NT(7-12) was substituted by cyclopentylalanine, Pro 7 and Pro 10 were replaced by allyl-glycine followed by side-chain to side-chain cyclization is the most selective analog targeting NTS2 identified to date (K i 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., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

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-β-Ala-NH 2 and NT(8-13) analogs optimized for NTS2 affinity. In the present work, the constrained dipeptide Aba-β-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 β 3 h Arg. 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 G αi1 and β-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 β-arrestin-2 ( E max = 17 and 12%, respectively), while both compounds were also found to be partial agonists at the G αi1 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., Competing Interests: The authors declare no competing financial interest., (© 2024 American Chemical Society.)

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., Competing Interests: Declaration of Competing Interest None., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

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 P2Y 6 . Despite the recent interest for this receptor in pathologies, such as gastrointestinal and neurological diseases, there is sparse information on the endosomal trafficking of P2Y 6 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 P2Y 6 . Interestingly, UDP induced clathrin-dependent P2Y 6 internalization, whereas receptor stimulation by MRS2693 endocytosis appeared to be associated with a caveolin-dependent mechanism. Internalized P2Y 6 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 P2Y 6 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 P2Y 6 receptor internalization and endosomal trafficking. These findings could guide the development of bias ligands that could influence P2Y 6 signaling., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

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 [Pyr 1 ]-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 Gα i1 activation (EC 50 Gα i1 0.4-0.9 nM vs Ape13 EC 50 1.1 nM), partial agonist behavior disfavoring β-arrestin 2 recruitment for positively charged ligands (e.g., 49 ( SBL-AP-058 ), EC 50 β-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 β-arrestin 2 signaling with partial maximal efficacy (EC 50 β-arr 864 nM, E max 48%), significantly reducing the hypotensive effect in vivo ., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

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., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

We previously reported a series of macrocyclic analogues of [Pyr 1 ]-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 ( K i 0.6 nM), which does not activate the Gα 12 signaling pathway while maintaining potency and efficacy similar to Ape13 to activate Gα i1 (EC 50 0.8 nM) and β-arrestin2 recruitment (EC 50 31 nM), still exerts cardiac actions. In addition, analogue 40 ( K i 5.6 nM), exhibiting a favorable Gα 12 -biased signaling and an increased in vivo half-life ( t 1/2 3.7 h vs <1 min of Ape13), produces a sustained cardiac response up to 6 h after a single subcutaneous bolus injection.

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 Tyr 11 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 Lys 8 and Trp 11 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 (K i 7.0 nM), with a more than 125-fold selectivity over NTS1, as well as an improved plasma stability profile (t 1/2 > 24 h) compared with NT (t 1/2 ~ 2 min). Following intrathecal administration, CR-01-64 exerted dose-dependent and long-lasting analgesic effects in acute (ED 50 = 4.6 µg/kg) and tonic (ED 50 = 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., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Sainsily, Coquerel, Giguère, Dumont, Tran, Noll, Ionescu, Côté, Longpré, Carpentier, Marsault, Lesur, Sarret and Auger-Messier.)

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 ( K i 0.08-0.18 nM vs Ape13 K i 0.7 nM). Besides, constrained (d-Tic) or α,α-disubstituted residues (Db z g; d-α-Me-Tyr(OBn)) were favorable for the Phe13 position. Compounds 47 (Pro12-Phe13 replaced by Aia-Phe, K i 0.08 nM) and 53 (Pro12-Phe13 replaced by 1Nal-Db z g, K i 0.08 nM) are the most potent Ape13 analogues activating the Gα 12 pathways ( 53 , EC 50 Gα 12 2.8 nM vs Ape13, EC 50 43 nM) known to date, displaying high affinity, resistance to ACE2 cleavage as well as improved pharmacokinetics in vitro ( t 1/2 5.8-7.3 h in rat plasma) and in vivo .

The endogenous tridecapeptide neurotensin (NT) has emerged as an important inhibitory modulator of pain transmission, exerting its analgesic action through the activation of the G protein-coupled receptors, NTS1 and NTS2. Whereas both NT receptors mediate the analgesic effects of NT, NTS1 activation also produces hypotension and hypothermia, which may represent obstacles for the development of new pain medications. In the present study, we implemented various chemical strategies to improve the metabolic stability of the biologically active fragment NT(8-13) and assessed their NTS1/NTS2 relative binding affinities. We then determined their ability to reduce the nociceptive behaviors in acute, tonic, and chronic pain models and to modulate blood pressure and body temperature. To this end, we synthesized a series of NT(8-13) analogs carrying a reduced amide bond at Lys 8 -Lys 9 and harboring site-selective modifications with unnatural amino acids, such as silaproline (Sip) and trimethylsilylalanine (TMSAla). Incorporation of Sip and TMSAla respectively in positions 10 and 13 of NT(8-13) combined with the Lys 8 -Lys 9 reduced amine bond (JMV5296) greatly prolonged the plasma half-life time over 20 h. These modifications also led to a 25-fold peptide selectivity toward NTS2. More importantly, central delivery of JMV5296 was able to induce a strong antinociceptive effect in acute (tail-flick), tonic (formalin), and chronic inflammatory (CFA) pain models without inducing hypothermia. Altogether, these results demonstrate that the chemically-modified NT(8-13) analog JMV5296 exhibits a better therapeutic profile and may thus represent a promising avenue to guide the development of new stable NT agonists and improve pain management., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Pain is reported as the leading cause of disability in the common forms of inflammatory arthritis conditions. Acting as a key player in nociceptive processing, neuroinflammation, and neuron-glia communication, the chemokine CCL2/CCR2 axis holds great promise for controlling chronic painful arthritis. Here, we investigated how the CCL2/CCR2 system in the dorsal root ganglion (DRG) contributes to the peripheral inflammatory pain sensitization., Methods: Repeated intrathecal (i.t.) administration of the CCR2 antagonist, INCB3344 was tested for its ability to reverse the nociceptive-related behaviors in the tonic formalin and complete Freund's adjuvant (CFA) inflammatory models. We further determined by qPCR the expression of CCL2/CCR2, SP and CGRP in DRG neurons from CFA-treated rats. Using DRG explants, acutely dissociated primary sensory neurons and calcium mobilization assay, we also assessed the release of CCL2 and sensitization of nociceptors. Finally, we examined by immunohistochemistry following nerve ligation the axonal transport of CCL2, SP, and CGRP from the sciatic nerve of CFA-treated rats., Results: We first found that CFA-induced paw edema provoked an increase in CCL2/CCR2 and SP expression in ipsilateral DRGs, which was decreased after INCB3344 treatment. This upregulation in pronociceptive neuromodulators was accompanied by an enhanced nociceptive neuron excitability on days 3 and 10 post-CFA, as revealed by the CCR2-dependent increase in intracellular calcium mobilization following CCL2 stimulation. In DRG explants, we further demonstrated that the release of CCL2 was increased following peripheral inflammation. Finally, the excitation of nociceptors following peripheral inflammation stimulated the anterograde transport of SP at their peripheral nerve terminals. Importantly, blockade of CCR2 reduced sensory neuron excitability by limiting the calcium mobilization and subsequently decreased peripheral transport of SP towards the periphery. Finally, pharmacological inhibition of CCR2 reversed the pronociceptive action of CCL2 in rats receiving formalin injection and significantly reduced the neurogenic inflammation as well as the stimuli-evoked and movement-evoked nociceptive behaviors in CFA-treated rats., Conclusions: Our results provide significant mechanistic insights into the role of CCL2/CCR2 within the DRG in the development of peripheral inflammation, nociceptor sensitization, and pain hypersensitivity. We further unveil the therapeutic potential of targeting CCR2 for the treatment of painful inflammatory disorders.

Neurotensin (NT) receptor type 2 (NTS2) represents an attractive target for the development of new NT-based analgesics. Here, we report the synthesis and functional in vivo characterization of the first constrained NTS2-selective macrocyclic NT analog. While most chemical optimization studies rely on the NT(8-13) fragment, we focused on NT(7-12) as a scaffold to design NTS2-selective macrocyclic peptides. Replacement of Ile 12 by Leu, and Pro 7 /Pro 10 by allylglycine residues followed by cyclization via ring-closing metathesis led to macrocycle 4 , which exhibits good affinity for NTS2 (50 nM), high selectivity over NTS1 (>100 μM), and improved stability compared to NT(8-13). In vivo profiling in rats reveals that macrocycle 4 produces potent analgesia in three distinct rodent pain models, without causing the undesired effects associated with NTS1 activation. We further provide evidence of its non-opioid antinociceptive activity, therefore highlighting the strong therapeutic potential of NTS2-selective analogs for the management of acute and chronic pain.

Cysteinyl leukotriene G protein-coupled receptors, CysLT1R and CysLT2R, regulate bronchoconstrictive and pro-inflammatory effects and play a key role in allergic disorders, cardiovascular diseases, and cancer. CysLT1R antagonists have been widely used to treat asthma disorders, while CysLT2R is a potential target against uveal melanoma. However, very few selective antagonist chemotypes for CysLT receptors are available, and the design of such ligands has proved to be challenging. To overcome this obstacle, we took advantage of recently solved crystal structures of CysLT receptors and an ultra-large Enamine REAL library, representing a chemical space of 680 M readily available compounds. Virtual ligand screening employed 4D docking models comprising crystal structures of CysLT1R and CysLT2R and their corresponding ligand-optimized models. Functional assessment of the candidate hits yielded discovery of five novel antagonist chemotypes with sub-micromolar potencies and the best Ki = 220 nM at CysLT1R. One of the hits showed inverse agonism at the L129Q constitutively active mutant of CysLT2R, with potential utility against uveal melanoma.

Cell migration is a ubiquitous process necessary to maintain and restore tissue functions. However, in cancer, cell migration leads to metastasis development and thus worsens the prognosis. Although the mechanism of cell migration is well understood, the identification of new targets modulating cell migration and deciphering their signaling events could lead to new therapies to restore tissue functions in diseases, such as inflammatory bowel disease, or to block metastatic development in different forms of cancer. Previous research has identified the G-protein-coupled P2Y 6 receptor as an innovative target that could dictate cell migration under normal and pathological conditions. Surprisingly, there is little information on the cellular events triggered by activated P2Y 6 during cell migration. Here, we demonstrated that P2Y 6 activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y 6 increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gα q /calcium/protein kinases C (PKC) and Gα 13 /RHO-associated protein kinase-dependent pathways that dictate the formation of the migratory structures. These pathways led to the stabilization of the actin cytoskeleton through a PKC-dependent phosphorylation of cofilin. These results support the idea that the P2Y 6 receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways., (© 2020 Wiley Periodicals LLC.)

Neurotensin (NT) exerts naloxone-insensitive antinociceptive action through its binding to both NTS 1 and NTS 2 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 Pro 10 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 NTS 1 activation, like hypothermia. In in vitro assays, JMV2009 exhibited high binding affinity for both NTS 1 and NTS 2 , 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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Long-term cognitive deficits are observed after treatment of brain tumors or metastases by radiotherapy. Treatment optimization thus requires a better understanding of the effects of radiotherapy on specific brain regions, according to their sensitivity and interconnectivity. In the present study, behavioral tests supported by immunohistology and magnetic resonance imaging provided a consistent picture of the persistent neurocognitive decline and neuroinflammation after the onset of irradiation-induced necrosis in the right primary somatosensory cortex of Fischer rats. Necrosis surrounded by neovascularization was first detected 54 days after irradiation and then spread to 110 days in the primary motor cortex, primary somatosensory region, striatum and right ventricle, resulting in fiber bundle disruption and demyelination in the corpus callosum of the right hemisphere. These structural damages translated into selective behavioral changes including spatial memory loss, disinhibition of anxiety-like behaviors, hyperactivity and pain hypersensitivity, but no significant alteration in motor coordination and grip strength abilities. Concomitantly, activated microglia and reactive astrocytes, accompanied by infiltration of leukocytes (CD45+) and T-cells (CD3+) cooperated to shape the neuroinflammation response. Overall, our study suggests that the slow and gradual onset of cellular damage would allow adaptation in brain regions that are susceptible to neuronal plasticity; while other cerebral structures that do not have this capacity would be more affected. The planning of radiotherapy, adjusted to the sensitivity and adaptability of brain structures, could therefore preserve certain neurocognitive functions; while higher doses of radiation could be delivered to brain areas that can better adapt to this treatment. In addition, strategies to block early post-radiation events need to be explored to prevent the development of long-term cognitive dysfunction., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Cell-based functional assays are an important part of compound screening and drug lead optimization, and they can also play a crucial role in the determination of the residues involved in ligand binding and signaling for a particular G-protein-coupled receptor. Conventional methods used for Gα q/15 -coupled receptors rely on the use of fluorescent probes for Ca ++ sensing (such as Fura-2 and Fluo-4) or on the incorporation of [ 3 H]-inositol into inositol 1,4,5- triphosphate (IP3). However, these methods are not suitable for screening large libraries of compounds or for screening several mutants of the same receptor. In contrast, the IP-One assay by Cisbio is a TR-FRET assay suitable for large compound library screening when using stable cell lines that express a specific 7TMR. However, when using transiently transfected mutants of a 7TMR, this assay is not ideal, as it requires a two-step protocol of cell culture. Therefore, we have optimized the IP-One assay protocol using the reverse transfection method in 384-well plates. This offers a time- and resource-efficient alternative to the two-step protocol previously used for the screening of several mutants of Gα q/15 -coupled 7TMRs., Competing Interests: Competing interestsThe authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (Copyright © 2020 The Authors; exclusive licensee Bio-protocol LLC.)

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 β-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., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships which have, or could be perceived to have, influenced the work reported in this article., (© 2020 The Author(s).)

Therapeutic hypothermia represents a brain-protective strategy for multiple emergency situations, such as stroke or traumatic injury. Neurotensin (NT), which exerts its effects through activation of two G protein-coupled receptors, namely NTS1 and NTS2, induces a strong and long-lasting decrease in core body temperature after its central administration. Growing evidence demonstrates that NTS1 is the receptor subtype mediating the hypothermic action of NT. As such, potent NTS1 agonists designed on the basis of the minimal C-terminal NT(8-13) bioactive fragment have been shown to produce mild hypothermia and exert neuroprotective effects under various clinically relevant conditions. The high susceptibility of NT(8-13) to protease degradation (half-life <2 min) represents, however, a serious limitation for its use in pharmacological therapy. In light of this, we report here a structure-activity relationship study in which pairs of NT(8-13) analogs have been developed, based on the incorporation of a reduced Lys 8 -Lys 9 bond. To further stabilize the peptide bonds, a panel of backbone modifications was also inserted along the peptide sequence, including Sip 10 , D-Trp 11 , Dmt 11 , Tle 12 , and TMSAla 13 . Our results revealed that the combination of appropriate chemical modifications leads to compounds exhibiting improved resistance to proteolytic cleavages (>24 h; 16 ). Among them, the NT(8-13) analogs harboring the reduced amine bond combined with the unnatural amino acids TMSAla 13 ( 4 ) and Sip 10 ( 6 ) or the di-substitution Lys 11 - TMSAla 13 ( 12 ), D-Trp 11 -TMSAla 13 ( 14 ), and Dmt 11 -Tle 12 ( 16 ) produced sustained hypothermic effects (-3°C for at least 1 h). Importantly, we observed that hypothermia was mainly driven by the increased stability of the NT(8-13) derivatives, instead of the high binding-affinity at NTS1. Altogether, these results reveal the importance of the reduced amine bond in optimizing the metabolic properties of the NT(8-13) peptide and support the development of stable NTS1 agonists as first drug candidate in neuroprotective hypothermia., (Copyright © 2020 Previti, Vivancos, Rémond, Beaulieu, Longpré, Ballet, Sarret and Cavelier.)

A comprehensive understanding of signalling downstream of GPCRs requires a broad approach to capture novel signalling modalities in addition to established pathways. Here, using an array of sixteen validated BRET-based biosensors, we analyzed the ability of seven different β-adrenergic ligands to engage five distinct signalling pathways downstream of the β 1 -adrenergic receptor (β 1 AR). In addition to generating signalling signatures and capturing functional selectivity for the different ligands toward these pathways, we also revealed coupling to signalling pathways that have not previously been ascribed to the βAR. These include coupling to G z and G 12 pathways. The signalling cascade linking the β 1 AR to calcium mobilization was also characterized using a combination of BRET-based biosensors and CRISPR-engineered HEK 293 cells lacking the Gαs subunit or with pharmacological or genetically engineered pathway inhibitors. We show that both G s and G 12 are required for the full calcium response. Our work highlights the power of combining signal profiling with genome editing approaches to capture the full complement of GPCR signalling activities in a given cell type and to probe their underlying mechanisms.

Pepducins are cell-penetrating, membrane-tethered lipopeptides designed to target the intracellular region of a G protein-coupled receptor (GPCR) in order to allosterically modulate the receptor's signaling output. In this proof-of-concept study, we explored the pain-relief potential of a pepducin series derived from the first intracellular loop of neurotensin receptor type 1 (NTS1), a class A GPCR that mediates many of the effects of the neurotensin (NT) tridecapeptide, including hypothermia, hypotension and analgesia. We used BRET-based biosensors to determine the pepducins' ability to engage G protein signaling pathways associated with NTS1 activation. We observed partial Gα q and Gα 13 activation at a 10 μM concentration, indicating that these pepducins may act as allosteric agonists of NTS1. Additionally, we used surface plasmon resonance (SPR) as a label-free assay to monitor pepducin-induced responses in CHO-K1 cells stably expressing hNTS1. This whole-cell integrated assay enabled us to subdivide our pepducin series into three profile response groups. In order to determine the pepducins' antinociceptive potential, we then screened the series in an acute pain model (tail-flick test) by measuring tail withdrawal latencies to a thermal nociceptive stimulus, following intrathecal (i.t.) pepducin administration (275 nmol/kg). We further evaluated promising pepducins in a tonic pain model (formalin test), as well as in neuropathic (Chronic Constriction Injury) and inflammatory (Complete Freund's Adjuvant) chronic pain models. We report one pepducin, PP-001, that consistently reduced rat nociceptive behaviors, even in chronic pain paradigms. Finally, we designed a TAMRA-tagged version of PP-001 and found by confocal microscopy that the pepducin reached the rat dorsal root ganglia post i.t. injection, thus potentially modulating the activity of NTS1 at this location to produce its analgesic effect. Altogether, these results suggest that NTS1-derived pepducins may represent a promising strategy in pain-relief., Competing Interests: Declaration of Competing Interest The authors declare no competing financial interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Improvements in the survival of breast cancer patients have led to the emergence of bone health and pain management as key aspects of patient's quality of life. Here, we used a female rat MRMT-1 model of breast cancer-induced bone pain to compare the effects of three drugs used clinically morphine, nabilone and zoledronate on tumor progression, bone remodeling and pain relief. We found that chronic morphine reduced the mechanical hypersensitivity induced by the proliferation of the luminal B aggressive breast cancer cells in the tumor-bearing femur and prevented spinal neuronal and astrocyte activation. Using MTT cell viability assay and MRI coupled to 18 FDG PET imaging followed by ex vivo 3D µCT, we further demonstrated that morphine did not directly exert tumor growth promoting or inhibiting effects on MRMT-1 cancer cells but induced detrimental effects on bone healing by disturbing the balance between bone formation and breakdown. In sharp contrast, both the FDA-approved bisphosphonate zoledronate and the synthetic cannabinoid nabilone prescribed as antiemetics to patients receiving chemotherapy were effective in limiting the osteolytic bone destruction, thus preserving the bone architecture. The protective effect of nabilone on bone metabolism was further accompanied by a direct inhibition of tumor growth. As opposed to zoledronate, nabilone was however not able to manage bone tumor-induced pain and reactive gliosis. Altogether, our results revealed that morphine, nabilone and zoledronate exert disparate effects on tumor growth, bone metabolism and pain control. These findings also support the use of nabilone as an adjuvant therapy for bone metastases.

Cysteinyl leukotriene G protein-coupled receptors CysLT 1 and CysLT 2 regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT 1 R has been used for treating asthma and associated diseases for over two decades, CysLT 2 R has recently started to emerge as a potential drug target against atopic asthma, brain injury and central nervous system disorders, as well as several types of cancer. Here, we describe four crystal structures of CysLT 2 R in complex with three dual CysLT 1 R/CysLT 2 R antagonists. The reported structures together with the results of comprehensive mutagenesis and computer modeling studies shed light on molecular determinants of CysLTR ligand selectivity and specific effects of disease-related single nucleotide variants.

Neurotensin (NT) exerts its analgesic effects through activation of the G protein-coupled receptors NTS1 and NTS2. This opioid-independent antinociception represents a potential alternative for pain management. While activation of NTS1 also induces a drop in blood pressure and body temperature, NTS2 appears to be an analgesic target free of these adverse effects. Here, we report modifications of NT at Tyr 11 to increase selectivity toward NTS2, complemented by modifications at the N-terminus to impair proteolytic degradation of the biologically active NT(8-13) sequence. Replacement of Tyr 11 by either 6-OH-Tic or 7-OH-Tic resulted in a significant loss of binding affinity to NTS1 and subsequent NTS2 selectivity. Incorporation of the unnatural amino acid β 3 hLys at position 8 increased the half-life to over 24 h in plasma. Simultaneous integration of both β 3 hLys 8 and 6-OH-Tic 11 into NT(8-13) produced a potent and NTS2-selective analogue with strong analgesic action after intrathecal delivery in the rat formalin-induced pain model with an ED 50 of 1.4 nmol. Additionally, intravenous administration of this NT analogue did not produce persistent hypotension or hypothermia. These results demonstrate that NT analogues harboring unnatural amino acids at positions 8 and 11 can enhance crucial pharmacokinetic and pharmacodynamic features for NT(8-13) analogues, i.e., proteolytic stability, NTS2 selectivity, and improved analgesic/adverse effect ratio.

The G protein-coupled cysteinyl leukotriene receptor CysLT 1 R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT 1 R antagonists are widely prescribed as antiasthmatic drugs; however, these drugs demonstrate low effectiveness in some patients and exhibit a variety of side effects. To gain deeper understanding into the functional mechanisms of CysLTRs, we determined the crystal structures of CysLT 1 R bound to two chemically distinct antagonists, zafirlukast and pranlukast. The structures reveal unique ligand-binding modes and signaling mechanisms, including lateral ligand access to the orthosteric pocket between transmembrane helices TM4 and TM5, an atypical pattern of microswitches, and a distinct four-residue-coordinated sodium site. These results provide important insights and structural templates for rational discovery of safer and more effective drugs., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Opioid and neurotensin (NT) receptors are expressed in both central and peripheral nervous systems where they modulate nociceptive responses. Nowadays, opioid analgesics like morphine remain the most prescribed drugs for the treatment of moderate to severe pain. However, despite their daily used, opioids can produce life-threatening side effects, such as constipation or respiratory depression. Besides, NT analogs exert strong opioid-independent analgesia. Here, we thus hypothesized that the combined use of opioid and NT agonists would require lower doses to produce significant analgesic effects, hence decreasing opioid-induced adverse effects. We used isobologram analyses to determine if the combination of a NT brain-penetrant analog, An2-NT(8-13) with morphine results in an inhibitory, synergistic or additive analgesic response. We found that intravenous administration of An2-NT(8-13) reduced by 90% the nocifensive behaviors induced by formalin injection, at the dose of 0.018 mg/kg. Likewise, subcutaneous morphine reduced pain by 90% at 1.8 mg/kg. Importantly, isobologram analyses revealed that the co-injection of An2-NT(8-13) with morphine induced an additive analgesic response. We finally assessed the effects of morphine and An2-NT(8-13) on the gastrointestinal tract motility using the charcoal meal test. As opposed to morphine which significantly reduced the intestinal motility at the analgesic effective dose of 1.8 mg/kg, An2-NT(8-13) did not affect the charcoal meal intestinal transit at 0.018 mg/kg. Interestingly, at the dose providing 90% pain relief, the co-administration of morphine with An2-NT(8-13) had a reduced effect on constipation. Altogether, these results suggest that combining NT agonists with morphine may improve its analgesic/adverse effect ratio., (Copyright © 2019 Elsevier B.V. All rights reserved.)

G protein-coupled receptors (GPCRs) are important therapeutic targets that exhibit functional selectivity (biased signaling), in which different ligands or receptor variants elicit distinct downstream signaling. Understanding all the signaling events and biases that contribute to both the beneficial and adverse effects of GPCR stimulation by given ligands is important for drug discovery. Here, we report the design, validation, and use of pathway-selective bioluminescence resonance energy transfer (BRET) biosensors that monitor the engagement and activation of signaling effectors downstream of G proteins, including protein kinase C (PKC), phospholipase C (PLC), p63RhoGEF, and Rho. Combined with G protein and β-arrestin BRET biosensors, our sensors enabled real-time monitoring of GPCR signaling at different levels in downstream pathways in both native and engineered cells. Profiling of the responses to 14 angiotensin II (AngII) type 1 receptor (AT1R) ligands enabled the clustering of compounds into different subfamilies of biased ligands and showed that, in addition to the previously reported functional selectivity between Gα q and β-arrestin, there are also biases among G protein subtypes. We also demonstrated that biases observed at the receptor and G protein levels propagated to downstream signaling pathways and that these biases could occur through the engagement of different G proteins to activate a common effector. We also used these tools to determine how naturally occurring AT1R variants affected signaling bias. This suite of BRET biosensors provides a useful resource for fingerprinting biased ligands and mutant receptors and for dissecting functional selectivity at various levels of GPCR signaling., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The neurotensin receptors are attractive targets for the development of new analgesic compounds. They represent potential alternatives or adjuvants to opioids. Herein, we report the structural optimization of our recently reported macrocyclic peptide analogues of NT(8-13). The macrocycle was formed via ring-closing metathesis (RCM) between an ortho allylated tyrosine residue in position 11 and the side chain of alkene-functionalized amino acid in position 8 of NT(8-13). Minute modifications led to significant binding affinity improvement ( K i improved from 5600 to 15 nM) with greatly improved plasma stability compared to NT(8-13). This study also delineates the structural features influencing these parameters. The signaling profiles of the new macrocycles were determined on the NTS1 receptor, and the physiological effects of the two most potent and stable analogues were assessed in vivo using rodent models. Both compounds displayed strong analgesic effects.

In this study, affinities and activities of derivatized analogues of Dmt-dermorphin[1-4] (i.e. Dmt-d-Ala-Phe-GlyNH 2 , Dmt = 2',6'-dimethyl-(S)-tyrosine) for the µ opioid receptor (MOP) and δ opioid receptor (DOP) were evaluated using radioligand binding studies, functional cell-based assays and isolated organ bath experiments. By means of solid-phase or solution-phase Suzuki-Miyaura cross-couplings, various substituted regioisomers of the phenylalanine moiety in position 3 of the sequence were prepared. An 18-membered library of opioid tetrapeptides was generated via screening of the chemical space around the Phe 3 side chain. These substitutions modulated bioactivity, receptor subtype selectivity and highly effective ligands with subnanomolar binding affinities, contributed to higher functional activities and potent analgesic actions. In search of selective peptidic ligands, we show here that the Suzuki-Miyaura reaction is a versatile and robust tool which could also be deployed elsewhere., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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 Phe 13 residue was replaced by natural or unnatural amino acids. In HEK293 cells expressing APJ, we evaluated the relative efficacy of these compounds to activate Gα i1 and Gα oA G-proteins, recruit β-arrestins 1 and 2 (βarrs), 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 βarrs. Finally, using Spearman correlations, we established that the hypotensive response was significantly correlated with βarr recruitment but not with G protein-dependent signaling. In conclusion, our results demonstrated that the βarr recruitment potency is involved in the hypotensive efficacy of activated APJ., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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, K i 0.15 nM and t 1/2 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.

Neurotensin exerts potent analgesic effects following activation of its cognate GPCRs. In this study, we describe a systematic exploration, using structure-based design, of conformationally constraining neurotensin (8-13) with the help of macrocyclization and the resulting impacts on binding affinity, signaling, and proteolytic stability. This exploratory study led to a macrocyclic scaffold with submicromolar binding affinity, agonist activity, and greatly improved plasma stability., Competing Interests: The authors declare no competing financial interest.

The epidemic of type 2 diabetes mellitus (T2DM) is fueled by added fructose consumption. Here, we thus combined high-fat/high-fructose diet, with multiple low-dose injections of streptozotocin (HF/HF/Stz) to emulate the long-term complications of T2DM. HF/HF/Stz rats, monitored over 56 weeks, exhibited metabolic dysfunctions associated with the different stages of the T2DM disease progression in humans: an early prediabetic phase characterized by an hyperinsulinemic period with modest dysglycemia, followed by a late stage of T2DM with frank hyperglycemia, normalization of insulinemia, marked dyslipidemia, hepatic fibrosis and pancreatic β-cell failure. Histopathological analyses combined to [ 18 F]-FDG PET imaging further demonstrated the presence of several end-organ long-term complications, including reduction in myocardial glucose utilization, renal dysfunction as well as microvascular neuropathy and retinopathy. We also provide for the first time a comprehensive µ-PET whole brain imaging of the changes in glucose metabolic activity within discrete cerebral regions in HF/HF/Stz diabetic rats. Altogether, we developed and characterized a unique non-genetic preclinical model of T2DM adapted to the current diet and lifestyle that recapitulates the major metabolic features of the disease progression, from insulin resistance to pancreatic β-cell dysfunction, and closely mimicking the target-organ damage occurring in type 2 diabetic patients at advanced stages.

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 β-arrestins. Using BRET-based biosensors, we found that neurotensin (NT), NT(8-13) and neuromedin N (NN) activated the Gα q -, Gα i1 -, Gα oA -, and Gα 13 -protein signaling pathways as well as the recruitment of β-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 Gα i1 and Gα 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 Gα s -dependent manner. Additionally, we demonstrated that the full activation of ERK1/2 required signaling through both a PTX-sensitive G i/o -c-Src signaling pathway and PLCβ-DAG-PKC-Raf-1-dependent pathway downstream of G q . 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Neurotensin exerts potent analgesia by acting at both NTS1 and NTS2 receptors, whereas NTS1 activation also results in other physiological effects such as hypotension and hypothermia. Here, we used molecular modeling approach to design highly selective NTS2 ligands by investigating the docking of novel NT[8-13] compounds at both NTS1 and NTS2 sites. Molecular dynamics simulations revealed an interaction of the Tyr 11 residue of NT[8-13] with an acidic residue (Glu 179 ) located in the ECL2 of hNTS2 or with a basic residue (Arg 212 ) at the same position in hNTS1. The importance of the residue at position 11 for NTS1/NTS2 selectivity was further demonstrated by the design of new NT analogues bearing basic (Lys, Orn) or acid (Asp or Glu) function. As predicted by the molecular dynamics simulations, binding of NT[8-13] analogues harboring a Lys 11 exhibited higher affinity toward the hNTS1-R212E mutant receptor, in which Arg212 was substituted by the negatively charged Glu residue.

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.

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.

Background: Accumulating evidence suggests that the C-C chemokine ligand 2 (CCL2, or monocyte chemoattractant protein 1) acts as a neuromodulator in the central nervous system through its binding to the C-C chemokine receptor 2 (CCR2). Notably, it is well established that the CCL2/CCR2 axis plays a key role in neuron-glia communication as well as in spinal nociceptive transmission. Gene silencing through RNA interference has recently emerged as a promising avenue in research and drug development, including therapeutic management of chronic pain. In the present study, we used 27-mer Dicer-substrate small interfering RNA (DsiRNA) targeting CCR2 and assessed their ability to reverse the nociceptive behaviors induced by spinal CCL2 injection or following intraplantar injection of complete Freund's adjuvant., Results: To this end, we first developed high-potency DsiRNAs designed to target different sequences distributed across the rat CCR2 (rCCR2) messenger RNA. For optimization, methyl groups were added to the two most potent DsiRNA candidates (Evader and M7 2'-O-methyl modified duplexes) in order to improve in vivo duplex stability and to reduce potential immunostimulatory activity. Our results demonstrated that all modified candidates formulated with the cell-penetrating peptide reagent Transductin showed strong RNAi activity following intrathecal delivery, exhibiting >50% rCCR2 knockdown in lumbar dorsal root ganglia. Accordingly, we found that these DsiRNA duplexes were able to reduce spinal microglia activation and were effective at blocking CCL2-induced mechanical hypersensitivity. Along with similar reductions of rCCR2 messenger RNA, both sequences and methylation patterns were similarly effective in inhibiting the CCL2 nociceptive action for the whole seven days testing period, compared to mismatch DsiRNA. DsiRNAs against CCR2 also reversed the hypernociceptive responses observed in the complete Freund's adjuvant-induced inflammatory chronic pain model., Conclusion: Altogether, these results validate CCR2 as a an appropriate molecular target for pain control and demonstrate that RNAi-based gene therapy represent an highly specific alternative to classical pharmacological approaches to treat central pathologies such as chronic pain., (© The Author(s) 2016.)

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 ELA's 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.

Clinical evidences suggest that an imbalance between descending inhibition and facilitation drives the development of chronic pain. However, potential mechanisms promoting the establishment of a persistent pain state and the increased pain vulnerability remain unknown. This preclinical study was designed to evaluate temporal changes in descending pain modulation at specific experimental endpoints (12, 28, 90 and 168 days) using a novel double-hit model of chronic/tonic pain (first hit: chronic constriction injury (CCI) model; second hit: tonic formalin pain in the contralateral hindpaw). Basal activity of bulbo-spinal monoaminergic systems was further assessed through liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) screening of cerebrospinal fluid (CSF). We found that CCI-operated rats exhibited a reduced nociceptive response profile, peaking on day 28, when subjected to tonic pain. This behavioral response was accompanied by a rapid increase in basal CSF serotonin and norepinephrine levels 12 days after neuropathy, followed by a return to sham levels on day 28. These molecular and behavioral adaptive changes in descending pain inhibition seemed to slowly fade over time. We therefore suggest that chronic neuropathic pain produces a transient hyperactivation of bulbo-spinal monoaminergic drive when previously primed using a tonic pain paradigm (i.e., formalin test), translating into inhibition of subsequent nociceptive behaviors. Altogether, we propose that early hyperactivation of descending pain inhibitory mechanisms, and its potential ensuing exhaustion, could be part of the temporal neurophysiological chain of events favoring chronic neuropathic pain establishment., (Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.)

The silylated amino acid (l)-(trimethylsilyl)alanine (TMSAla) was incorporated at the C-terminal end of the minimal biologically active neurotensin (NT) fragment, leading to the synthesis of new hexapeptide NT[8-13] analogues. Here, we assessed the ability of these new silylated NT compounds to bind to NTS1 and NTS2 receptors, promote regulation of multiple signaling pathways, induce inhibition of the ileal smooth muscle contractions, and affect distinct physiological variables, including blood pressure and pain sensation. Among the C-terminal modified analogues, compound 6 (JMV2007) carrying a TMSAla residue in position 13 exhibits a higher affinity toward NT receptors than the NT native peptide. We also found that compound 6 is effective in reversing carbachol-induced contraction in the isolated strip preparation assay and at inducing a drop in blood pressure. Finally, compound 6 produces potent analgesia in experimental models of acute and persistent pain.

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.

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., (© 2014 Wiley Periodicals, Inc.)

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.

ADAMTS5 (aggrecanase-2), a key metalloprotease mediating cartilage destruction in arthritis, is synthesized as a zymogen, proADAMTS5. We report a detailed characterization of the propeptide excision mechanism and demonstrate that it is a major regulatory step with unusual characteristics. Using furin-deficient cells and a furin inhibitor, we found that proADAMTS5 was processed by proprotein convertases, specifically furin and PC7, but not PC6B. Mutagenesis of three sites containing basic residues within the ADAMTS5 propeptide (RRR(46), RRR(69) and RRRRR(261)) suggested that proADAMTS5 processing occurs after Arg(261). That furin processing was essential for ADAMTS5 activity was illustrated using the known ADAMTS5 substrate aggrecan, as well as a new substrate, versican, an important regulatory proteoglycan during mammalian development. When compared to other ADAMTS proteases, proADAMTS5 processing has several distinct features. In contrast to ADAMTS1, whose furin processing products were clearly present intracellularly, cleaved ADAMTS5 propeptide and mature ADAMTS5 were found exclusively in the conditioned medium. Despite attempts to enhance detection of intracellular proADAMTS5 processing, such as by immunoprecipitation of total ADAMTS5, overexpression of furin, and secretion blockade by monensin, neither processed ADAMTS5 propeptide nor the mature enzyme were found intracellularly, which was strongly suggestive of extracellular processing. Extracellular ADAMTS5 processing was further supported by activation of proADAMTS5 added exogenously to HEK293 cells stably expressing furin. Unlike proADAMTS9, which is processed by furin at the cell-surface, to which it is bound, ADAMTS5 does not bind the cell-surface. Thus, the propeptide processing mechanism of ADAMTS5 has several points of distinction from those of other ADAMTS proteases, which may have considerable significance in the context of osteoarthritis.

ADAMTS9 is a secreted, cell-surface-binding metalloprotease that cleaves the proteoglycans versican and aggrecan. Unlike most precursor proteins, the ADAMTS9 zymogen (pro-ADAMTS9) is resistant to intracellular processing. Instead, pro-ADAMTS9 is processed by furin at the cell surface. Here, we investigated the role of the ADAMTS9 propeptide in regulating its secretion and proteolytic activity. Removal of the propeptide abrogated secretion of the ADAMTS9 catalytic domain, and secretion was inefficiently restored by expression of the propeptide in trans. Substitution of Ala for Asn residues within each of three consensus N-linked glycosylation sites in the propeptide abrogated ADAMTS9 secretion. Thus, the propeptide is an intramolecular chaperone whose glycosylation is critical for secretion of the mature enzyme. In addition to two previously identified furin-processing sites (Arg74 downward arrow and Arg287 downward arrow) the ADAMTS9 propeptide was also furin-processed at Arg209. Substitution of Ala for Arg74, Arg209, and Arg287 resulted in secretion of an unprocessed zymogen. Unexpectedly, versican incubated with cells expressing this pro-ADAMTS9 was processed to a greater extent than when incubated with cells expressing wild-type, furin-processable ADAMTS9. Moreover, cells and medium treated with the proprotein convertase inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone had greater versican-cleaving activity than untreated cells. Following furin processing of pro-ADAMTS9, propeptide fragments maintained a non-covalent association with the catalytic domain. Collectively, these observations suggest that, unlike other metalloproteases, furin processing of the ADAMTS9 propeptide reduces its catalytic activity. Thus, the propeptide is a key functional domain of ADAMTS9, mediating an unusual regulatory mechanism that may have evolved to ensure maximal activity of this protease at the cell surface.

Processing of polypeptide precursors by proprotein convertases (PCs) such as furin typically occurs within the trans-Golgi network. Here, we show in a variety of cell types that the propeptide of ADAMTS9 is not excised intracellularly. Pulse-chase analysis in HEK293F cells indicated that the intact zymogen was secreted to the cell surface and was subsequently processed there before release into the medium. The processing occurred via a furin-dependent mechanism as shown using PC inhibitors, lack of processing in furin-deficient cells, and rescue by furin in these cells. Moreover, down-regulation of furin by small interference RNA reduced ADAMTS9 processing in HEK293F cells. PC5A could also process pro-ADAMTS9, but similarly to furin, processed forms were absent intracellularly. Cell-surface, furin-dependent processing of pro-ADAMTS9 creates a precedent for extracellular maturation of endogenously produced secreted proproteins. It also indicates the existence of a variety of mechanisms for processing of ADAMTS proteases.