Publications by authors named "Jean-Michel Longpre"

50 Publications

Mechanistic insights into the role of the chemokine CCL2/CCR2 axis in dorsal root ganglia to peripheral inflammation and pain hypersensitivity.

J Neuroinflammation 2021 Mar 23;18(1):79. Epub 2021 Mar 23.

Département de Pharmacologie & Physiologie, Institut de Pharmacologie de Sherbrooke, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Quebec, J1H 5N4, Canada.

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.
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http://dx.doi.org/10.1186/s12974-021-02125-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7986025PMC
March 2021

Assessing Gα-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay.

Bio Protoc 2020 Aug 20;10(16):e3715. Epub 2020 Aug 20.

Dept. of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada.

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-coupled receptors rely on the use of fluorescent probes for Ca sensing (such as Fura-2 and Fluo-4) or on the incorporation of [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-coupled 7TMRs.
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http://dx.doi.org/10.21769/BioProtoc.3715DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7842692PMC
August 2020

Metabolically stable neurotensin analogs exert potent and long-acting analgesia without hypothermia.

Behav Brain Res 2021 May 17;405:113189. Epub 2021 Feb 17.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada; Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada. Electronic address:

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-Lys 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-Lys 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.
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http://dx.doi.org/10.1016/j.bbr.2021.113189DOI Listing
May 2021

Design, Structural Optimization, and Characterization of the First Selective Macrocyclic Neurotensin Receptor Type 2 Non-opioid Analgesic.

J Med Chem 2021 Feb 4;64(4):2110-2124. Epub 2021 Feb 4.

Department of Pharmacology and Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001 12e Avenue Nord, Sherbrooke, Quebec J1H 5N4, Canada.

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 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 by Leu, and Pro/Pro by allylglycine residues followed by cyclization via ring-closing metathesis led to macrocycle , which exhibits good affinity for NTS2 (50 nM), high selectivity over NTS1 (>100 μM), and improved stability compared to NT(8-13). profiling in rats reveals that macrocycle 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.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01726DOI Listing
February 2021

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

J Med Chem 2021 Feb 1. Epub 2021 Feb 1.

Département de Pharmacologie-Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke J1H 5N4, Québec, Canada.

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 ( 0.08-0.18 nM vs Ape13 0.7 nM). Besides, constrained (d-Tic) or α,α-disubstituted residues (Dbg; d-α-Me-Tyr(OBn)) were favorable for the Phe13 position. Compounds (Pro12-Phe13 replaced by Aia-Phe, 0.08 nM) and (Pro12-Phe13 replaced by 1Nal-Dbg, 0.08 nM) are the most potent Ape13 analogues activating the Gα pathways (, EC Gα 2.8 nM vs Ape13, EC 43 nM) known to date, displaying high affinity, resistance to ACE2 cleavage as well as improved pharmacokinetics ( 5.8-7.3 h in rat plasma) and .
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http://dx.doi.org/10.1021/acs.jmedchem.0c01941DOI Listing
February 2021

Structure-Based Virtual Screening of Ultra-Large Library Yields Potent Antagonists for a Lipid GPCR.

Biomolecules 2020 12 3;10(12). Epub 2020 Dec 3.

Michelson Center for Convergent Biosciences, Department of Quantitative and Computational Biology, University of Southern California, Los Angeles, CA 90089, USA.

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.
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http://dx.doi.org/10.3390/biom10121634DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7761830PMC
December 2020

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

Data Brief 2020 Aug 20;31:105884. Epub 2020 Jun 20.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada.

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 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 [1]. The reader is directed to the associated article for results interpretation, comments, and discussion.
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http://dx.doi.org/10.1016/j.dib.2020.105884DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7327804PMC
August 2020

Insightful Backbone Modifications Preventing Proteolytic Degradation of Neurotensin Analogs Improve NT-Induced Protective Hypothermia.

Front Chem 2020 5;8:406. Epub 2020 Jun 5.

Institut des Biomolécules Max Mousseron, IBMM, UMR-5247, CNRS, Université de Montpellier, ENSCM, Montpellier, France.

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-Lys bond. To further stabilize the peptide bonds, a panel of backbone modifications was also inserted along the peptide sequence, including Sip, D-Trp, Dmt, Tle, and TMSAla. Our results revealed that the combination of appropriate chemical modifications leads to compounds exhibiting improved resistance to proteolytic cleavages (>24 h; ). Among them, the NT(8-13) analogs harboring the reduced amine bond combined with the unnatural amino acids TMSAla () and Sip () or the di-substitution Lys - TMSAla (), D-Trp-TMSAla (), and Dmt-Tle () 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.
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http://dx.doi.org/10.3389/fchem.2020.00406DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291367PMC
June 2020

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

Eur J Pharmacol 2020 Sep 10;882:173174. Epub 2020 Jun 10.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12thAvenue North, Sherbrooke, Québec, J1H5N4, Canada; Department of Anesthesiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, 3001 12thAvenue North, Sherbrooke, Québec, J1H5N4, Canada; Institut de pharmacologie de Sherbrooke, Université de Sherbrooke, 3001 12thAvenue North, Sherbrooke, Québec, J1H 5N4, Canada; Centre de recherche du Centre hospitalier universitaire de Sherbrooke, CIUSSS de l'Estrie - CHUS, 3001 12thAvenue North, Sherbrooke, Québec, J1H5N4, Canada. Electronic address:

Neurotensin (NT) exerts naloxone-insensitive antinociceptive action through its binding to both NTS and NTS 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 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 activation, like hypothermia. In in vitro assays, JMV2009 exhibited high binding affinity for both NTS and NTS, 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.
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http://dx.doi.org/10.1016/j.ejphar.2020.173174DOI Listing
September 2020

Signal profiling of the βAR reveals coupling to novel signalling pathways and distinct phenotypic responses mediated by βAR and βAR.

Sci Rep 2020 05 29;10(1):8779. Epub 2020 May 29.

Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada.

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-adrenergic receptor (βAR). 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 and G pathways. The signalling cascade linking the βAR 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 and G 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.
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http://dx.doi.org/10.1038/s41598-020-65636-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260363PMC
May 2020

The P2Y receptor signals through Gα /Ca /PKCα and Gα /ROCK pathways to drive the formation of membrane protrusions and dictate cell migration.

J Cell Physiol 2020 12 18;235(12):9676-9690. Epub 2020 May 18.

Department of Immunology and Cell Biology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Quebec, Canada.

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 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 during cell migration. Here, we demonstrated that P2Y activation stimulated A549 human lung cancer cells and Caco-2 colorectal cancer cell migration. Activated P2Y increased the number of filopodia and focal adhesions; two migratory structures required for cell migration. The generation of these structures involved Gα /calcium/protein kinases C (PKC) and Gα /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 receptor represents a target of interest to modulate cell migration and revealed an intricate dialogue between two Gα-protein signaling pathways.
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http://dx.doi.org/10.1002/jcp.29779DOI Listing
December 2020

Brain irradiation leads to persistent neuroinflammation and long-term neurocognitive dysfunction in a region-specific manner.

Prog Neuropsychopharmacol Biol Psychiatry 2020 08 29;102:109954. Epub 2020 Apr 29.

Department of Pharmacology-Physiology, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.pnpbp.2020.109954DOI Listing
August 2020

Cell-penetrating pepducins targeting the neurotensin receptor type 1 relieve pain.

Pharmacol Res 2020 05 6;155:104750. Epub 2020 Mar 6.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, QC, Canada; Institut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada. Electronic address:

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 and Gα 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.
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http://dx.doi.org/10.1016/j.phrs.2020.104750DOI Listing
May 2020

Publisher Correction: Dichotomic effects of clinically used drugs on tumor growth, bone remodeling and pain management.

Sci Rep 2020 Feb 14;10(1):2979. Epub 2020 Feb 14.

Département de Pharmacologie-Physiologie/Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Québec, Canada.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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http://dx.doi.org/10.1038/s41598-020-59781-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021783PMC
February 2020

Dichotomic effects of clinically used drugs on tumor growth, bone remodeling and pain management.

Sci Rep 2019 12 27;9(1):20155. Epub 2019 Dec 27.

Département de Pharmacologie-Physiologie/Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Québec, Canada.

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 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.
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http://dx.doi.org/10.1038/s41598-019-56622-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6934511PMC
December 2019

Structural basis of ligand selectivity and disease mutations in cysteinyl leukotriene receptors.

Nat Commun 2019 12 6;10(1):5573. Epub 2019 Dec 6.

Research Сenter for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny, Russia, 141701.

Cysteinyl leukotriene G protein-coupled receptors CysLT and CysLT regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLTR has been used for treating asthma and associated diseases for over two decades, CysLTR 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 CysLTR in complex with three dual CysLTR/CysLTR 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.
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http://dx.doi.org/10.1038/s41467-019-13348-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6897958PMC
December 2019

Structure-based mechanism of cysteinyl leukotriene receptor inhibition by antiasthmatic drugs.

Sci Adv 2019 10 9;5(10):eaax2518. Epub 2019 Oct 9.

Research Center for Molecular Mechanisms of Aging and Age-Related Diseases, Moscow Institute of Physics and Technology, Dolgoprudny 141701, Russia.

The G protein-coupled cysteinyl leukotriene receptor CysLTR mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLTR 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 CysLTR 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.
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http://dx.doi.org/10.1126/sciadv.aax2518DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6785256PMC
October 2019

Neurotensin Analogues Containing Cyclic Surrogates of Tyrosine at Position 11 Improve NTS2 Selectivity Leading to Analgesia without Hypotension and Hypothermia.

ACS Chem Neurosci 2019 11 21;10(11):4535-4544. Epub 2019 Oct 21.

Département de pharmacologie et physiologie, Institut de pharmacologie de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Faculté de médecine et des sciences de la santé , Université de Sherbrooke , Sherbrooke , Québec J1H 5H4 , Canada.

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 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 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 βhLys at position 8 increased the half-life to over 24 h in plasma. Simultaneous integration of both βhLys and 6-OH-Tic 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 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.
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http://dx.doi.org/10.1021/acschemneuro.9b00390DOI Listing
November 2019

The combination of opioid and neurotensin receptor agonists improves their analgesic/adverse effect ratio.

Eur J Pharmacol 2019 Apr 29;848:80-87. Epub 2019 Jan 29.

Département de pharmacologie-physiologie, Institut de pharmacologie de Sherbrooke, Centre de Recherche du Centre Hospitalier Universitaire de Sherbrooke, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ejphar.2019.01.048DOI Listing
April 2019

Functional selectivity profiling of the angiotensin II type 1 receptor using pathway-wide BRET signaling sensors.

Sci Signal 2018 12 4;11(559). Epub 2018 Dec 4.

Department of Medicine, Research Institute of the McGill University Health Center (RI-MUHC), McGill University, Montréal, QC H4A 3J1, Canada.

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 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.
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http://dx.doi.org/10.1126/scisignal.aat1631DOI Listing
December 2018

Structural Optimization and Characterization of Potent Analgesic Macrocyclic Analogues of Neurotensin (8-13).

J Med Chem 2018 08 13;61(16):7103-7115. Epub 2018 Aug 13.

Department of Pharmacology and Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke , Université de Sherbrooke , 3001 12e Avenue Nord , Sherbrooke , Quebec J1H 5N4 , Canada.

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 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.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00175DOI Listing
August 2018

Chemical space screening around Phe in opioid peptides: Modulating µ versus δ agonism by Suzuki-Miyaura cross-couplings.

Bioorg Med Chem Lett 2018 07 12;28(13):2320-2323. Epub 2018 May 12.

Research Group of Organic Chemistry, Departments of Bioengineering Sciences and Chemistry, Vrije Universiteit Brussel, Pleinlaan 2, 1050 Brussels, Belgium. Electronic address:

In this study, affinities and activities of derivatized analogues of Dmt-dermorphin[1-4] (i.e. Dmt-d-Ala-Phe-GlyNH, 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 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.
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http://dx.doi.org/10.1016/j.bmcl.2018.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6005765PMC
July 2018

In Search of the Optimal Macrocyclization Site for Neurotensin.

ACS Med Chem Lett 2018 Mar 29;9(3):227-232. Epub 2018 Jan 29.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, 3001, 12e Avenue Nord, Sherbrooke, Quebec J1H 5N4, Canada.

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.
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http://dx.doi.org/10.1021/acsmedchemlett.7b00500DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5846049PMC
March 2018

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

Pharmacol Res 2018 05 9;131:7-16. Epub 2018 Mar 9.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada; Institut de pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec J1H 5N4, Canada. Electronic address:

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 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 and Gα 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.
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http://dx.doi.org/10.1016/j.phrs.2018.02.032DOI Listing
May 2018

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

J Med Chem 2018 03 26;61(6):2266-2277. Epub 2018 Feb 26.

Département de Pharmacologie-Physiologie, Faculté de Médecine et des Sciences de la Santé , Université de Sherbrooke , Sherbrooke J1H 5N4 , Québec , Canada.

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 0.15 nM and t 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.
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http://dx.doi.org/10.1021/acs.jmedchem.7b01353DOI Listing
March 2018

Combination of high-fat/high-fructose diet and low-dose streptozotocin to model long-term type-2 diabetes complications.

Sci Rep 2018 01 11;8(1):424. Epub 2018 Jan 11.

Département de Pharmacologie et Physiologie/Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Québec, Canada.

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 [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.
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http://dx.doi.org/10.1038/s41598-017-18896-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5765114PMC
January 2018

Use of Molecular Modeling to Design Selective NTS2 Neurotensin Analogues.

J Med Chem 2017 04 10;60(8):3303-3313. Epub 2017 Apr 10.

Institut des Biomolécules Max Mousseron, IBMM, UMR-5247, CNRS, Université Montpellier, ENSCM , Place Eugène Bataillon, 34095 Montpellier cedex 5, France.

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 residue of NT[8-13] with an acidic residue (Glu) located in the ECL2 of hNTS2 or with a basic residue (Arg) 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 exhibited higher affinity toward the hNTS1-R212E mutant receptor, in which Arg212 was substituted by the negatively charged Glu residue.
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http://dx.doi.org/10.1021/acs.jmedchem.6b01848DOI Listing
April 2017

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

Eur J Pharmacol 2017 Jun 22;805:1-13. Epub 2017 Mar 22.

Department of Pharmacology-Physiology, Faculty of Medicine and Health Sciences, Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4. Electronic address:

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-, Gα-, Gα-, and Gα-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 and Gα, 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-dependent manner. Additionally, we demonstrated that the full activation of ERK1/2 required signaling through both a PTX-sensitive G-c-Src signaling pathway and PLCβ-DAG-PKC-Raf-1-dependent pathway downstream of G. 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.
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http://dx.doi.org/10.1016/j.ejphar.2017.03.046DOI Listing
June 2017

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

Org Biomol Chem 2017 Jan;15(2):449-458

Département de Pharmacologie-Physiologie, Faculté de Médecine et des Sciences de la Santé, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada. and Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC J1H 5N4, Canada.

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.
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http://dx.doi.org/10.1039/c6ob02247bDOI Listing
January 2017