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

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

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation.

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca2+ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gαq led to a Gαq-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT1R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT1R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gαq-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.

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

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 Gαq and Gα13 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.

Apelin-13 Regulates Vasopressin-Induced Aquaporin-2 Expression and Trafficking in Kidney Collecting Duct Cells.

BACKGROUND/AIMS: Apelin and its G protein-coupled receptor APJ (gene symbol Aplnr) are strongly expressed in magnocellular vasopressinergic neurons suggesting that the apelin/APJ system plays a key role at the central level in regulating salt and water balance by counteracting the antiduretic action of vasopressin (AVP). Likewise, recent studies revealed that apelin exerts opposite effects to those of vasopressin induced on water reabsorption via a direct action on the kidney collecting duct. However, the underlying mechanisms of the peripheral action of apelin are not clearly understood. Here, we thus investigated the role of the apelin/APJ system in the regulation of water balance in the kidney, and more specifically its involvement in modulating the function of aquaporin-2 (AQP2) in the collecting duct. METHODS: Mouse cortical collecting duct cells (mpkCCD) were incubated in the presence of dDAVP and treated with or without apelin-13. Changes in AQP2 expression and localization were determined by immunoblotting and confocal immunofluorescence staining. RESULTS: Herein, we showed that the APJ was present in mpkCCD cells. Treatment of mpkCCD with apelin-13 reduced the cAMP production and antagonized the AVP-induced increase in AQP2 mRNA and protein expressions. Immunofluorescent experiments also revealed that the AVP-induced apical cell surface expression of AQP2, and notably its phosphorylated isoform AQP2-pS269, was considerably reduced following apelin-13 application to mpkCCD cells. CONCLUSION: Our data reinforce the aquaretic role of the apelin/APJ system in the fine regulation of body fluid homeostasis at the kidney level and its physiological opposite action to the antiduretic activity of AVP.

Label-free cell signaling pathway deconvolution of angiotensin type 1 receptor reveals time-resolved G-protein activity and distinct AngII and AngIIIIV responses.

Angiotensin II (AngII) type 1 receptor (AT1R) is a G protein-coupled receptor known for its role in numerous physiological processes and its implication in many vascular diseases. Its functions are mediated through G protein dependent and independent signaling pathways. AT1R has several endogenous peptidic agonists, all derived from angiotensinogen, as well as several synthetic ligands known to elicit biased signaling responses. Here, surface plasmon resonance (SPR) was used as a cell-based and label-free technique to quantify, in real time, the response of HEK293 cells stably expressing the human AT1R. The goal was to take advantage of the integrative nature of this assay to identify specific signaling pathways in the features of the response profiles generated by numerous endogenous and synthetic ligands of AT1R. First, we assessed the contributions of Gq, G12/13, Gi, Gßγ, ERK1/2 and ß-arrestins pathways in the cellular responses measured by SPR where Gq, G12/Rho/ROCK together with ß-arrestins and ERK1/2 were found to play significant roles. More specifically, we established a major role for G12 in the early events of the AT1R-dependent response, which was followed by a robust ERK1/2 component associated to the later phase of the signal. Interestingly, endogenous AT1R ligands (AngII, AngIII and AngIV) exhibited distinct responses signatures with a significant increase of the ERK1/2-like components for both AngIII and AngIV, which points toward possibly distinct physiological roles for the later. We also tested AT1R biased ligands, all of which affected both the early and later events. Our results support SPR-based integrative cellular assays as a powerful approach to delineate the contribution of specific signaling pathways for a given cell response and reveal response differences associated with ligands with distinct pharmacological properties.

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

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

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

Apelin is the endogenous ligand for the G protein-coupled receptor APJ and exerts a key role in regulating cardiovascular functions. We report herein a novel series of macrocyclic analogues of apelin-13 in which the N- and C-terminal residues as well as the macrocycle composition were chemically modified to modulate structure-activity relationships on the APJ receptor. To this end, the binding affinity and the ability to engage G protein-dependent and G protein-independent signalling pathways of the resulting analogues were assessed. In this series, the position and the nature of the C-terminal aromatic residue is a determinant for APJ interaction and ß-arrestin recruitment, as previously demonstrated for linear apelin-13 derivatives. We finally discovered compounds 1, 4, 11 and 15, four potent G protein-biased apelin receptor agonists exhibiting affinity in the nanomolar range for APJ. These macrocyclic compounds represent very useful pharmacological tools to explore the therapeutic potential of the apelinergic system.

Design, synthesis, and biological evaluation of CXCR4 ligands.

A combination of the CXCR4 inverse agonist T140 with N-terminal CXCL12 oligopeptides has produced the first nanomolar synthetic CXCR4 agonists. In these agonists, the inverse agonistic portion provides affinity whereas the N-terminal CXCL12 sequence induces receptor activation. Several CXCR4 crystal structures exist with either CVX15, an inverse agonist closely related to T140 and IT1t, a small molecule; we therefore attempted to produce another CXCL12 oligopeptide combination with IT1t. For this purpose, a primary amino group was introduced by total synthesis into one of the methyl groups of IT1t, serving as an anchoring point for the oligopeptide graft. The introduction of the oligopeptides on this analog however yielded antagonists, one compound displaying high affinity. On the other hand, the amino-substituted analogue itself proved to be an inverse agonist with a binding affinity of 2.6 nM compared to 11.5 nM for IT1t. This IT1t-like analog is hitherto one of the most potent non-peptidic CXCR4 inverse agonists reported.

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

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

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

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 Lys8-Lys9 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 Lys8-Lys9 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.

Structural insights on ligand recognition at the human leukotriene B4 receptor 1.

The leukotriene B4 receptor 1 (BLT1) regulates the recruitment and chemotaxis of different cell types and plays a role in the pathophysiology of infectious, allergic, metabolic, and tumorigenic human diseases. Here we present a crystal structure of human BLT1 (hBLT1) in complex with a selective antagonist MK-D-046, developed for the treatment of type 2 diabetes and other inflammatory conditions. Comprehensive analysis of the structure and structure-activity relationship data, reinforced by site-directed mutagenesis and docking studies, reveals molecular determinants of ligand binding and selectivity toward different BLT receptor subtypes and across species. The structure helps to identify a putative membrane-buried ligand access channel as well as potential receptor binding modes of endogenous agonists. These structural insights of hBLT1 enrich our understanding of its ligand recognition and open up future avenues in structure-based drug design.

Assessing Gαq/15-signaling with IP-One: Single Plate Transfection and Assay Protocol for Cell-Based High-Throughput Assay.

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 Gαq/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 [3H]-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 Gαq/15-coupled 7TMRs.

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

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.

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

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

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

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

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

Cysteinyl leukotriene G protein-coupled receptors CysLT1 and CysLT2 regulate pro-inflammatory responses associated with allergic disorders. While selective inhibition of CysLT1R has been used for treating asthma and associated diseases for over two decades, CysLT2R 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 CysLT2R in complex with three dual CysLT1R/CysLT2R 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.

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

The G protein-coupled cysteinyl leukotriene receptor CysLT1R mediates inflammatory processes and plays a major role in numerous disorders, including asthma, allergic rhinitis, cardiovascular disease, and cancer. Selective CysLT1R 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 CysLT1R 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.

In Search of the Optimal Macrocyclization Site for Neurotensin.

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.

Angiotensin II cyclic analogs as tools to investigate AT1R biased signaling mechanisms.

G protein coupled receptors (GPCRs) produce pleiotropic effects by their capacity to engage numerous signaling pathways once activated. Functional selectivity (also called biased signaling), where specific compounds can bring GPCRs to adopt conformations that enable selective receptor coupling to distinct signaling pathways, continues to be significantly investigated. However, an important but often overlooked aspect of functional selectivity is the capability of ligands such as angiotensin II (AngII) to adopt specific conformations that may preferentially bind to selective GPCRs structures. Understanding both receptor and ligand conformation is of the utmost importance for the design of new drugs targeting GPCRs. In this study, we examined the properties of AngII cyclic analogs to impart biased agonism on the angiotensin type 1 receptor (AT1R). Positions 3 and 5 of AngII were substituted for cysteine and homocysteine residues ([Sar1Hcy3,5]AngII, [Sar1Cys3Hcy5]AngII and [Sar1Cys3,5]AngII) and the resulting analogs were evaluated for their capacity to activate the Gq/11, G12, Gi2, Gi3, Gz, ERK and ß-arrestin (ßarr) signaling pathways via AT1R. Interestingly, [Sar1Hcy3,5]AngII exhibited potency and full efficacy on all pathways tested with the exception of the Gq pathway. Molecular dynamic simulations showed that the energy barrier associated with the insertion of residue Phe8 of AngII within the hydrophobic core of AT1R, associated with Gq/11 activation, is increased with [Sar1Hcy3,5]AngII. These results suggest that constraining the movements of molecular determinants within a given ligand by introducing cyclic structures may lead to the generation of novel ligands providing more efficient biased agonism.

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

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 ( Ki 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.