Modulation of the Neurotensin Receptor Type I by Pepducins Mimicking Its First Intracellular Loop”

Université de Sherbrooke

“Modulation of the neurotensin receptor type I by pepducins mimicking its first intracellular loop”

By German de Armas-Guitart Pharmacology Program

Thesis presented to the Faculty of Medicine and Health Sciences for the obtention of the Master of Science (M. Sc.) degree in Pharmacology

Sherbrooke, Quebec, Canada April 2021

Members of evaluation jury Philippe Sarret, Pharmacology Michel Grandbois, Pharmacology Mannix Auger-Messier, Pharmacology Xavier Roucou, Biochemistry

German de Armas-Guitart, 2021

RÉSUMÉ Modulation du récepteur de la neurotensine de type 1 par des pepducines mimant sa première boucle intracellulaire.’’

Par German de Armas-Guitart Programme de pharmacologie

Mémoire présenté à la Faculté de médecine et des sciences de la santé en vue de l’obtention du diplôme de maîtrise en pharmacologie, Faculté de médecine et des sciences de la santé, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4 Le récepteur de la neurotensine de type 1 (NTS1) appartient à la superfamille des récepteurs couplés aux protéines G (RCPG), qui après son interaction avec son ligand peut exercer différents effets physiologiques tels qu’analgésie, hypothermie et hypotension. La diversité des réponses physiologiques possibles indique que plusieurs voies de signalisation sont activées et que plusieurs effecteurs cytosoliques sont impliqués. Des études précédentes menées avec des conjugués lipopeptidiques, dont la partie peptidique imite la première boucle intracellulaire (ICL1) de NTS1 (également appelées pepducines), ont montré que certaines voies de signalisation sont favorisées ou inhibées, démontrant l'influence de ces conjugués sur l'interaction avec les effecteurs cytosoliques et sur la modulation des voies de signalisation. De plus, l'évaluation in vivo de cette pepducine dans des modèles de douleur aiguë, douleur tonique et douleur chronique a révélé un effet antinociceptif ainsi qu'une forte capacité à diminuer la pression artérielle. Dans cette étude, nous avons évalué l'effet de l'augmentation de l’hydrophobicité des pepducines sur leurs activités biologiques. En conséquence, nous avons généré une série de pepducines basées sur la séquence de ICL1 de NTS1 dans laquelle chaque acide aminé a été remplacé par un tryptophane. L'impact de l’introduction du tryptophane dans la séquence lipopeptidique de nos pepducines a été évalué en mesurant la réponse cellulaire globale par un essai d’impédance électrique. Le profil de signalisation de nos pepducines a aussi été déterminé à l'aide de biosenseurs spécifique BRET (Gα13, Gαq et β-arrestines 1 et 2), qui ont révélé une probable modulation allostérique biaisée. En effet, la substitution de certains résidus semble favoriser l'activation des voies de signalisation Gα13 et Gαq sans recrutement des β-arrestines 1 et 2. De façon intéressante, en utilisant la neurotensine radiomarquée dans un test de liaison compétitif sur le récepteur hNTS1, nous avons également démontré que nos pepducines pouvaient induire le déplacement d’un ligand orthostérique via un site allostérique probablement placé à la surface intracellulaire du récepteur. Enfin, les composés les plus puissants des tests in vitro ont été évalués dans un modèle de douleur aiguë (test de retrait de la queue). L'administration intrathécale des pepducines PP-W11 à 275 nmol/kg a induit une réponse antinociceptive puissante qui a surpassé toutes les réponses analgésiques rapportées par toutes pepducines synthétisées jusqu'à présent. Ces résultats ont confirmé que l'augmentation du potentiel hydrophobe de PP-ICL1 pourrait constituer une approche intéressante dans le raffinement des propriétés des pepducines de NTS1 ainsi que dans la découverte de nouveaux candidats médicaments.

SUMMARY “Modulation of the neurotensin receptor type I by pepducins mimicking its first intracellular loop”

By German de Armas-Guitart Pharmacology Program

Thesis presented to the Faculty of Medicine and Health Sciences for the obtention of the Master of Science (M. Sc.) degree in Pharmacology, Faculty of Medicine and Health Sciences, Université de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4 The neurotensin receptor type I (NTS1) belongs to the G protein coupled receptor (GPCR) superfamily, which induces after ligand-receptor interaction different physiological effects, such as analgesia, hypothermia, and hypotension. The diversity in the physiological response reveals that several pathways are being activated and that several cytosolic effectors are also involved. Previous studies conducted with lipopeptide conjugates whose peptide portion mimics the first intracellular loop (ICL1) of NTS1 (also known as pepducins) have shown that some pathways are favored or inhibited, demonstrating the influence of these ICLs on the interaction with the cytosolic effectors and in the modulation of signaling pathways. The evaluation of this pepducin (PP-ICL1) in acute, tonic, and chronic pain models has revealed that the activation of the Gα13 and Gαq proteins signaling pathway could be associated with an antinociceptive action. In this study, we have evaluated the effect of enhancing the hydrophobic properties in punctual positions of PP-ICL1 sequence on its biological potential. Accordingly, we synthetized a series of pepducins based on the sequence of PP-ICL1 in which each amino acid was substituted by tryptophan. The whole cellular response induced by our pepducins was monitored using electrical cell impedance sensing and used to evaluate the effect of the tryptophan introduction. The signaling profile of the pepducins was determined using BRET biosensors, which revealed a biased modulation. Indeed, the substitution of certain residues seemed to favor the engagement of the Gα13 and Gαq signaling pathways without causing any effect on the recruitment of β- arrestins 1 and 2. Using a radiolabeled probe in a competitive radioligand binding assay on the hNTS1 receptor, we demonstrated that our pepducins could displace the bounded orthosteric ligand by probably acting in an allosteric site. Finally, the most potent compounds of this series tested in vitro were screened in a model of an acute pain model (tail-flick test). Intrathecal administration of pepducins PP-W11 at 275 nmol/kg induced a potent antinociceptive response which outperformed all the analgesic responses reported by any pepducin synthesized so far. Taken together, these results confirm that enhancing selectively the hydrophobic potential of PP-ICL1 could constitute a valid approach in the refinement of these pepducin properties as well as in the discovery of new drugs candidates.

III

TABLE OF CONTENTS

RÉSUMÉ ...... II SUMMARY ...... III TABLE OF CONTENTS ...... IV LIST OF FIGURES ...... VII LIST OF TABLES ...... VIII LIST OF ABBREVIATIONS ...... IX Remerciement ...... XI INTRODUCTION ...... 1 1.1 Transduction of biochemical signals by cell membrane receptors ...... 1 1.1.1 G protein-coupled receptors (GPCRs) ...... 1 1.1.2 GPCR structures and classification ...... 1 1.1.3 GPCR activation ...... 3 1.1.4 Rearrangement associated with GPCR activation ...... 5 1.1.5 Intracellular structural elements involved in the activation of the G proteins ...... 6 1.1.6 Heterotrimeric G proteins and functional cycle ...... 7 1.1.7 Canonical G protein signaling pathways ...... 8 1.1.8 β-arrestin signaling ...... 9 1.1.9 Receptor oligomerization ...... 11 1.2 The neurotensinergic system and the NTS1 receptor ...... 12 1.2.1 Neurotensin discovery, structure, and biosynthesis ...... 12 1.2.2 Discovery and distribution ...... 13 1.2.3 NT receptors ...... 14 1.2.4 NTS1 signaling ...... 15 1.2.5 Physiological effects of the NT ...... 17 1.2.6 Agonist modulators of NT receptors ...... 18 1.2.7 Neurotensin antagonists ...... 20 1.3 Pepducins ...... 21 1.3.1 Pepducin generalities ...... 21 1.3.2 Pepducins structural elements ...... 21 1.3.3 Mechanism of action of pepducins ...... 23 1.3.4 Mandatory presence of GPCR for pepducin mechanisms of action ...... 24

1.3.5 Further insight in pepducins mechanism of action ...... 25 1.3.6 Specificity and selectivity of pepducins for its receptor ...... 26 1.3.7 Agonist effects of pepducins ...... 26 1.3.8 Antagonist effects of pepducins ...... 27 1.3.9 Therapeutics applications of pepducins ...... 28 1.3.10 Pharmacokinetic and biodistribution of pepducins ...... 29 1.4 Basic principles of solid-phase peptide synthesis ...... 30 1.4.1 The strategy of Fmoc/t‑Bu solid-phase peptide synthesis ...... 31 1.4.2 Solid supports and linkers used in SPPS ...... 32 1.4.3 Side chain protecting groups ...... 33 1.4.4 Amino acid coupling step ...... 34 1.4.5 Racemization ...... 35 1.4.6 Aggregation ...... 35 1.4.7 Fmoc deprotection ...... 36 1.4.8 Peptide precipitation ...... 36 1.5 Problematic, hypothesis, and objectives ...... 37 MATERIALS AND METHODS ...... 39 2.1 Materials ...... 39 2.2 Peptides synthesis ...... 39 2.3 Peptides purification ...... 40 2.4 Cell culture and transfections ...... 40 2.5 Whole cellular response measured through a label-free phenotypical assay ...... 41 2.6 Bioluminescence Resonance Energy Transfer (BRET) ...... 42 2.7 Competitive Radioligand Binding Assay on the hNTS1 receptor ...... 44 2.8 In vivo experiments ...... 45 2.8.1 Animals, housing, and habituation ...... 45 2.8.2 Intrathecal administration ...... 45 2.8.3 Tail-flick test ...... 45 2.8.4 Body temperature measurements ...... 46 2.8.5 Blood pressure monitoring ...... 46 2.9 Data and statistical analysis ...... 47 RESULTS ...... 48 3.1 Pepducins design and synthesis ...... 48

3.2 Cell response measured through a label-free phenotypical assay ...... 49 3.3 Signaling profiles of pepducins derived from the ICL1 of hNTS1 ...... 52

3.3.1 Signaling pathways associated to the Gα13 subunit ...... 52

3.3.2 Signaling pathways linked to the Gαq subunit ...... 54 3.3.3 Non-canonical G protein signaling pathways ...... 55 3.4 Effect of pepducins on NT binding to NTS1 ...... 57 3.5 In vivo physiological effects of pepducins ...... 58 3.5.1 In vivo physiological effects, analgesic effects ...... 58 3.5.2 In vivo physiological effects, hypothermic effects ...... 61 3.5.3 In vivo physiological effects, hypotensive effects ...... 62 DISCUSSION ...... 63 4.1 Synthesis of pepducins derived from the ICL1 of hNTS1 ...... 63 4.2 Cell response measured through a label-free phenotypical assay ...... 64

4.3 Signaling pathways associated to G proteins and β-arrestins 1 and 2 ...... 65 4.4 Effect of pepducins on NT binding to NTS1 ...... 67 4.5 In vivo physiological effects of pepducins ...... 68 4.5.1 In vivo physiological effects of pepducins: Analgesic effects ...... 68 4.5.2 In vivo physiological effects of pepducins: Hypotensive effects ...... 70 4.5.3 In vivo physiological effects of pepducins: Hypothermic effects ...... 71 4.5.4 Further insight into the tryptophane derivatives ...... 71 CONCLUSIONS ...... 73 LIST OF REFERENCES ...... 74

LIST OF FIGURES

Figure 1: Structure and topology of GPCRs ...... 2 Figure 2: Schema of the ligand-receptor interaction for the two-states receptor theory ...... 4 Figure 3: Schema of the ligand-receptor interaction for the ternary complex theory ...... 5 Figure 4: Active and inactive conformation of β2AR ...... 6 Figure 5: Schematic representation of the canonical GPCRs signaling pathway ...... 9 Figure 6: Schematic representation of β-arrestin recruitment and GPCR endocytosis ...... 11 Figure 7: Schematic representation of the lipopeptide structure of a pepducin ...... 21 Figure 8: Actual model proposed to explain the mechanism of action of pepducins ...... 24 Figure 9: Schematic representation of Fmoc/t‑Bu strategy on SPPS ...... 32 Figure 10: Amine nucleophilic attack of on the acyl moiety to produce an amide...... 34 Figure 11: Mechanisms of base-catalyzed racemization during the activation ...... 35 Figure 12: Deprotection mechanism of the Fmoc group ...... 36 Figure 13: BRET principle applied to study the interaction between GPCRs and heterotrimeric proteins ...... 43 Figure 14: Electric cell-substrate impedance sensing assays exhibiting the global morphological changes in response to treatment with pepducins derived from the ICL1 of hNTS1 ...... 51

Figure 15: Gα13 subunit engagement induced by pepducins derived from the ICL1 of hNTS1 ...... 53

Figure 16: Gαq subunit engagement induced by pepducins derived from the ICL1 of hNTS1 ...... 55 Figure 17: β-arrestin 1 and 2 recruitment induced by pepducins derived from the ICL1 of hNTS1 . 56 Figure 18: Displacement curves of [125I] Neurotensin on hNTS1 by NT (8-13) and pepducins...... 57 Figure 19: Analgesic effect of acute intrathecal injection of SCR peptide, PP-ICL1, PP-W5, PP-W11, PP-W13 at 275 nmol/kg (A) and 100 nmol/kg (B) on tail-flick latencies in Sprague-Dawley rats ...... 59 Figure 20: Analgesic effect of acute intrathecal injection of increasing doses of PP-W11 on tail-flick latencies in Sprague-Dawley rats ...... 60 Figure 21: Variation of body temperature monitored after intrathecal injection of pepducins ...... 61 Figure 22: Delta mean arterial blood pressure ...... 62

VII

LIST OF TABLES

Table 1: Position of the amino acid substitution into the peptide sequence of pepducins derived from the ICL1 of hNTS1 ...... 49 Table 2: Maximal response of pepducins derived from the ICL1 of hNTS1 in the electric cell- substrate impedance sensing assay ...... 52

Table 3: Efficacy and potency of pepducins derived from the ICL1 of hNTS1 to engage the Gα13 subunit...... 54

Table 4: Efficacy and potency of pepducins derived from the ICL1 of hNTS1 to engage the Gαq subunit...... 55

Table 5: NT (8-13) and pepducins binding affinity on hNTS1. IC50 was derived from the resulting dose-response curves ...... 58 Table 6: Physiologic effect of pepducins in vivo...... 72

VIII

LIST OF ABBREVIATIONS

AC Alternating current α2-AR α2- adrenergic receptor ACN Acetonitrile ATP Adenosine triphosphate β2AR β2-adrenergic receptor BRET Bioluminescence resonance energy transfer BSA Bovine serum albumin cAMP Cyclic adenosine monophosphate Ca2+ Calcium CHO Chinese hamster ovarian cells CNS Central nervous system CXCR1/2 C-X-C chemokine receptor type 1, type 2 CXCR4 C-X-C chemokine receptor type 4 DAG Diacylglycerol DCM Dichloromethane DIPEA Diethylisopropylamine DMEM-F12 Dulbecco’s modified eagle medium: Nutrient mixture F-12 DMF Dimethylformamide DMR Dynamic mass redistribution DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid EC50 Half-maximal effective concentration ECIS Electric cell-substrate impedance sensing ECL Extracellular loop EDTA Ethylenediaminetetraacetic acid ERK Extracellular signal-regulated kinase FBS Fetal bovine serum FMOC Fluorenylmethyloxycarbonyl chloride FRET Fluorescence resonance energy transfer FLC Phospholipase C GEF Guanine nucleotide exchange factor GDP Guanosine diphosphate GFP1 Green fluorescent protein variant 1 GPCR G protein-coupled receptor GRK GPCR kinase GTP Guanosine triphosphate HATU 1-[Bis(dimethylamino)methyle]-1H-1,2,3-triazolo[4,5-b] pyridinium 3-oxide hexafluorophosphate HBSS Hank’s balanced salt solution HCL Hydrochloric acid HEK293 Human endothelial kidney cells HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid HPLC High performance liquid chromatography ICL Intracellular loop iNOS Nitric oxide synthase i.v. Intravenous

i.t. Intrathecal i.v.c. Intracerebroventricular IP1 Inositol monophosphate IP3 Inositol triphosphate K+ Potassium MAPK Mitogen-activated protein kinase MS Mass spectroscopy NAM Negative allosteric modulator NN Neuromedin N NPxxY Asparagine-Proline-xx-Tyrosine motif NT Neurotensin NT (1-13) Neurotensin 1-13 NT (8-13) Neurotensin 8-13 NTS1 Neurotensin receptor type 1 NTS2 Neurotensin receptor type 2 NTS3 Neurotensin receptor type 3 NTS4 Neurotensin receptor type 4 PAM Positive allosteric modulator PAR1 Protease-activated receptor type 1 PAR2 Protease-activated receptor type 2 PAR4 Protease-activated receptor type 4 PBS Phosphate-buffered solution PEI Polyethylenimine PIP2 Phosphatidylinositol biphosphate PKA Protein kinase A PKC Protein kinase C PKD Protein kinase D PLC Phospholipase C PSG Penicillin-streptomycin-glutamine RCPG Récepteur couplé à une protéine G RhoGEF Rho guanine nucleotide exchange factor RlucII Renilla luciferase II enzyme S.E.M. Standard error of the mean SSTR2 Somatostatin receptor type 2 TBME Tert-butyl methyl ether TFA Trifluoroacetic acid TIPS Triisopropylsilane TM Transmembrane helical domain UPLC Ultraperformance liquid chromatography WxP Tryptophan-x-Proline motif

Remerciement Tout d’abord, j’aimerais exprimer ma profonde gratitude à mon directeur de recherche, le professeur Eric Marsault, pour m’avoir permis de travailler sur ce projet et dans son laboratoire. Eric, même si tu n’es plus là pour te remercier pour ta supervision et tes conseils, merci pour la confiance que tu m’as toujours accordée, ma progression scientifique et personnelle ces dernières années a été largement possible grâce à toi. Travailler avec toi a été une expérience enrichissante et grandissante. Merci, Eric, pour ton amitié avant tout, te rencontrer a été un grand honneur, tu nous manqueras mon ami. Je tiens également à remercier mes deux autres directeurs de recherche, les professeurs Michel Grandbois et Philippe Sarret, vous avez été un roc solide sur lequel j’ai pu m’appuyer à la mort d’Eric. Merci d’être là pour moi. Philippe merci de m’avoir permis de faire des recherches dans votre laboratoire, vos conseils m’ont toujours aidé à être meilleur, merci surtout pour votre attitude cordiale. Michel honnêtement, si j’écris ça aujourd’hui, c’est grâce à chaque instant que vous m’avez accordé. Sur le plan académique, vous êtes l’une des personnes qui ont le plus contribué à ma formation, merci beaucoup pour cela. Je suis également extrêmement reconnaissant à chacune des personnes qui ont contribué d’une manière ou d’une autre à cette thèse: Alex Murza pour les premiers conseils en chimie peptidique, Rebecca Brouillette et Magali Chartier tout au long des analyses in vitro et in vivo ainsi que pour votre soutien inconditionnel, Ulrike Fröhlich et Elie Besserer-Offroy. Je tiens également à remercier les gars du laboratoire Marion, Julien, Iryna, Runjun, Huy, Etienne, Abdou et Sandra. Vous avez rendu les choses plus faciles et plus amusantes. Merci aussi aux professeurs Mannix Auger-Messier et Xavier Roucou d’avoir accepté d’évaluer ce mémoire. Enfin, je tiens à remercier trois personnes extraordinaires, mes parents et ma petite sœur: Mama, papa y Agnie. Vosotros no por ser los últimos sois menos importantes. En realidad, sois quienes más habéis contribuido, todo cuanto soy y podre ser os lo debo. Os debo tanto que siempre os estaré agradecido. Vosotros sois el centro de mi universo. Os quiero mucho.

Deus meus, gratias ago tibi, quia sapientiam et fortitudinem dedisti mihi.

INTRODUCTION 1.1 Transduction of biochemical signals by cell membrane receptors 1.1.1 G protein-coupled receptors (GPCRs) Biosignaling in cells is largely mediated by protein-based membrane structures responsible for the transduction of extracellular stimuli. These supramolecular transductors, commonly named membrane receptors, can be classified in one of the following four protein families: G protein-coupled receptors, ionotropic receptors, kinase linked receptors, and nuclear hormone receptors (Nelson and Cox, 1993). GPCRs represent the largest family of proteins in vertebrate species, exceeding the number of 800 members encoded in the human genome (Fredriksson et al., 2003). These receptors recognize a large variety of ligands including ions, organic odorants, amines, peptides, proteins, lipids, nucleotides, and photons and act by prompting signaling cascades in the cytosol (Schiöth and Fredriksson, 2005). GPCRs modulate several intracellular signaling cascades linked to numerous human pathophysiological processes, which make of GPCRs an important pharmacological target. Accordingly, 34% of all the drugs approved by the FDA have been designed to act over a GPCRs family member (Hauser et al., 2017).

1.1.2 GPCR structures and classification GPCRs display some similarities across all the family members, despite the great diversity of ligands and responses triggered following their activation. The most common feature in the structure of GPCRs is the presence of a hydrophobic core formed by seven transmembrane α-helices which are constituted of approximately 25 to 35 amino acids (Figure 1A) (Kobilka et al., 1987). In this arrangement, the C-terminus is in the intracellular compartment whereas the N-terminus faces the extracellular compartment. The α-helix are inter-connected by three intracellular loops (ICL) and three extracellular loops (ECL) in a counterclockwise manner (Figure 1B) (Schiöth and Fredriksson, 2005). The ECL and the external portion of the membrane bundle form a cavity responsible for ligand recognition. Some key residues in ICL and the C-terminal sequence recognize cytoplasmic effectors like heterotrimeric G proteins, β-arrestins, and dissimilar receptor kinases (GRKs) through which the regulation of the second messengers occurs (Lefkowitz, 2007).

Figure 1: Structure and topology of GPCRs A. GPCRs are made of seven transmembrane helices (gray), three extracellular loops (ECLs) and an amino terminus (orange), and three intracellular loops (ICLs) and a carboxyl terminus (purple). B. Cartoon representation of the β2 adrenergic receptor (β2AR). Figure reproduced from (Latorraca et al., 2017)

GPCRs also present highly preserved structural motifs that play an essential role in the transition from an inactive conformation to an active one upon binding of the ligand. D[E]RY is a key peptide sequence located in the helix III which forms a salt bridge with a residue of glutamate or aspartate in the helix VI. This salt bond is associated with an inactive conformation of GPCRs since it acts by hindering the interaction between the receptor and the heterotrimeric G proteins (Palczewski, 2000). Two other highly conserved motifs located in the transmembrane core, the WxP sequence in helix VI and the sequence NPxxY in helix VII, are also involved in the generation of a GPCRs active state (Zhang et al., 2014). Conserved motifs are not solely restricted to transmembrane domains, but also found in extracellular space. The cysteine (Cys) in the extra-cellular tip of helix III and a Cys in ECL2, delimit the ligand-binding site through the formation of a disulfide bond. In addition, the [F(RK)xx(FL)xxx] sequence located in the C-terminus of the receptors has been found in almost all GPCRs characterized so far (Zhang et al., 2014).

GPCRs can be classified in 5 distinct categories based in the sequence homology: Glutamate, Rhodopsin, Adhesion, Frizzled/Taste2, and Secretin (Fredriksson et al., 2003). Although all

the GPCRs members share structural features, the homology of their transmembrane domains is less than 20% which is reflected in different angles of inclination adopted by the transmembrane segments or in the dimensions and shapes that their helixes can exhibit (Katritch et al., 2013). The intracellular region is characterized by a high degree of variability mainly manifested at the level of the secondary structures that the ICLs show. Similarly, the ligand pocket and the N-terminus are variable throughout the subfamilies (Zhang et al., 2014).

1.1.3 GPCR activation Ligand binding and subsequent receptor activation is a complex process that has been explained using several models. GPCRs activation considers the existence of two well- defined receptor states; an active state (R*), producing an intracellular response and an inactive state or GPCR basal state (R) which elicits no cellular response (Figure 2). The two- state receptor theory assumes that the stimulator effect performed by the ligand is an essential condition to ensure the transition from one state to the other (Leff, 1995). According to this model, the ligands are classified in three categories; agonists which are molecules that bind the receptor in the orthosteric site and act by inducing a conformational change that leads the receptor to an active state. Antagonists, which block the orthosteric site, thus preventing other compounds from accessing this cavity and keep the receptor in the basal state. Finally, inverse agonists which promote the switch from the active state, that may arise spontaneously, to the GPCRs basal configuration (Bridges and Lindsley, 2008).

Figure 2: Schema of the ligand-receptor interaction for the two-states receptor theory The binding of an agonist (A) to the receptor (R) leads to the complex (AR). This complex evolves to (AR*), which represents the active state of the receptor (R*). The binding of an antagonist (B) does not lead to a receptor active state. k+1, k-1, α and β are the rate constants for the binding and activation steps. β =0 when the molecules are antagonists.

The diversity in the mechanisms of interactions between ligand and receptor gave rise to a more complete model to describe the effects of a second molecule on the complex ligand- receptor. The ternary complex model (Figure 3) describes the presence of other binding sites in the GPCRs structure named allosteric binding sites (Kenakin, 2001). An allosteric modulator may either enhance a G protein signaling pathway or completely inhibit it. In the first case the compound would be classified as a positive allosteric modulator (PAM) and in the second case a negative allosteric modulator (NAM). This model also suggests the existence of an allosteric agonist capable of triggering a complete activation of a signaling pathway in the absence of the GPCRs orthosteric natural ligand (Bridges and Lindsley, 2008). As presented above, the two-state receptor model implies the existence of only two possible GPCR conformations. However, the ternary complex model relates the number of conformational states to the number of allosteric modulators and endogenous ligands that can bind to the GPCR. Similarly, in this model the prevalence of a GPCR conformation and therefore of a G protein signaling pathway, would be determined by the combination of the bound ligand and the allosteric modulator (Bridges and Lindsley, 2008). Currently, GPCRs are conceived as highly dynamic structural entities in which a vast range of conformations coexists, each of which can contribute to a given cellular response. Accordingly, a new type of ligand called biased modulator has been proposed. A biased modulator can bind to a

GPCR and induces a conformational change capable of activating not only one signaling pathway, but several of them with various efficacies (Luttrell, 2014).

Figure 3: Schema of the ligand-receptor interaction for the ternary complex theory The binding of an agonist (A) to the receptor (R) leads to the active receptor state (AR*), which can be modulated by allosteric ligand (G). The factor α designates the effect of an allosteric modulator on the equilibrium dissociation constant (K). Generally, α>1.0 refers to PAM, α<1.0 to NAM, whereas α=1 refers to an allosteric modulator that do not affect the equilibrium dissociation constant. Figure reproduced from (Bridges and Lindsley, 2008).

1.1.4 Rearrangement associated with GPCR activation The transition of the GPCRs from an inactive to an active state is characterized by highly preserved changes in the spatial arrangement of certain GPCR structural motifs. Specifically, the most marked feature of this transition is the displacement undergone by the transmembrane helices (Schwartz et al., 2006). Crystallographic studies have confirmed that helix 5 and helix 6 leaning towards the intracellular cell surface during the receptor activation. Indeed, the α-helices displacement from the inactive state to the active was found to be around 3.5 Å in the A2A adenosine receptor (A2AAR); approximately 6 Å in opsin receptor and between 11 Å and 14 Å in beta 2-adrenergic receptor (β2AR) in complex with nanobody and G proteins (Figure 4a and 4b)(Rasmussen et al., 2011b). Similarly, TM3 and TM7 experience an appreciable shift in A2AAR when the receptor changes to the active state. Specifically, the conserved motif NPxxY located in the helix 7 undergoes considerable changes at the level of its secondary structure which could be associated with the modulation of the β-arrestins pathway (Katritch et al., 2013).

Figure 4: Active and inactive conformation of β2AR a) Superposition of the conformational changes undergone by complex β2AR-Gs (green) with the inactive carazolol-bound β2AR structure (blue). b) Superposition of the β2AR-Gs structure with the nanobody-stabilized active state of β2AR-Nb80. Figure reproduced from (Rasmussen et al., 2011b)

1.1.5 Intracellular structural elements involved in the activation of the G proteins Binding of the orthosteric ligand to the receptor binding site triggers a conformational change which is transmitted to the transmembrane helices. Those changes affect the conformation of the intracellular surface of the receptor, in particular the surface residues involved in the interaction with heterotrimeric G proteins. Structural studies have highlighted the engagement of the C-terminal sequences of the ICL2 and the ICL3 in the interaction with G proteins (Wess, 1997). Likewise, the ICL1 as well as the intracellular C- terminus has been implicated in the interface of contact between the G protein and the receptor (Oldham and Hamm, 2008).

In the crystal structure obtained from the complex formed between α2-AR and the subunit

Gαs, the contact interface is made of a contribution of the whole ICL2 and a portion of the α-helix 5 and 6 (TM5 and TM6) provided by the receptor. The heterotrimeric G protein contributes to this interface with the α -helix 4 and 5 and the domain of interchange of GDP

(Rasmussen et al., 2011a). Additionally, the α2-AR-Gαs complex is stabilized by electrostatic and polar interaction of the acid, basic and polar residues placed in the contact interface of the aforementioned structural elements (Syrovatkina et al., 2016).

1.1.6 Heterotrimeric G proteins and functional cycle Heterotrimeric G proteins work as mediators between an active conformation of the receptor and cytosolic effectors (Gilman, 1987). G proteins are heterocomplex consisting of three subunits α, β, and γ. The Gα subunits can be classified into four main families Gαs, Gαi,

Gαq, and Gα12 which is based on the homology they show in their sequences and their effect on the signaling pathways. In each family, subclassifications give a measure of complexity, as well as the variety of second effectors that they can modulate (Syrovatkina et al., 2016). Gα subunits have been extensively studied, crystallography analysis has revealed the presence of two main domains: a GTPase domains which has a Ras-like structure and whose function is precisely associated with the hydrolysis and exchange of this nucleotide, and an α-helical rich domain which serves as a scaffold for recognition and interaction between GPCRs and βγ subunits (Sprang et al., 2007).

In contrast, the subunits β and γ do not exhibit GTPase activity but they can regulate the downstream signaling in a binary complex form. The β subunits are represented in the human genome by five different proteins subfamilies named Gβ1, Gβ2, Gβ3, Gβ4, and Gβ5. There are a total of 12 genes coding for the Gγ subunits (Cherfils and Chabre, 2003). Interestingly, even if the different subfamilies of subunits β and γ can combine in different manners, this does not seem to significantly affect the signaling profile. Crystallographic studies of the β-subunits have demonstrated the prevalence of β-sheets which fold into a heptagonal arrangement. Each side of the heptagon is made up of an average of 4 strands of beta-sheets. The Gγ subunits are mainly made of two α-helixes which show a potent affinity for the Gβ subunits (Katritch et al., 2013). In the basal state of the receptor, the three subunits of the G proteins are assembled as a heterogenous trimeric complex (αβγ complex) which binds tightly the GDP molecule (Kobilka, 2007). The transition to the GPCRs' active conformational state implies a loss of affinity of the complex GPCR-αGDPβγ for the GDP, which releases the nucleotide from its binding site. The ligand-receptor interaction triggers the dissociation of GDP, thus acting as a nucleotide exchanging factor (Syrovatkina et al., 2016). Subsequently, a new GTP molecule binds the α subunit, which displays a high affinity for GTP. This binding destabilizes the complex receptor-αGTPβγ which dissociates in

the αGTP and the βγ binary subunits (Oldham and E. Hamm, 2006). After dissociation, the

αGTP subunit recovers the GTPase activity and hydrolyzes the bound GTP to GDP. Finally,

αGDP reassociates with the βγ subunit, which leads to reassembly of the initial complex

GPCR-αGDPβγ (Syrovatkina et al., 2016).

1.1.7 Canonical G protein signaling pathways GPCR-dependent signaling is not monodirectional and a specific receptor conformation can activate distinct heterotrimeric G proteins pathways. The G-proteins have been linked to the presence of specific second messengers and to the subsequent activation of downstream effectors (Wettschureck and Offermanns, 2005). The classification of the α- subunit as Gαs and Gαi was assigned considering their effects on the adenyl cyclase, which is their common natural target (Figure 5). The subunit Gαs stimulates the adenyl cyclase located on the cell membrane to hydrolyze the adenosine triphosphate (ATP) to 3’,5’- cyclic AMP (cAMP) and pyrophosphate. cAMP acts as an intracellular second messenger activating other cAMP effector proteins like the protein kinase A, cyclic nucleotide-gated channels, and exchange factors. In contrast, the subunit Gαi is associated with the inhibition of the adenyl cyclase and provokes a reduction in the intracellular concentrations of cAMP (Syrovatkina et al., 2016; Wettschureck and Offermanns, 2005).

The common target of the members of the Gαq/Gα11 family is the phospholipase C (PLC), specifically the β-isoform of the enzyme. Besides, PLC works in the interface cytosol- membrane hydrolyzing the phosphodiester bond of the phosphatidylinositol-4,5- bisphosphate (PIP2) and producing diacylglycerol (DAG) and inositol-1,4,5-trisphosphate

(IP3). DAG and IP3 are the second messengers of Gαq/Gα11 which modulate the ionic

2+ channels of Ca sensitive to IP3 located on the surface of the endoplasmic reticulum (ER) and the protein kinase C activated by DAG (Rhee and Bae, 1997).

The subunits Gα12/Gα13 perform their modulator effect on a wide variety of downstream targets including the Na/H exchanger, the c-jun NH2-terminal kinase, and the phospholipase

A2 which is responsible for the cleavage of membrane phospholipids at the second ester bond producing arachidonic acid and lysophosphatidic acid. Besides, Gα12/Gα13 has been

associated with the dynamism of the cytoskeleton by activating on the GTPase RhoA which is associated with the contractility of the actomyosin filament. However, the effect of

Gα12/Gα13 on RhoA is not direct and the nucleotide exchange factors Gα12:RhoGEF and

Gα13:RhoGEF are required for its activation (Ritchie et al., 2013)

On the other hand, the binary complex Gβγ has been linked to the modulation of various downstream effectors including the phospholipase Cβ, adenylyl cyclase, voltage-gated Ca2+ channels, and rectifying K+ channels. (Khan et al., 2013).

Figure 5: Schematic representation of the canonical GPCRs signaling pathway Upon activation by a GPCR, the subunits Gα dissociate from Gβγ, mediating the regulation of downstream effectors. Figure reproduced from (Wootten et al., 2018)

1.1.8 β-arrestin signaling The signal transduction cascades linked to the activation of GPCRs occurs in a bimodal way which can be dependent on G proteins or dependent on β-arrestins (Figure 6) (Jean-Charles et al., 2017). Initially, it was thought that the main function of β-arrestins was to mediate GPCRs desensitization by acting as an adaptor in the process of internalization. According to this view, β-arrestins sterically hinder the interaction between a target GPCR and G proteins. Moreover, they may also disrupt the engaged pathways by recruiting

diacylglycerol kinases and cyclic nucleotide phosphodiesterases which cleave the second messenger (Reiter et al., 2012; Shenoy and Lefkowitz, 2011).

β-arrestins also participate in downstream signaling pathways independently of G protein signaling activities. The complexity of β-arrestins regulation is due to different patterns of GRK-dependent phosphorylation of Ser and Thr residues in the C-terminus of the target GPCR, but also in the ICLs. This phosphorylation code determines the specificity of the binding performed by β-arrestins on a target GPCR, as well as the conformation adopted by the β-arrestins which regulates the coupling with cytosolic effectors (Lefkowitz, 2005). Accordingly, β-arrestins can exert their effect directly by scaffolding and binding components of the kinase cascade, among which can be cited the mitogen-activated protein kinases (MAPKs) and serine/threonine kinases including ERK1/2, p38 kinases, and the c-Jun N-terminal kinases that are activated by an upstream protein kinase which mediates their phosphorylation (DeWire et al., 2007).

β-arrestins can also contribute to the maintenance of the signal transduction cascades induced by the complex ligand-receptor after its internalization following an endosomal pathway. Luttrell et al. demonstrated the activation of ERK induced by the non-receptor tyrosine kinase protein, c-Src, after the endocytosis of the complex β2AR-β-arrestin. To confirm the endosomal β-arrestin-dependent activation of ERK they used a mutant of β- arrestin 1 which retained the binding capacity without causing internalization, which was not able to activate the MAPK protein (Luttrell et al., 1999). Altogether, these results suggested that β-arrestins trigger changes by recruiting signalosome complexes, which are

supramolecular arrangements of GPCRs and other proteins responsible of regulating the activity of effectors (Eichel et al., 2016).

Figure 6: Schematic representation of β-arrestin recruitment and GPCR endocytosis (1) Upon ligand activation, the subunits Gα and Gβγ dissociate, and (2) the GPCR is phosphorylated by GRK. (3) The phosphorylation induces the recruitment of β-arrestin which serves as scaffolding for the assembly of the clathrin and the endocytosis machinery. (4) Endocytosis and endosomal signaling. Figure reproduced from (Tian et al., 2014).

1.1.9 Receptor oligomerization Our conception about GPCRs as monomeric units capable to accomplish all the molecular events ranging from the ligand's recognition to the transduction of the signal has changed during the two decades. GPCRs can form oligomer structures throughout the GPCR families (Gahbauer and Böckmann, 2016). Indeed, within certain families, the presence of dimers, homo-oligomers, hetero-oligomers, and other products of GPCRs oligomerization is a general feature. GPCR family C members stand out for the ability to form oligomeric units and the formation of this supramolecular complex is essential to ensure their functionality (Kniazeff et al., 2011). Mutagenesis studies have demonstrated the obligatory dimeric existence of the metabotropic glutamate receptor. The substitution of the Cys 121 which is essential in the process of homodimerization by an Ala residue produced a mutant unable to couple with the G proteins, which corroborated the importance of a dimeric arrangement to ensure the receptor activity (El Moustaine et al., 2012; Kniazeff et al., 2004). The γ- aminobutyric acid (GABA) receptor exists as a heterodimeric entity formed by the GABA-R1 and GABA-R2. Angers et al, making use of the bioluminescence resonance energy transfer

(BRET) were the first to report that the β2-adrenergic receptor, as a member of the family A of GPCRs, also possessed the ability to form dimeric structures in living cells (Angers, 2000). Interestingly, the formation of dimeric structures appears to be associated with an allosteric effect of each monomeric unit on the other. This allosteric influence has been confirmed in the heterodimer formed by the ligand-free ghrelin receptor (apo-GHSR1a) and the dopamine D2 receptor. In a monomeric state, the activation of the dopamine D2 receptor is associated with the coupling to the Gαi/o units. However, upon apo-ghrelin GHSR1a receptor allosteric modulation, the signal transduction cascade of the D2 receptor

2+ leads to the modulation of the Ca concentration in an independent Gαi/o units manner (Kern et al., 2012). It's worth mentioning that this allosteric crosstalk between two different receptor systems has constituted an indication of the possible rearrangement of GPCRs as heteromeric complex. The hetero-oligomers discovered this way include the dopamine D2/adenosine A2A receptor, the heterodimers between the subtypes δ and κ of the opioid receptors, and the pair serotonin 5-HT2A receptor- mGlu2 (Gaitonde and González-Maeso, 2017).

1.2 The neurotensinergic system and the NTS1 receptor 1.2.1 Neurotensin discovery, structure, and biosynthesis Neurotensin (NT) is the endogenous ligand of the neurotensin receptors (NTS), which are members of the family of GPCRs. The biosynthesis of the NT starts with an initial protein precursor whose sequence shows a high degree of homology in different animal species. This 170-amino acid precursor displays two functional units. The NT sequence is located at the C-terminus of the precursor and it follows the sequence of another peptide, neuromedin (NN) (de Nadai et al., 1989). Both peptides partake the last four residues and their sequences homology suggests that they arose from a tandem duplication of an ancestral genetic sequence (Kislauskis et al., 1988). During the maturation process, both peptides are separated by the action of specific tissue endopeptidases belonging to the convertase family, which recognize a cleavage point marked by the pair Arg-Lys at the beginning of each peptide sequence (Vincent et al., 1999). Posterior studies have revealed that the tissue expression levels of NT and NN, will depend on the cleavage point recognized

by the endopeptidase which will be prone to favor the prevalence of one peptide over the other. However, the co-expression in the same tissue remains high (Kitabgi, 2006).

1.2.2 Discovery and distribution NT was first isolated as a peptide of 1.6 kDa being a concomitant product in the purification of the substance P extracted from bovine hypothalami. The elucidation of the NT structure was done during the earlies 1970 by Carraway and Leeman. Using an approach that involved the Edman degradation, they identified the following sequence (pGlu-Leu-Tyr-Glu-Asn-Lys- Pro-Arg-Arg-Pro-Tyr-Ile-Leu). The structure-activity relationships (SAR) studies have confirmed that most, if not all, the activity relies on the last six amino acids NT(8–13), even though this fragment is not found in nature. (Carraway and Leeman, 1975). NT induces appreciable vasodilatation in the cutaneous region when is administrated i.v. in anesthetized rats (Carraway and Leeman, 1973). NT is equally capable of causing a strong contraction of the rat uterus and guinea pig ileum, as well as a relaxation of the rat duodenum. Those features allows to classify it within the family of kinin peptides (Webster, 1966). NT presence has been reported in all the distinct animal models where it has been sought. Using immunohistochemical and in situ localization experiments Kislauskis et al., have mapped the tissue-specific expression of NN and NT. NT has been identified in the periphery as well as in the central nervous system (Kislauskis et al., 1988). The expression in the hypothalamus is 30 times higher compared to the cortex and 5,7 greater than in the brain stem. In the central nervous system (CNS) NT serves as a neurotransmitter or neuromodulator specifically in the dopaminergic transmission (Geisler et al., 2006). NT is equally associated with hypothermia, antinociception, and stimulation of anterior pituitary hormone (Nemeroff et al., 1982). Peripherally, NT is abundant in the gastrointestinal tract, specifically in endocrine-like cells localized in the intestinal mucosa, whereby it is secreted due to the luminal presence of lipids after food ingestion. NT effects are also linked to the paracrine and endocrine regulation of gastrointestinal motility and secretion (Kalafatakis and Triantafyllou, 2011).

1.2.3 NT receptors The physiological effects mediated by neurotensin involves four subtype receptors (Kleczkowska and Lipkowski, 2013). Radioligand binding assays evidenced the existence of these NT subtypes. Cellular membranes extracted from brain and gastrointestinal tissue show two NT binding sites (Uhl and Snyder, 1977). The first NT binding site displayed a high affinity for its ligand (Kd = 0.1–0.3 nM) and the incubation with GTP and Na1+ reduced its affinity for the NT (Tanaka et al., 1990; Vita et al., 1993).The other NT binding site exhibits a lower affinity for the NT (Kd = 3–5 nM) and it is less sensitive to Na1+ ions and GTP (Chalon et al., 1996; Mazella et al., 1996). The selective antagonist toward the lower-affinity site, levocabastine became an useful tool in the identification of both NT binding sites by demonstrating that the dissimilar affinity values are due to the existence of two receptors NTS1 and NTS2 (Kitabgi et al., 1987).

Hitherto, three of the four subtypes have been cloned and named NTS1, NTS2, and NTS3 (Pelaprat, 2006). A common characteristic shared for all of them is their binding to the NT(8- 13) (Tyler-McMahon et al., 2000). NTS1 and NTS2 have seven transmembrane spanning domains flanked by three intracellular and extracellular loops, this typical structure allows their classification into the family of the GPCRs. Nakanishi et al., were the first to clone a NT receptor using cDNA extracted from rat brains. This cDNA encoded a 424 amino acid protein. The human 418 amino acid NT receptor is 84% homolog with the rat NTS1. This receptor account for less than 30% of the NT binding sites in adult rats' brains (Chalon et al., 1996). In situ hybridization studies demonstrated that it is highly expressed in the hippocampus, in the piriform cortex, and the cerebellar cortex. NTS1 is present in the olfactory cortex, Island of Calleja, lateral septum, suprachiasmatic nucleus, anterodorsal thalamic nucleus, and dorsal motor nucleus of the vagus nerve (Hermans and Maloteaux, 1998).

NTS2 has been cloned in rodents and in humans. It has a higher sensitivity for levocabastine and a lower affinity for NT compared to NTS1 (Mazella et al., 1996). NTS2 has 416 amino acids in rats and mice and 410 amino acids in humans. It shares only around 40% amino acid identity and a 64% amino acid homology with NTS1. Structurally, the ICL3 of the NTS2 is 14

longer than its counterpart in NTS1. The N-terminal extracellular tail of NTS2 has not putative N-glycosylation sites, therefore it is shorter than that of NTS1. Another important difference between both receptor is the replacement of a very conserved Asp in the second transmembrane domain by an Ala or by a Gly residue (Vincent et al., 1999). This modification is the main reason for the lower sensitivity of the NTS2 to Na1+ ions and GTP. Northern-blot studies in rats have shown that NTS2 is expressed in the brain, mainly in the cerebral and cerebellar cortices the olfactory system, the hippocampal formation, and selective hypothalamic nuclei (Sarret et al., 1998).

NTS3 and NTS4 partake the capacity to bind NT with high affinity values (Geisler et al., 2006). However, these two proteins don’t belong to the GPCR family. NTS3 is a member of the type I amino acid receptor single transmembrane-spanning region (NTS3/sortilin) family (Kleczkowska and Lipkowski, 2013). Likewise, NTS4 or SorLA/LR11 is a single transmembrane amino acid receptor (Dobner, 2005; McMahon et al., 2002). Both receptors are poorly expressed on the cell surface (5–10% of total). The main cellular compartment where NTS3 and NTS4 are predominantly expressed is in the Golgi apparatus and the ER (Geisler et al., 2006). In terms of the receptor localization NTS3 and NTS4 are spread throughout the CNS (Geisler et al., 2006; Sarret et al., 2003).

1.2.4 NTS1 signaling The earliest experimental evidence of G proteins coupling to NTS1 came from the detection of second messengers. NTS1 activation leads to PLC activation, which hydrolyzes the membrane phosphatidylinositol 4,5-bisphosphate to diacylglycerol and inositol 1,4,5- trisphosphate. These two intracellular effectors will finally increase the intracellular Ca2+ concentration by acting on a ligand gated Ca2+ channel that is found on the surface of the ER and activates the PKC. It was subsequently established that the presence of these second messengers is linked to the activation of the Gαq/11 subunit (Kitabgi, 2002).

On the other hand, NTS1 activation in CHO cells transfected with either rat or human NTS1 (Gailly et al., 2000; Najimi et al., 2002) and in rat neuroblastoma N1E115 cell line endogenously expressing NTS1 (Bozou et al., 1986), stimulates the production of

arachidonic acid and inhibits the adenylyl cyclase by engaging the Gαi/o type G proteins. Conversely, in human pancreatic cancer cells endogenously expressing NTS1, its activation stimulates the adenylyl cyclase in a Gαs dependent manner, which provokes an increase of the intracellular concentration of cAMP. The disparity in these responses suggests a diversity of pathways modulated by the same ligand and a relationship between the activated pathway and the cell type.

PLC activation by the Gαq/11 subunits, is linked to two cellular events: the activation of the guanylyl cyclase (Snider et al., 2006) and the calcium-dependent adenylyl cyclase activation which leads to an increased cAMP in a Gαs-independent mechanism (Wettschureck and Offermanns, 2005). This increase in cAMP suggests the existence of converging pathways dependent on two different heterotrimeric G proteins. BRET demonstrated the engagement of the Gαq, Gαi1, GαoA, and Gα13 subunits on the NTS1 intracellular surface after its stimulation with 3 different ligands, NN, NT, and NT (8-13). The use of UBOQIC, a specific

Gαq blocker, revealed that the cAMP increase is due to this pathway. In addition, the measurement of the effect of NT on the recruitment of β-arrestins 1 and 2 demonstrated the engagement of this pathway (Besserer-Offroy et al., 2017).

Interactions between NTS1 and heterotrimeric G proteins involves the ICL3 and the C- terminal region. NTS1 mutants lacking the ICL3 sequence in CHO transfected cells show a detrimental effect on ability to activate the PLC without having any effect on cAMP levels

(Yamada et al., 1994). These results suggest that ICL3 is specifically engaged in the interaction with the Gαq subunit. On the other hand, the removal of the C-terminal receptor domain does not affect phospholipase C activity; however, it prevents adenylyl cyclase activation. Najimi et al. confirmed the preferential coupling of Gαi/o and Gαs to the first amino acids in the C-terminus of the NTS1 (Najimi et al., 2002). The intracellular injection of antibodies targeting the C-terminal sequence of the Gαq and Gα11 subunits prevents the calcium mobilization revealing that the C-terminal of the Gα subunits is involved with the ICL3 in the coupling between GPCR and G proteins (Katritch et al., 2013).

1.2.5 Physiological effects of the NT 1.2.5.1 Neurotensin and pain modulation NT central administration induces an antinociceptive response in a μ-opioid receptor- independent way (Clineschmidt and McGuffin, 1977). The microinjection of NT in CNS structures involved in the pain modulatory circuit inhibits nociception, confirming the brain distribution of NT in these cerebral structures (Dobner, 2005). Moreover, the NT antinociceptive effects have been found after testing the colorectal distension, which demonstrates that the regulation of the NT system may also be associated with the modulation of visceral pain (Urban et al., 1999).

Regarding the different NT receptor subtypes involved in the NT’s antinociceptive response, it was demonstrated that the microinjection of an antisense nucleic acid-peptide which targets the coding region of NTS1 in the CNS can reduce NTS1 receptor expression by 35– 46%. This decrease in the receptor expression results in a reduction in NT-induced analgesia supporting the role of NTS1 in NT’s antinociceptive effects (Tyler et al., 1999, 1998). NTS1 specific antagonists, like SR 48692, inhibit the NT-mediated antinociception in a tail-flick test performed in rats (Smith et al., 1997). Nonetheless, the use of NTS2-selective antagonists have also revealed that NTS2 participates in nociceptive modulation. Accordingly, levocabastine which blocks NT binding to NTS2 produces a partial inhibition of the NT’s induced analgesia in the writhing test, which consist of the intraperitoneal administration of acetic acid to evaluate the pain killer effect of a co-administrated compound. These results corroborate that a NT receptor other than NTS1 participates in the antinociceptive response triggered by NT (Dubuc et al., 1999; Tyler et al., 1998).

1.2.5.2 Neurotensin and cardiovascular system The link between the cardiovascular system and the neurotensinergic system in humans and various animal species has been confirmed by immunohistochemical studies. The presence of nerve fibers enriched in immunoreactive NT-containing vesicles in structural components of the heart demonstrates the possible modulation of NT over cardiac function (Osadchii, 2015). NT systemic administration produces heart rates acceleration in rats (Oishi et al., 1983). This acceleration is due to an increase in the contractions of heart structures

like atria and ventricles (Bachelard et al., 1987; Nisato et al., 1994). The use of the NT receptor antagonist SR48692, as well as histamine and β-adrenoreceptor blockers, has revealed that both contractions are due to the release of histamine and noradrenaline induced by the NT (Osadchii, 2015).

NT also induces a drop in blood pressure that varies depending on the animal model studied. NT describes a biphasic, triphasic, and even dose-response hypotensive effect. The biphasic and triphasic hypotensive effect appears as dissimilar patterns of decrease and increase in arterial blood pressure describing different phases (Osadchii, 2015). The response elicited by NT on the vascular smooth muscle tone, seems to be ascribed to some endogenous modulator like histamine (Miller et al., 1995), norepinephrine (Osadchii et al., 2006b, 2006a, 2005), epinephrine (Oishi et al., 1983) and prostaglandins (Schaeffer et al., 1997). NT affects the blood supply of other organs. In the intestine, NT causes a reduction in blood irrigation to the gastric mucosa and physiological changes like gastrointestinal vasodilatation and constriction of the portal vein (Fletcher et al., 1985; Harper et al., 1984; Onarheim et al., 1982). Other places where NT induces appreciable vasodilatation include the skin and brain (White and Robertson, 1987).

1.2.5.3 Neurotensin and hypothermia Bissette et al., were the first to report the hypothermic dose-response effects linked to NT intracisternal administration in mice (Bissette et al., 1976). Similar results have been obtained with other NT analogs like the Eisai compound, JMV-449, and NT69L when they are injected centrally. Among these compounds, the most promising is NT69L, which decreases the body temperature by 5°C (rectal) and its effect extends for more than 4 hours after its infusion (Tyler-McMahon et al., 2000). Remaury et al using NTS1 knockout mice demonstrated that thermal regulation is performed by a mechanism involving NTS1 without any participation of NTS2 (Remaury et al., 2002).

1.2.6 Agonist modulators of NT receptors The design and synthesis of molecules targeting the NT receptors represent a promising approach in the development of new drugs to modulate pain, blood pressure, and body

temperature. This should consider two main aspects. First, (SAR) studies have revealed that the last six amino acids in the NT C-terminus NT(8–13) (Arg8–Arg9–Pro10–Tyr11–Ile12– Leu13) induce a response even more potent than the NT(1-13) (Lambert et al., 1995). Secondly, since the NT is a peptide, its blood-brain barrier (BBB) penetration is not favored. Therefore, an optimized agonist must be delivered peripherally and able to reach the central nervous system and to exhibit a sequence homology with the NT(8–13) (Tyler- McMahon et al., 2000). Eisai Co. Ltd. was the first to be successfully synthesized as an agonist of NT that fulfilled the previous two conditions and that displayed a neurochemical, behavioral, and physiological profile like that of the NT. However, it showed a notable difference in its binding affinity toward the human (Kd ~ 130 nM) and the rat (Kd ~ 5nM) NTS receptors (Tyler-McMahon et al., 2000). In the search for new compounds with higher affinities for human NTS, NT66L, NT67L, NT69L were synthesized. They cross the BBB and also exhibit higher potencies in causing hypothermia and antinociception regarding the Eisai compound (Boules et al., 2006). NT69L has only one structural difference with the Eisai compound, a tryptophan analog, L-neo-Trp, at position 11 which is the cause of the best analgesic properties. (Tyler-McMahon et al., 2000).

The resistance to degradation by brain peptidases is another property that has been optimized in the search for new NT peptide analogs (Einsiedel et al., 2008). [D-Tyr11] NT is an analog resistant to hydrolysis, whose agonist action has been already confirmed. Accordingly, systemic treatment of HT-29 cells with [D-Tyr11] NT, showed superior intracellular Ca2+ levels and an augmentation in the cyclic GMP concentration. The changes caused by [D-Tyr11] NT have been ulteriorly associated with the induction of a hypothermic effect (Checler et al., 1983). JMV-449 is a pseudo peptide analog of NT, resistant to proteolytic degradation, which provokes hypothermia and analgesia (tail-flick test) in mice when it is injected i.c.v. JMV-449 can also restrain the binding of radio-labeled NT in homogenates of the mouse brain and displays a neuroprotective effect in models of permanent middle cerebral ischemia (Lugrin et al., 1991).

1.2.7 Neurotensin antagonists SR 48692 was the first selective non-peptide antagonist of the NT identified. Its discovery was the result of an optimization process of the best hit of an aleatory screened library of compounds (Dubuc et al., 1994). SR 48692 prevents [125I]-NT binding in cellular models expressing either NTS1 or NTS2. The inhibitory values obtained (IC50) for each receptor are IC50 = 5.6 nM for NTS1 and IC50 = 300 nM for NTS2, which demonstrates a higher selectivity towards NTS1 (Labbé-Jullié et al., 1995). The co-administration of SR 48692 also affect the hypothermic response caused by the NT or by the Eisai compound, and likewise, it affect the antinociception induced by i.c.v. injection of NT (Gully et al., 1995). The binding site of this competitive antagonist involves residues in the TM6 and TM7. Specifically, Arg327 and Tyr351 in TMs 6 and 7, are essentials for its binding. The NT binding site overlaps with the SR48692 binding pocket. Some residues placed in both overlapping areas are equally crucial for the binding of both compounds, which is consistent with the competitive nature of SR48692 (Kitabgi, 2006).

SR142948A is a compound chemically related to SR48692, developed by the Sanofi group, that restricts the binding of NT (in the nanomolar range) to NTS1 and NTS2 (Betancur et al., 1998). Likewise its predecessor, SR142948A antagonizes the hypothermic and analgesic properties of the NT in a dose-response manner (Gully et al., 1997). Interestingly, the antagonist binding site targeted by SR142948A seems to slightly diverge from that of SR48692. Experiments of saturation and competition performed with levocabastine demonstrated that [3H]SR 142948A still binds with high affinity to NTS1 and NTS2 ( IC50 =3.4 and 8.5 nM, respectively) (Betancur et al., 1998; Tyler-McMahon et al., 2000). Finally, the aforementioned levocabastine is an antihistaminic drug void of any of the pharmacological properties of the NT. Binding experiments have shown that levocabastine can hamper the binding of NT to NTS2 inhibiting its signaling (Kleczkowska and Lipkowski, 2013). Besides, its i.c.v. administration has demonstrated an antinociceptive effect in the writhing test.

1.3 Pepducins 1.3.1 Pepducin generalities Pepducins are cell-penetrating lipopeptides that were originally designed to interfere with the interaction between GPCRs and heterotrimeric G proteins to modulate GPCR signaling. Structurally, the peptide moiety of pepducins comes from the whole or truncated amino acid sequences of one of the three intracellular loops (ICL1, ICL2, and ICL3) or from the C- terminus of the GPCRs that they modulate, as shown in figure 7 (L. Covic et al., 2002). These peptide moieties are tethered to a lipid component at their N-terminus. The lipids part is made up of carbon backbones between 12 and 16 carbon atoms, such as palmitate and myristate, but also of steroid backbones like in the case of lithocholic acid pepducins (L. Covic et al., 2002; Wielders et al., 2007). The coexistence in their structures of a lipid and a peptidic moiety impacts the pepducins conformation, their allosteric modulation, pharmacology profile as well as their biodistribution (Tressel et al., 2011).

Figure 7: Schematic representation of the lipopeptide structure of a pepducin The peptide moiety based in the ICL1 of the hNTS1 is represented by circles. The lipid moiety is represented by a winding line.

1.3.2 Pepducins structural elements 1.3.2.1 Lipid moiety The first pepducins to be synthesized were a succession of lipopeptides derived from the third intracellular loop (ICL3) of the protease-activating receptor PAR1. In the initial design, the ICL3 of PAR1 was attached to peptidyl fragments of different sizes whose sequence

mimicked the adjacent hydrophobic transmembrane α-helix. The hydrophobic part of this molecule was intended to facilitate the anchoring to the membrane as well as its translocation through the cell bilayer. In an optimization process aiming at reducing the length of the hydrophobic core, the peptidyl fraction was substituted for a palmitate residue. The new palmitate tethered peptides triggered a similar cytosolic response to that generated by the extracellular ligand, measured in terms of Ca2+ intracellular concentration and a full physiological effect measured as the platelet aggregation (L. Covic et al., 2002).

The relationship between palmitoylation, biological activity, and cell-penetrating properties have been confirmed by several groups. Pepducins based on the ICL3 of the β2-adrenergic receptor increase transiently the intracellular concentration of cAMP which is due to the

Gαs pathway activation. The peptide sequence lacking palmitoylation does not affect cAMP intracellular levels, which is associated with the inability to reach the inner leaflet of the membrane and to produce the receptor activation (Carr et al., 2014). Similarly, P1pal-19 a pepducin derived from the ICL3 of PAR1 and its non-palmitoylated homologous were tagged with fluor and infused i.v. in mice. The cytometric analysis of purified platelet revealed 5- fold higher fluorescence values in mice treated with the palmitoylated version of the peptide than with the non-palmitylated. This confirmed the membrane penetrating properties conferred by the acyl function (L. Covic et al., 2002; Lidija Covic et al., 2002).

1.3.2.2 Peptide moiety Pepducins peptide moiety is primarily responsible for modulating the receptor's activity. The introduction of specific modifications in this region was the initial used approach to get insight about the pepducin’s mechanism of action and how to regulate the targeted receptor. Covic et al. decided to assess the impact of modifying the peptide moiety of a series of pepducins derived of different regions of the ICL3 of PAR1 by removing the first N- terminal amino acids. The deletion of the first seven N-terminal residues in the ICL3 did not affect the platelet aggregation nor the increase in the Ca2+concentration, revealing the importance of the amino acids placed in the central region of ICL3 for the pepducin activity (L. Covic et al., 2002; Lidija Covic et al., 2002). Brouillette et al., have also performed SAR studies in pepducins derived from the ICL1 of the NTS1. In these compound structures, the

last two C-terminal amino acids were deleted successively until generating a total of 5 truncated pepducins. The compounds evaluation in an acute pain model in rats (tail-flick test) demonstrated that the progressive deletion was associated with a diminution in the antinociceptive effect. This result suggests that the length and the residues essential in the peptide sequence for the pepducin activity vary and rely on the chosen GPCR (Brouillette et al., 2020).

Another SAR study has demonstrated the effect of the peptide moiety on the signal transduction cascades associated to the β2-adrenergic receptor (β2AR). In this functional assay, Carr III et al., decided to analyze whether pepducins synthesized from different ICL of β2AR could modulate differently the downstream pathways associated to G protein coupling or to β-arrestins recruitment (Carr et al., 2014). Pepducins whose sequence comes from the ICL1 of β2AR stimulated the recruitment of β-arrestin 2 on the receptor. Conversely, pepducins derived from the ICL2 and ICL3 induced an augmentation in the cytosolic cAMP concentration in a Gαs protein-dependent manner, without having any effect on the β-arrestin 2 engagement. Regarding to punctual modification introduced in the peptide portion, the amination of the pepducin C-terminal has proven to be essential for the functionality of pepducins. ICL3(3-9), a pepducin based on the ICL3 of β2AR induces an increase in the intracellular concentration of cAMP. However, after removing its amination in the C-terminal, any effect on the variation in the cytosolic content of cAMP was abolished (Carr et al., 2014).

1.3.3 Mechanism of action of pepducins The approach of the pepducin to the outer leaflet of the plasma membrane results in a partition from the aqueous phase into the hydrophobic phase of the cell bilayer, due to the amphiphilic nature of pepducins (Figure 8A) (Tressel et al., 2011). Once the molecule is embedded into the outer leaflet, it reversibly translocates to the inner monolayer of the plasma membrane through a flip-flop process (Figure 8B). The monitoring of the trajectory using the FRET methodology of a labeled pepducin derived of PAR1 has demonstrated the delivery of the pepducin to the inner side of the membrane which confirms the flip-flop process (Wielders et al., 2007). Subsequently, pepducins target an allosteric binding site

placed in the intracellular surface of the receptor (Figure 8C) which contributes to the stabilization of a particular conformation of the GPCR and to the activation of a specific pathway (Figure 8D) (Tressel et al., 2011).

Figure 8: Actual model proposed to explain the mechanism of action of pepducins a) Membrane interaction. b) Translocation. c) Interaction with the receptor. d) Receptor modulation.

1.3.4 Mandatory presence of GPCR for pepducin mechanisms of action Another important fact regarding pepducin is whether their mechanism of action necessarily involves the receptor. Two possible mechanisms had been proposed. In the first model pepducins can directly interact with and activate the heterotrimeric G proteins. The second mechanism states that pepducins bind to an allosteric site on the receptor structure inducing an optimal conformation which causes the interaction with G proteins and their activation (L. Covic et al., 2002). The first evidence in this regard was obtained using Rat1 fibroblasts which were transfected to express PAR1. Consequently, only in the transfected cells was detected the inositol triphosphate in response to pepducin treatment. Inositol triphosphate was not found in non-transfected cells. Nonetheless, even when the general mechanism of action seems to involve an interaction with the receptor, an exception to this generality has been reported. ICL3-8, a pepducin based on a fragment of the ICL3 of β2AR induces the exchange of GTP in Gαs subunits isolated from cells in a total absence of β2AR.

Further crystallographic studies revealed a close contact between the pepducin and the Gαs subunit in the β2AR-Gs complex (Carr et al., 2014).

A shred of conclusive evidence about the indirect interaction of pepducins with G proteins was obtained using a mutant of PAR1 in COS7 cells. PAR1's active state is characterized by a big conformational change that brings TM3 closer to TM6. This variation in PAR1 architecture leads to the activation of the heterotrimeric G proteins (Rasmussen et al., 2011b; Scheerer et al., 2008). The substitution of the Ser in position 309 by a Pro alters the TM6 structure, which prevents the transition to an active conformation. P1pal-19, whose C-terminal includes the mutated residue, does not restore the normal level of IP3 abrogated by the mutation. This confirms that the pepducin mechanism of action is not based on the formation of an alternative scaffold that facilitates the direct interaction between pepducin and G proteins. In contrast, P1pal-19 seems to interact with a site on PAR1 structure that enables a transition to an active conformation in the absence of the orthosteric ligand (L. Covic et al., 2002).

1.3.5 Further insight in pepducins mechanism of action The mechanism of action of pepducins occurs conventionally in a receptor-dependent way. However, the question about how they can induce an optimal conformation that favors the G proteins coupling remains unclear. In this sense, the exclusion of the possibility that pepducins exert their activity through the interaction with the orthosteric ligand-binding pocket needed to be corroborated. BMS-200661 is a competitive inhibitor of the PAR1 which hinders platelet activation. Its inhibitory effect is quantified in terms of the reduction of Ca2+ fluxes linked to the stimulation of the receptor by its natural ligand. Interestingly the coincubation of platelet with BMS-200661 and P1pal-19 does not affect the physiological effects induced by the pepducin on the receptor (L. Covic et al., 2002). Furthermore, binding assays using [125I] iodocyanopindolol revealed that the pepducins ICL3-8 or ICL3-9 do not displace the radiomarked agonist from β2AR confirming that they act in an allosteric site different from the orthosteric ligand-binding pocket. This allosteric binding site is likely to be located on the intracellular surface of the receptor, given the presence of an acyl residue in the ICL3-8 or ICL3-9 structure which provokes the translocation of the pepducins through the membrane (Carr et al., 2014).

1.3.6 Specificity and selectivity of pepducins for its receptor Pepducins display a marked specificity and selectivity towards their cognate receptors. The measurement of the variation in the concentration of IP3 in COS7 cells transfected with plasmids coding for PAR2, PAR4, CCKA, cholecystokinin B (CCKB), somatostatin (SSTR2), and substance P (SubP) was taken as a proof of the pepducin specificity and selectivity. This study revealed that pepducins derived of PAR1 activated the receptors that shared a high amino acids homology (100% of activation for the high homologous receptor PAR2 and only 30% for CCKB). Indeed, the higher the differences between the receptors, the less was the induced activation (L. Covic et al., 2002). Comparable results were obtained with ICL3-9, a pepducin whose sequence is based on the β2AR. In this case, the presence of the second messenger cAMP was taken as evidence of the receptor activation in CHO-K1 cells expressing β1AR, β2AR, and prostaglandin E2 receptor (EP2R). Since ICL3-9 shares a high amino acid homology and identity with the ICL3 of β1AR, it can induce an increase in cAMP almost at the same level showed by β2AR. Contrarily, ICL3-9 has no effect on EP2R which does not belong to the same family of receptors as β1AR and β2AR (Carr et al., 2014). Therefore, pepducins only cross-activate GPCRs which share high sequence homology with their cognate receptors.

1.3.7 Agonist effects of pepducins Since their discovery, pepducins have been employed as allosteric agonists or antagonists to modulate receptors. Besides, their simple structures make it easy to generate libraries of compounds based on different intracellular loops of GPCRs as well as modify chemically their structures. P1pal-19 and P1pal-13 were the first pepducins showing a full agonist behavior toward PAR1. Both pepducins can produce full activation of platelets and an augmentation in the intracellular concentration of Ca2+ (Lidija Covic et al., 2002). P2pal-21 a pepducin derived of the ICL3 of PAR2 behaves as a partial agonist, showing efficacies values of 18%. The optimization of the P2pal-21 structure by introducing a Phe makes its sequence like that of P1pal-19 and produced P2pal-21F. For the time being P2pal-21F is the most potent agonist discovered for PAR2 which operates as a full agonist indistinctly for PAR1 and PAR2. (Lidija Covic et al., 2002). MC4pal-14 is a pepducin synthesized to target

the melancortin-4 (MC4) receptor involved in obesity which displays a potent agonist effect and an efficacy of 40% compared to the α-melanocyte-stimulating hormone, the natural ligand of the receptor. Similarly, ATI-2341, ATI-2342, and ATI-2347 are three pepducins derived from the ICL1 of the chemokine CXC-type receptor 4 (CXCR4) capable to induce an intense chemotactic reaction in absence of the orthosteric ligands the CXC chemokine, CXCL12, or stromal cell-derived factor 1α (SDF-1α). ATI-2341 stands out by activating with higher values of potency and efficacy the Gαi-dependent pathway. This activation was determined by the inhibition of the production of cAMP and an increase in the cytosolic level of Ca2+. Moreover, the effect of ATI-2341 was also linked to receptor internalization (Tchernychev et al., 2010).

1.3.8 Antagonist effects of pepducins Pepducins were early designed and conceived as natural receptor antagonists, which acted hindering the interaction between the receptor and their cytosolic counterparts (L. Covic et al., 2002). P1pal-12 exerts an antagonist effect on PAR1 when it’s co-stimulated with the peptide ligand SFLLRN, by inhibiting the Ca2+ efflux and the soluble inositol phosphate production coupled to Gαq-PLC-β activation (L. Covic et al., 2002; Lidija Covic et al., 2002). In addition, P4pal-10 a pepducin derived from the ICL3 of the human PAR4 reduces the signaling associated to the receptor after incubation with the peptide ligand AYPGKF. Mechanistically, P4pal-10 can also exert an antagonist effect on PAR1. P4pal-10 antagonist effect is detected as an inhibition of the Gαq signaling pathway that affects the second messengers including Ca2+ and soluble inositol phosphate. The antagonist effect of P4pal- 10 absent in P1pal-12, rely on the homology of the sequence of the C terminal region in PAR1 and PAR4 (Lidija Covic et al., 2002)

β2AR produces an increase in the cytosolic cAMP concentration following by its internalization in a β-arrestin dependent manner followed by the stimulation with isoproterenol. However, when β2AR is co-stimulated with isoproterenol and ICL3-9, the β- arrestin pathway is blocked and the receptor is not internalized, which demonstrates the marked biased effect of ICL3-9 (Carr et al., 2014). In addition, PZ-218 a pepducin based on the ICL1 of CXCR4 and PZ-210 a derivate from the receptor ICL3, inhibit the chemotaxis

effect induced by SDF-1, revealing an antagonist effect (Kaneider et al., 2005; Tchernychev et al., 2010). Another example of antagonist effect was described by Shpakov et al., with a pepducin derived of the C-terminus of the type 1 relaxin receptor (LGR7). This pepducin antagonizes the adenylyl cyclase activity induced by the natural ligand of the receptor LGR7 (Shpakov et al., 2007).

1.3.9 Therapeutics applications of pepducins GPCRs constitute one of the main targets in the design and synthesis of new drugs due to the role they play in human pathophysiology. Several pepducin treatments have been developed to modulate receptors like PAR1, PAR2, PAR4, CXCR1, CXCR2, and CXCR4 which are involved in physiological disorders like cancer, coagulation diseases, sepsis, systemic inflammation, and angiogenesis (Tressel et al., 2011). The thrombin receptor PAR1 and PAR2 are co-expressed in human platelets where they are engaged in the thrombin signaling pathway and the aggregation of platelets depends on their presence, which make them a subject of research in the treatment of thrombotic diseases and acute coronary syndromes. P1pal-7 a molecule derived from ICL3 of the PAR1 has proven its effect on the reduction of platelet aggregation (Trivedi et al., 2009). Similarly, P4pal-10 which targets PAR4 is associated with a protective effect in sepsis against the systematic inflammation and the intravascular coagulation (Slofstra et al., 2007). In addition, P4pal-10 has proven to reduce the nociception in a model of rheumatoid arthritis in mice and to relieve joint inflammation (McDougall et al., 2009). PAR1 and PAR2 are also linked to pathologies associated with cancer such as metastasis formation and angiogenesis (Dorsam and Gutkind, 2007). Furthermore, P1pal-7 has demonstrated inhibitory properties against angiogenesis and proliferation of peritoneal ovarian and breast cancers (Agarwal et al., 2008; Boire et al., 2005). Likewise, P1pal-i1, a pepducin derived from ICL3 of PAR1 reduced the metastases of lung cancer at the same levels as gene therapy by silencing PAR1 expression (Cisowski et al., 2011)

On the other hand, the chemokine receptors CXCR1, CXCR2, and CXCR4 have a close relationship with the immunological system. x1/2pal-i3 and x1/2LCA-i1, two pepducins based on the ICL1 of CXCR1 and the ICL3 of CXCR2 can reduce the disseminated 28

intravascular coagulation associated with sepsis. This effect is mediated by the inhibition of the chemotaxis of the neutrophils. Additionally, x4pal-i1 and i3 x4pal-i3 are implicated in the SDF-1α neutrophil homeostasis and antagonize CXCR4 by producing leukocytosis (Kaneider et al., 2005). The overexpression of CXCR2 provokes tumorigenesis induced by inflammation in skin and intestine and is associated with the prevalence of adenocarcinoma. The use of a pepducin antagonist like x1/2pal-i3 is associated with a diminution in the rate of occurrence of tumors linked to CXCR2 (Mantovani et al., 2008). x4pal-i1, a pepducin derived from CXCR4 has demonstrated an inhibitor effect in the metastasis of lymphocytic leukemia and lymphoma (O’Callaghan et al., 2012). While pepducins acting on CXCR1 and CXCR2 respectively cause a reduction of IL-8 angiogenesis linked to ovarian cancer.

1.3.10 Pharmacokinetic and biodistribution of pepducins Studies of pharmacokinetic and pharmacodynamic have been performed with a fluorescent-labeled variant of P4pal-10 (P4pal-10-Alexafluor), whose fluorescence signal was detected in plasma and platelet 5 hours after its i.v. infusion in mice. Moreover, its half- life clearance time is approximate 3.5 hours (L. Covic et al., 2002; Lidija Covic et al., 2002). Similarly, the Fluor-PAR1-Pal-i3 was also identified in platelet 15 minutes after administration, which suggest that pepducin distribution does not seem to be tissue- specific since mice platelets don’t express PAR1 (Lidija Covic et al., 2002). Otherwise, the time of bleeding after the i.v. and s.c. injection of P4pal-10 has been measured to establish the pharmacodynamic profile of pepducins. It has been reported a prolongation of the time of bleeding estimably in twofold, 4 hours after the i.v. injection and in sixfold after s.c. injection of 3 mg/kg P4pal-10 (Tressel et al., 2011).

The radio-labeled pepducin [14C]-P4pal-10C, was also used to analyze the pepducin distribution into mouse body. The highest radio-ligand signals were detected in organs with high vascularity, like liver, kidneys, lungs, and spleen after i.v. administration in a therapeutic dose. A dosage above the therapeutic margin is associated with higher radioactivity levels in the mentioned tissues without having any apparition in heart, blood,

muscle, and fat. Furthermore, [14C]-P4pal-10C was never detected in the brain suggesting that the molecule is not able to cross the BBB. In terms of partition into the blood, almost 50% is transported associated to the red blood cell, 40% to plasma, and only 10% was identified linked to white blood cells and platelets 4h after i.v. or s.c. injection. Regarding its clearance, the first radioactive signal was detected 1h after the compound i.v. infusion, with a fivefold increase in urine at 4 h (Tressel et al., 2011).

1.4 Basic principles of solid-phase peptide synthesis Pepducins are cell penetrating lipopeptides whose structures are easily synthetized in laboratories following the basic principle of solid-phase peptide synthesis. The SPPS is a chemical method developed by (Merrifield, 1969) that consists of the progressive growth of an amino acidic peptide chain on a solid support. In this method, the solid and immiscible support acts anywise as a ribosome providing the surface on which occurs the growth of the peptide chain. Similarly, to the peptide biosynthesis, the Merrifield method follows an obligatory direction dictated by binding of the first amino acidic residue to the resin through its carboxyl group. However, in this sense, it differs from the peptide biosynthesis associated to ribosomes which starts from the peptide N-terminus (amino function). Since the solid-phase peptide synthesis progresses from the C-terminal (carboxyl group) to the N- terminal (amino group) the protection and masking of the amino groups are essential to avoid unwanted reactions between the amino function and the side chains of the second amino acid (Merrifield, 1969). Many functional groups in the lateral chains of amino acids should be covered up with protective groups that are not affected by the reaction conditions used during the coupling step, but they are finally released during the resin cleavage. After the resin loading with the first amino acid, the labile protecting group of the α-amino function is removed by a deprotection step and the second amino acid is introduced in excess. The formation of amide binding does not occur spontaneously, therefore this method requires a catalyst capable of activating the carboxyl group of the incoming amino acid (Jaradat, 2018). The ''activated'' intermediate is immediately attacked by the amino function of the amino acid linked to the resin creating a new peptide bond.

Since the reactions occur on a solid and immiscible support, all the subproducts and the excess of reactants are removed by filtration and washing. Accordingly, the SPPS follows a general pattern of repetitive coupling-washing-deprotection-washing cycles, where each amino acid is added in a precise order until the desired peptide sequence is obtained. The final step involves the peptide cleavage from its support and the protective groups of the side chains are removed under the same conditions that favor the peptide release (Carpino and Han, 1972).

1.4.1 The strategy of Fmoc/t‑Bu solid-phase peptide synthesis SPPS is performed by two fundamental methods which are classified in function of the protective groups employed. The first method was developed by (Merrifield, 1969) and make use of tertbutyloxycarbonyl (Boc)/benzyl (Bzl) as masking groups, whereas the second method and most widespread one, employs 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (t-Bu) as protecting groups (Carpino and Han, 1972). In both methods, the protecting groups are chosen following an orthogonal approach which consists of the protection and masking of the main chemical functions present in the amino acids with different groups that can be eliminated in a logical order under specific conditions. This methodology helps to ensure that the elongation phase of the peptide chain occurs in the direction from the C-terminal end to the N-terminal (Barany et al., 2009). In Fmoc/t-Bu SPPS, the protector group of the amino function is the Fmoc whose bound with the amino is easily cleavable under mild basic conditions compared to harsh acid conditions used in the Merrifield methods. The removal step of the Fmoc is repeated before the new amino-acid coupling with 20% piperidine in N,N-dimethylformamide (DMF). The fact that the Fmoc protecting group needs to be removed to make way for the next coupling makes it a temporary protecting group. After the deprotection of the first amino acid follows a washing stage in which the DMF is generally used as a solvent (Jaradat, 2018). This is followed by the activation of the carboxyl group of the incoming amino acid by an activating reagent, whose purpose is the formation of a temporary amide between the amino acid in solution and the activator. This coupling step eventually involves the nucleophilic attack of the Fmoc-deprotected amino group to the intermediate amide yielding a new dipeptide. On the other hand, the removal of the

tert-butyl from amino acid side chains is accomplished at the end of the synthesis, making it a sort of permanent protecting groups (Carpino, 1987; Carpino and Han, 1972). Empirically, the protecting groups of the amino acid side chains were conceived to be removed with trifluoroacetic acid (TFA) at 95% at the final step of the synthesis. In this sense, TFA is an excellent reagent since it produces the detachment of the peptides from the resin and the deprotection of tert-butyl groups from the side chains. Besides, TFA is a good solvent for peptides, and it is eliminated by evaporation since it is very volatile (Figure 9)(Fields and Noble, 2009).

Figure 9: Schematic representation of Fmoc/t‑Bu strategy on SPPS In peptide synthesis, a Fmoc-protected amino acid is bound to the resin, by an amide bond between the carboxyl group of the Fmoc-protected amino acid and the free amine function of the resin. The Fmoc protecting group is cleaved from the first coupled amino acid to make it react with the upcoming amino acid. The deprotecting and coupling cycles are repeated to form the desired peptide chain. After the completion of all the reactions, the synthesized peptide is cleaved from the resin. Figure reproduced from (Jaradat, 2018)

1.4.2 Solid supports and linkers used in SPPS Historically, peptide synthesis is performed through two main methods that differ in the phase on which occurs the growth of the peptide chain. Solution phase peptide synthesis demands protecting groups that remain throughout the process without favoring a

transition of phase during the reactions (Merrifield, 1969). Nonetheless, SPPS requires chemically functionalized surfaces on which the reaction of elongation of the peptide chain occurs (Figure 9). These surfaces received the name of solid supports and they meet a set of requirements. The resins should be chemically inert to all reagents used in the synthesis and insoluble in the used solvents, which allow their removal by filtration and washing (Merrifield, 1969). The resins are made up of solid polymers which display a high surface ensuring a greater area of contact between the solid phase and solvents. The most exhaustively exploited solid support is the 1–2% divinylbenzene-crosslinked polystyrene (Blackburn, 1998; Vaino and Janda, 2000). Solid supports should also show maximized swelling properties since the elongation reaction occurs in the crisscross of polymeric units of the resin. The better the capacity of the solid support to expand, the greater is the accessibility of solvents and reagents inside the resin and consequently, the peptide yield is increased (Wieland et al., 1969). The anchoring of the first amino acid to the solid support is mediated by the linkers or handles. In Fmoc/t-Bu SPPS, most linkers are designed to release the peptide chain after treatment with TFA. TFA concentration dictates whether the cleavage will lead to a C-terminal completely unprotected or retaining the side-chain protective groups. This way, the purpose of a linker is not just to provide a reversible bond with the peptide chain but also to protect the C-terminal of the amino acid during the chain extension process (Merrifield, 1969).

1.4.3 Side chain protecting groups The reaction conditions used in the Fmoc/t-Bu SPPS method are mild with regard the harsh acid deprotection cycles utilized in the Boc/Bzl strategy. However, the side chains of most amino acids contain reactive functional groups which can undergo unspecific reactions leading to the obtention of undesirable coproducts (Jaradat, 2018). The use of protecting groups whose main function is to mask the chemical function of the amino acid side chains is obligatory in peptide synthesis. In the Fmoc/t-Bu SPPS strategy, those protecting groups have been conceived to be released under TFA at 95% treatment when the cleavage of the peptide from the resin is produced. Currently, the side chain protective groups used are t- butyl steres for negative charged amino acids like Asp and Glu, as well as for other amino

acid residues like Ser, Thr, and Tyr. Boc chemistry is widely used to protect the amino function of Lys and Orn but also to mask the side chain of the Trp. Finally, the trityl group is usually used to bind and to mask the side chain of His, Asn, Gln, and Cys, whereas the 2,2,4,6,7-pentamethyl-dihydro benzofuran-5-sulfonyl groups protect the Arg residue (Isidro-Llobet et al., 2009).

1.4.4 Amino acid coupling step Most of the methods of amide bond formation involve the chemical activation of the carboxyl group by enhancing the leaving properties of the hydroxyl function. This favors the nucleophilic attack on the carboxy group by the amino function (Merrifield, 1969). The activators used in organic synthesis are too potent to be used in SPPS and lead to overactive intermediaries. Therefore, the activators or coupling agents commonly used in SPPS guarantee smoother reaction conditions, based on the formation of active esters performed or generated in situ. The most used coupling agents in SPPS are carbodiimides, active esters, uronium/aminium salts, and phosphonium salts (Jaradat, 2018). Hexafluorophosphate azabenzotriazole tetramethy uronium (HATU) stands out within the category uronium/aminium salts and it is the chosen activator used in the coupling steps presented in this work.

Figure 10: Amine nucleophilic attack of on the acyl moiety to produce an amide. Coupling reagents activate the carboxyl group by increasing the leaving properties of the hydroxyl group. Thus, the carboxy group is better attacked by a nucleophilic amine of another residue. Figure reproduced from (Jaradat, 2018)

1.4.5 Racemization The presence of chiral centers in the α carbon is a characteristic shared by all amino acids apart from the glycine. However, during the coupling step, the activation of the carboxyl group provokes an increase in the acidity of the proton attached to the α carbon. Under this condition, the proton can be easily released, which represents a potential racemization mechanism (Figure 10). Another well-described mechanism involves the deprotonation and opening of an oxazolone intermediary structure (Goodman and Stueben, 1962).

Figure 11: Mechanisms of base-catalyzed racemization during the activation Basic conditions used during the carboxyl activation increase the acids properties of the proton placed in the α carbon. Proton output results in two possible racemization mechanisms: formation of 5(4H)-oxazolone (Route A) or enolization and the formation of an enantiomeric activated peptide (Route B). Figure reproduced from (Jaradat, 2018)

1.4.6 Aggregation The progressive growth of the peptide chain is associated with the appearance of secondary structural elements. These secondary structures often arise from the coupling of the fifth amino acid and are prone to hinders the elongation process. The hindrance is due to the formation of β folded sheet structures where the amino is engaged in a hydrogen bond with the carbonyl group (-C=0) of other amino acids (Meli et al., 2008). This way the activated acyl function cannot be targeted by the amino groups which are occluded in the aggregate (Krchnak et al., 1993). The aggregation hampers the solvation of the peptide-resin, causing

a shrink of the polymeric matrix which reduces the penetration of the reagents (Miranda et al., 2011).

1.4.7 Fmoc deprotection Fmoc group elimination is achieved under a mild basic condition that does not affect the labile amide bond between the peptide, the resin and the rest of protecting group attached to the amino acid side chains (Figure 11). The elimination involves the deprotonation of the fluorene ring to produce an aromatic intermediary. The dibenzofulvene undergoes an attack by the piperidine producing an adduct fulvene–piperidine and the deprotected amino function. The reaction product absorbs in the UV light, allowing the monitoring of the reaction (Carpino, 1987; Carpino and Han, 1972).

Figure 12: Deprotection mechanism of the Fmoc group The fluorene ring system has strong electron-withdrawing properties and makes the hydrogen highly acidic. Piperidine attack generates a stabilized anion which moves to the adjacent atoms triggering the production of CO2 and dibenzofulvene. The amino group becomes fully deprotected. Finally, the amine used for Fmoc cleavage neutralizes the reactive dibenzofulvene. Figure reproduced from (Jaradat, 2018)

1.4.8 Peptide precipitation Following the cleavage step, the peptide is precipitated by the action of cold diethyl ether. Precipitation can be made directly from the TFA solution or after the evaporation of TFA and neutralizing agents (Merrifield, 1969).

1.5 Problematic, hypothesis, and objectives So far, my description has addressed topics related to GPCRs modulation and how the signal transduction cascades can be regulated in dissimilar ways using endogenous and exogenous ligands. Inside the category of exogenous ligand, I have discussed how GPCRs and most precisely NTS1 can be targeted by different modulators that will bind on different sites of its structures. Previous researches conducted in our group have provided insight into how a new sort of allosteric modulator called pepducins can influence the interaction between the NTS1 and the heterotrimeric G proteins. Maybe the most intriguing feature of these molecules was their ability of triggering the activation of signaling pathway-dependent of the Gαq and Gα13 subunits in the absence of NT. Additionally, due to the chemical properties of these compounds, their mechanisms of modulation suggest the possible existence of an allosteric site placed on the surface of the NTS1 exposed to the intracellular environment.

Initially, these pepducins were conceived to mimic the ICL1 of NTS1 even though the interaction between the GPCR and G proteins occurs mainly by events involving the ICL3 and the C-terminus of the targeted receptor. Several reasons led our group to choose this structural element. First, the high homology and identity in the sequence among the ICL1 in different species including humans and rodents. Second, the limited information collected to date about the modulation of pepducins derived from the ICL1 on NTS1. Additionally, early SAR studies performed in our laboratories demonstrated that progressive truncations in the pepducin sequences from their C-terminus are linked to a gradual loss in their biological activities. Our group has performed two scans by substituting each amino acid in the compound sequence with the purpose to determine how relevant they are for the pepducins' activity. In both cases, the results led us to keep immutable the residues close to the N-terminus, since most of the substitutions were bound to losing the biological activity.

To get further insight into the allosteric mechanism of modulation of pepducins on NTS1 we have decided to conceive and synthesize a new series of pepducins. The new compounds mimic the ICL1 from NTS1 and each position has been replaced by tryptophan. The augmentation of the hydrophobic properties leads to a progressive improvement in the

affinity and increase the global interactions between modified ligands and their targeted receptors. Thus, we hypothesize that increasing the hydrophobic properties in punctual positions of the pepducins sequence may represent a new strategy to enhance the interactions between them and NTS1, which contributes to the improvement of their therapeutic potential to relieve pain. This hypothesis will be tested through the following objectives:

Aim 1: To design and to synthesize new pepducins targeting hNTS1

Aim 2: To characterize the global cellular responses induced by pepducin treatment

Aim 3: To establish the signaling profiles of these pepducins targeting NTS1

Aim 4: To assess the physiological effects of pepducins in vivo.

MATERIALS AND METHODS 2.1 Materials The Fmoc protected amino acids and all the reagents used in the peptide and pepducin synthesis were acquired from Sigma-Aldrich (Canada) or Fisher Scientific (USA). Resins were obtained from Rapp Polymere (TentaGel SRAM). NT (1-13) was procured from R & D Systems, Inc. (Minneapolis, MN, USA). Coelenterazine 400A was purchased from Gold Biotechnology Inc. (St. Louis, MO, USA), for the BRET experiments. HEPES (4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid), DMEM-F12, fetal bovine serum (FBS), penicillin-streptomycin-glutamine, phosphate-buffered solution (PBS), Hank’s balanced salt solution (HBSS), gentamycin G418, trypsin + EDTA 0,25 % were all obtained from Wisent (St. Bruno, QC, Canada). Opti-MEM was bought from Invitrogen (Burlington, ON, Canada). The plasmids encoding for Gαq-RlucII, Gα13-RlucI, GFP10-Gγ1, Gβ1, RlucII-β-arrestin 1 or 2 and hNTS1-GFP10 were kindly provided by Dr. Michel Bouvier (Dept. of Biochemistry and IRIC, Université de Montréal, Montréal, QC, Canada).

2.2 Peptides synthesis The synthesis of the new pepducin library derived from the ICL1 of the hNTS1 was performed using the TentaGel S-RAM resin (0.24mmol/g). As a pre-synthesis stage, the resin was pre-swelled and its N-terminal group deprotected with 20% piperidine/DMF under agitation on an orbital shaker at room temperature (RT) for 20 to 30 minutes. After the initial Fmoc deprotection, the resin was thoroughly washed with dichloromethane (DCM) (2x5 mL), 2-propanol (1x5 mL), DCM (1x5 mL), 2-propanol (1x 5 mL), DCM (2x 5 mL) during a period of 5 minutes. For the coupling of the first Fmoc-protected amino-acid residue, a solution containing 5 equivalent of the residue and the activators HATU (5 equiv) and N,N- diisopropylethylamine (DIPEA) (10 equiv) in DMF (5 mL) were prepared and added to the reactor. The first coupling step was kept under agitation for 20 minutes at RT. Then the coupling solution and solvents were filtered, and the resin was washed thoroughly under the same condition described above. In a similar way to the initial step of deprotection of the resin, piperidine (20% in DMF) was employed to remove the Fmoc protecting group

after each coupling step during the elongation process. The resin was washed after each coupling and deprotection step with the above sequence of solvents. The correct elimination of the protecting group, as well as the introduction of the new amino acid, was monitored through the Keiser test (Kaiser et al., 1970). These series of stages were repeated until obtaining the desired peptide sequence. The palmitate coupling step was performed under the same condition used with other amino acids at 0.2M with HATU (5 equiv) and DIPEA (10 equiv) in DMF (5 mL). Final resin cleavage was performed using a mixture of TFA/H2O/TIPS (triisopropylsilane), 95/2.5/2.5, v/v (4 mL / 0.4 g of resin) for 2 h at RT. After filtration, the peptide was precipitated in tert-butyl methyl ether (TBME) at 0°C, the suspension was centrifuged, the supernatant removed and the crude product re-dissolved and lyophilized.

2.3 Peptides purification The crude product was redissolved in acetonitrile/water (3/7), filtered, and purified on a reverse-phase preparative HPLC-UV system from Waters (Milford) (C4 column [XBridge Protein BEHC4 OBD Prep Column, 300 Å, 5 μm, 19 mm x 150 mm] using acetonitrile and water + 0.1% TFA as eluents. Pure fractions were lyophilized to give a white solid final product. For purity assessment, compounds were analyzed on a UPLC-MS system from Waters (Milford) [Acquity UPLC Protein BEH C4 Column, 300 Å, 1.7 μm, 2.1 mm x 50 mm] with the following gradient: acetonitrile and water with 0.1% formic acid (0 → 0.2 min: 5% acetonitrile; 0.2 → 1.5 min: 5 → 95%; 1.5 → 1.8 min: 95%; 1.8 → 2.0 min: 95 → 5%; 2.0 → 2.5 min: 5%). High-resolution mass spectrometry (HRMS) of all analogs was performed using electrospray infusion on a maXis ESI-Q-Tof apparatus from Bruker (Billerica).

2.4 Cell culture and transfections The in vitro effects of pepducins were evaluated in Human Embryonic Kidney 293 (HEK293). This cell line has been extensively used in our laboratory to study different signaling pathways associated with hNTS1 activation. The cells were cultured in Dulbecco’s Modified Eagle’s Medium-F12 (DMEM-F12) supplemented with 10% (vol/vol) FBS, 100 Units of penicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine. They were then incubated at 37

°C and 5 % CO2, in a humidified atmosphere. The HEK cells were used between passages 5 and 20. The in vitro experiment performed in this research required the transfection of cDNA plasmids, for transient expression of recombinant proteins. The transfection procedure was as follows: 1.5 × 106 cells were seeded onto 100 mm2 cell culture dishes, and 24 h later, received a total of 12 μg cDNA, prepared in Opti-MEM serum-free media along with the transfection agent polyethylenimine (PEI) at a 3:1 ratio (PEI: DNA).

2.5 Whole cellular response measured through a label-free phenotypical assay The whole cellular responses induced by pepducins based on the ICL1 of the hNTS1 were monitored by electric cell-substrate impedance sensing (ECIS). ECIS is a label-free technique developed to measure the dynamic mass redistribution (DMR) of living cells in real-time. ECIS used a simple and sensitive model based on two electrodes placed on the bottom of a cell culture plate. These electrodes detect any change undergone by the cell monolayer in terms of resistance or impedance to the electric flow. As a previous step to the cell monolayer resistivity measurements, 96-well plates (96w20idf furnished by Applied Biophysics, Troy, NY, USA) were prepared and stabilized. Each well was covered by a cysteine solution (10 nM sterile L-Cysteine, 100 μL) for 10 min., washed two times with sterile water, and let to air-dry for 10 min. A solution of Poly-L-Lysine (0.01 mg/mL in sterile water, 100 μL) was then added for 10 min, drained, and washed two times with sterile water as recommended by the manufacturer. 36,000 CHO-K1 cells stably expressing hNTS1 (ES-690- C) purchased from PerkinElmer (Montréal, Canada) were plated into each well with 300 μL of complete DMEM-F12 medium and allowed to grow for 24 h, before being washed with PBS and serum-starved for 16–18 h before the experiment, which contributed to aligning them in the same cell cycle for 24 h. On the day of the experiment, 10 μL of 100 μM pepducin solution was added to 90 μL HBSS already in plate wells, giving a final concentration of 10 μM pepducin per well. The electrical resistance was measured at 4000 Hz single frequency, recorded on an ECIS Zθ instrument linked to a 96-well array station (from Applied Biophysics). The baseline was measured 1 h before compound addition and subsequent global cellular responses induced by pepducins were recorded for 3 h. The experiments

were carried out three times, in duplicate. Results were normalized by dividing the recorded resistance by the resistance before pepducin stimulation and plotted as the mean ± S.E.M. in Graphpad Prism 7.

2.6 Bioluminescence Resonance Energy Transfer (BRET)

The signaling pathway linked to Gα13, Gαq, and the recruitment of β-arrestins 1 and 2 in response to pepducins treatment was monitored using BRET-based biosensors. BRET represents a particular case of Föster resonance energy transfer, in which the radiation less energy is transferred from the Renilla luciferase to green fluorescent protein (GFP) when they are at a distance lower than 100Å (Lohse et al., 2012). In BRET assays the efficacy of the transference relies on three factors: the overlapping between the emission-absorption spectra of the molecules engaged, the orientation of their dipole moment, and the distance between them. The modification of those factors by using substrates that are targeted by the luciferase, as well as a better selection of the donor-acceptor system led to the development of the following generation of BRETs (BRET1, BRET2, eBRET2, BRET3, and QD- BRET)(Bacart et al., 2008). In BRET2 which is the chosen method in this study, the substrate that undergoes oxidative hydrolysis responsible for the light peak emission at approximately 400nm is the DeepBlueC or the bisdeoxycoelenterazine CT. Additionally, the acceptor of the resonance energy transfer is the GFP10 which has values of excitation and emission of 400 and 510 nm (Figure 12).

Figure 13: BRET principle applied to study the interaction between GPCRs and heterotrimeric proteins (A) In the complex GPCR-heterotrimeric G proteins the Gα subunit is tagged to the luminescent donor and the Gγ subunit to the fluorophore acceptor. Substrate coelenterazine 400A application (Phase 1). Endogenous ligand application NT (8-13) triggers the dissociation of the complex and a drop in the signal (Phase 2). (B) The decrease in the amount of light emitted followed the dissociation of the GPCR-heterotrimeric G proteins complex due to the activation can provide insight about which G protein subunits can be coupled to a particular active state of the receptor.

In this experiment 1.5 × 106 HEK293 cells were seeded into 100 mm2 cell culture dishes and transfected 24 h later according to the procedure described above. For the experiment aiming to assess pepducins’ ability to activate NTS1-associated G protein signaling pathways, the cells were transfected with the following biosensor couples: hNTS1, Gαq-

RlucII, Gβ1 and Gγ1-GFP10; or hNTS1, Gα13-RlucII, Gβ1, and Gγ1-GFP10. For the experiment aiming to assess pepducins’ ability to induce β-arrestin recruitment, cells were transfected with either RlucII-β-arrestin 1 or 2 and hNTS1-GFP10. Cells were detached 24 h post- transfection using trypsin-EDTA 0.25 % and were seeded into white opaque 96-well plates (BD Falcon, Corning, NY, USA) at a concentration of 35 000 cells/well. At 48 h post- transfection, the cells were washed once with 100 µL of PBS, and then 90 μL of HBSS containing 20 mM HEPES was added. The wells were then treated with 10 µL of 10X NT (1- 13) concentrations (final concentrations ranging from 10-12 M to 10-6 M) or 10 µL of 10X pepducin concentrations (final concentrations 10-7 M to 10-4 M, at half-log concentration intervals), which were previously dissolved in HBSS containing 20 mM HEPES. For the experiment aiming to assess the pepducins’ ability to activate NTS1-associated G protein signaling pathways, the cells were incubated at 37 °C for 30 min, before receiving 5 μM of coelenterazine 400A. For the experiment aiming to assess the pepducins’ ability to induce

the recruitment of β-arrestins 1 and 2, the cells were incubated at 37 °C for 50 min, before the stimulation with 5 μM of coelenterazine 400A. The lecture was performed on a GENios Pro plate reader (Tecan, Durham, NC, USA) using a BRET2 filter set (400−450 nm and 500−550 nm emission filters). The determination of the BRET2 ratio for each well was done by dividing the GFP10-associated light emission by RlucII-associated light emission. The data was subsequently normalized relative to NT (8–13); values for non-treated cells were set as 0 % pathway activation, and those for cells treated with 1 μM NT (8–13) were set as 100 % pathway activation.

2.7 Competitive Radioligand Binding Assay on the hNTS1 receptor CHO-K1 cells stably expressing hNTS1 (ES-690-C) purchased from PerkinElmer (Montréal, Canada) were cultured in DMEM-F12 culture medium at 37°C in a humidified chamber under 5% CO2. The culture medium was supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Cells were rinsed with PBS, and frozen when they reached 80% confluency. They were scrapped off the dish with 10 mM Tris-buffer, 1 mM EDTA, pH 7.5 and centrifuged at 15,000 g for 5 min at 4°C. The pellet was re-suspended in 1 mL binding buffer. Competitive radioligand binding experiments were performed by incubating 15 μg of cell membranes expressing the hNTS1 receptor with 45 pM of [125I]-[Tyr3]-NT (2200 Ci/mmol) in binding buffer (50 mM Tris-HCl, pH 7.5, 0.2% BSA) with increasing concentrations of pepducins ranging from 10−11 to 10−4 M for 60 min at 25°C. After incubation, the binding reaction mixture was filtered in PEI-coated 96-well filter plates (glass fiber filters GF/B, Millipore, Billerica, MA) to end the reaction. Plates were washed three times with 200 μL ice-cold binding buffers. Glass filters were then counted using a γ- counter (2470 Wizard2, PerkinElmer, Mississauga, Ontario, Canada). Non-specific binding was measured in the presence of 10−5 M unlabeled NT (8−13) and represented less than 5% of total binding. Data were normalized compared to NT (8-13) to control for the unwanted source of variation. IC50 values were determined from competition curves as the unlabeled ligand concentration inhibiting 50% of [125I]-[Tyr3]-NT-specific binding. Competitive radioligand binding data were plotted using the nonlinear regression One-site-Fit Log (IC50)

and represented the mean ± S.E.M. of two independent experiments. The binding assay was performed in triplicate to ensure the reliability of the single values.

2.8 In vivo experiments 2.8.1 Animals, housing, and habituation Experiments were performed with adult male Sprague-Dawley rats, weighing 250-300 g, (Charles River laboratories, St-Constant, Canada). Rats were housed two per cage on Aspen shavings in a quiet room and kept on a 12 h light/dark cycle and allowed ad libitum access to food and water. All experimental procedures in this study were approved by the Animal Care Committee of Université de Sherbrooke following the policies and directives of the Canadian Council on Animal Care. Furthermore, they were performed in agreement with the principles of ARRIVE and United States NIH (Animals in Research: Reporting In Vivo Experiments) (Kilkenny et al., 2010). Before the experiments, rats were acclimatized for 4 consecutive days to the animal facility and 3 consecutive days to the experimental conditions of each test. Animals were euthanized following prior anesthesia with isoflurane by using carbon dioxide inhalation.

2.8.2 Intrathecal administration Rats were lightly anesthetized with a flow of isoflurane/oxygen (Baxter Corporation, Mississauga, ON, Canada; 2 L/min). 25 µL of pepducins at concentrations ranging from 50 to 275 nmol/kg were injected intrathecally at the L5-L6 intervertebral space using a 27 G 1/2 needle. Pepducins were diluted in a vehicle composed of physiological 0.9% saline, 10% DMSO, and 20% polyethylene glycol 4000 (PEG4000). Control animals were injected with the vehicle alone.

2.8.3 Tail-flick test The Tail flick test (Tail Flick Analgesia meter V2.00, Columbus Instruments, Columbus, Ohio, USA) was used to assess pepducins’ analgesic efficacy in acute pain. The test measures heat- induced pain using a high-intensity light beam focused on the rat tail (Kallina and Grau, 1995). The tail-flick apparatus was set at a light intensity of 6 and a cutoff of 10 seconds.

The latency to flick the tail out of the path of the light beam corresponds to the measure of pain sensitivity or analgesia. Before testing, animals were individually acclimatized to manipulations and behavioral apparatus 5min/day for three consecutive days. On the test day, a latency baseline measure was taken before drug injection. The pepducins were diluted in the vehicle (0.9% saline, 10% DMSO, 20% PEG4000) and intrathecally injected at increasing doses ranging from 50 to 275 nmol/kg. The effects of the pepducin or vehicle on thermal nociception were assessed every 10 min for up to 60 min following i.t. administration. Tail flick latencies were then converted into the percent maximal possible effect (% MPE) at the time of the maximal peak of analgesia. % MPE was calculated according to the following formula: %MPE = [(Test latency) – (Saline latency)] / [(Cutoff) – (Saline latency)] × 100. Data are expressed as mean ± S.E.M. of 5 animals for each dose. The half-maximal effective dose (ED50) of the compound was determined on the % MPE at 10 min, calculated for each dose in the tail-flick test. Then, ED50 values were determined using the dose-response stimulation log (agonist) vs response (four parameters).

2.8.4 Body temperature measurements Body temperature was measured using a thermistor probe inserted into the rectum of the rat. Animals were individually acclimatized to manipulations and thermistor probe 5 min/day for three consecutive days. On the test day, temperatures were measured before (baseline) and each 10 min for up to 60 min following intrathecal administration of the pepducins. The pepducins were diluted in the vehicle (0.9% saline, 10% DMSO, 20% PEG4000) and intrathecally injected at 100 and/or the highest dose at 275 nmol/kg. Changes in body temperature (Δ body temperature) were determined from baseline for each time and each animal. Data represent the mean ± S.E.M. of 5 rats for each condition.

2.8.5 Blood pressure monitoring Rats were anesthetized with ketamine/xylaxine (87 mg/kg: 13 mg/kg, intramuscular) and placed in a supine position on a heating pad. Mean, systolic and diastolic arterial blood pressure and heart rate were measured through a catheter (PE 50 filled with heparinized saline) inserted in the right carotid artery and connected to a Micro-Med transducer (model TDX-300, USA) linked to a blood pressure Micro-Med analyzer (model BPA-100c). Another

catheter (PE 10 filled with heparinized saline) was inserted in the left jugular vein for injections of the pepducins at 55 nmol/kg (volume 1 mL/kg, 5–10 s) diluted in 0.9% saline and 10% DMSO (PEG4000 as solvent was not used here, since it is not recommended for i.v. administration). Blood pressure was recorded each second for up to 1000 seconds following i.v. injection. Changes in mean arterial blood pressure (Δ MABP) were determined from the basal pressure of rats. Data represent the mean ± S.E.M. of 5 rats for each condition.

2.9 Data and statistical analysis All experimental protocols, data, and statistical analysis in this study comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2018). Animals were randomized to treatment groups. For the tail-flick test and the body temperature monitoring, the baseline measures followed a normal (or Gaussian) distribution determined by the Shapiro-Wilk normality test. Parametric tests were then performed to increase statistical power. Power analysis estimated sample sizes of 2.2 rats per group. For ANOVA analysis, posthoc tests were conducted only if F was significant and there was no variance in homogeneity. Data are expressed as mean ± S.E.M. All graphs and statistical analysis were performed using GraphPad Prism 7 (GraphPad Software, La Jolla, CA, USA). A two-way ANOVA followed by Dunnett's multiple comparisons test was used to determine the significant differences in tail-flick latencies and body temperature between the vehicle and the different pepducins. %MPE was analyzed using a one-way ANOVA followed by Dunnett's multiple comparison test to compare the effects of vehicle and

11 pepducins. To determine the half-maximal effective dose (ED50) of PP-W , the %MPE in acute pain was calculated for each dose of the pepducin. Then, ED50 values were determined using the dose-response stimulation log(agonist) vs response (four parameters). A difference in response between vehicle and pepducins was considered significant with p-values *p<0.05, **p<0.01, ***p<0.001 and ****p<0.0001.

RESULTS 3.1 Pepducins design and synthesis Pepducins were synthesized by SPPS following the Fmoc/t‑Bu approach. The Fmoc- protecting group of the resin TentaGel S-RAM was initially removed by treatment with 20% piperidine in DMF. The first Fmoc protected amino acid was subsequently coupled to the resin with HATU and DIPEA. During the elongation steps, the cycles of Fmoc cleavage and the attachment of the inward amino acid were repeated stepwise. Palmitate incorporation to the pepducin’s N-terminus was performed under the same reaction condition heretofore mentioned. Sidechain protecting groups removal and pepducins final cleavage from the resin was simultaneously accomplished using a mixture of TFA/TIPS/water (95/2.5/2.5). The compounds were precipitated in TBME and later purified using a reverse-phase preparative HPLC. The purity was estimated by UPLC-MS which was in all the cases superior to 95%.

The sequence of the ILC1 of the hNTS1 was obtained from Uniprot (Uniprot website, Ref: P30989) (Figure 7, page 23), and it is shown in the table 1 as NP-ICL1. The ICL1 of hNTS1 was used as a template for the design and synthesis of PP-ICL1 which was taken as our pepducin control. Pepducins derived from the ICL1 of the NTS1 have been previously involved in allosteric agonist modulation of signal transduction cascades coupled to Gαq and

Gα13 (Brouillette et al., 2020). However, the G protein activation was shown to occur at a high concentration of 10 µM which is 10 fold superior to the NT concentration tested (Brouillette et al., 2020). Therefore, a new pepducin series was generated by substituting each amino acid position into the ICL1 with tryptophan. Tryptophan was chosen in order to enhance protein-protein interaction and thus the affinity between pepducins and hNTS1 since this residue promotes pepducin receptor interaction through hydrophobic interactions (Bissantz et al., 2010). It should be noted that the first four residues were not replaced since previous SAR studies have revealed that they are essential for the pepducin activity. The pepducins sequence and the position of the tryptophan substitution are represented in table 1.

Table 1: Position of the amino acid substitution into the peptide sequence of pepducins derived from the ICL1 of hNTS1 Compound Sequence 1 2 3 4 5 6 7 8 9 10 11 12 13 14 NP-ICL1 H2N- A -R -K -K -S -L -Q -S -L -Q -S -T -V -H -CO-NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-ICL1 Palmitoyl-A -R -K -K -S -L -Q -S -L -Q -S -T -V -H -CO-NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-ICL1-SCR Palmitoyl-L -V -Q -R -L -T -A -K -S -S -K -Q -H -S -CO-NH2 5 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -W -L -Q -S -L -Q -S -T -V -H -CO-NH2 6 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -W -Q -S -L -Q -S -T -V -H -CO-NH2 7 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -L -W -S -L -Q -S -T -V -H -CO-NH2 8 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -L -Q -W -L -Q -S -T -V -H -CO-NH2 9 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -L -Q -S -W -Q -S -T -V -H -CO-NH2 10 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -L -Q -S -L -W -S -T -V -H -CO-NH2 11 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -L -Q -S -L -Q -W -T -V -H -CO-NH2 12 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -L -Q -S -L -Q -S -W -V -H -CO-NH2 13 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -L -Q -S -L -Q -S -T -W -H -CO-NH2 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 PP-W Palmitoyl-A -R -K -K -S -L -Q -S -L -Q -S -T -V -W -CO-NH2 NT (1-13) H2N-E-L-Y-E-N-K-P-R-R-P-Y-I-L-COOH NT (8-13) H2N-R-R-P-Y-I-L-COOH

3.2 Cell response measured through a label-free phenotypical assay As a first attempt to evaluate in vitro the biological effects triggered by our compounds, we decided to measure the cell response followed by stimulation with different pepducins and peptides using ECIS. Impedance monitoring is a non-invasive label-free technique that allows the real-time measurement of DMR in living cells grown on gold-plated electrodes and exposed to different ligands. The Figure 13 shows the variation of the normalized resistance which is plotted as a function of the time which represents the phenotypical changes undergone by the cells' monolayer after the stimulation with pepducins. Three distinct global response patterns were observed as described in Figure 13A, 13B, and 13C. Consistently, the first group designates compounds capable of inducing an abrupt increase in the cellular response, which reaches a transient maximal value and then descends sharply underneath the baseline. The transient maximal value was reached at approximately 10 minutes and varied slightly from one compound to another with maximal resistance values of 1.618±0.009 for PP-W9, 1.399±0.008 for PP-W12, and 1.729±0.01 for PP-W13 as shown in Table 2. Besides, in all cases, their higher maximal response outweighed the higher effect induced by NT and PP-ICL1, respectively (Figure 13A). The second group is characterized by

a gradual augmentation in the global cellular responses. The maximal response exhibited by the pepducins in this group is variable but quantitatively lower than those of the first group. Nonetheless, the maximal values were in all cases quantitatively similar to or greater than those displayed by NT and PP-ICL1 which were 1.183±0.002, and 1.132±0.007, respectively (Table 2). Furthermore, the maximal response value is reached nearly 10 minutes after pepducins application and decreases transiently underneath the baseline (Figure 13B). Finally, two pepducins provoke a moderate increase in cellular response that was sustained for a long period compared to the other two groups of pepducins. The decrease in such resistance values follows a smooth descent (Figure 13C). The non- palmitoylated peptide (NP-ICL1), the scrambled peptide (PP-ICL1-SCR), and the palmitate were used as negative controls and they behaved like the non-stimulated cells which did not induce any response.

A PP-ICL1 PP-W9 2.0 PP-W12 13 1.5 PP-W NT(1-13) 1.0 NP-ICL1 PP-ICL1-SCR 0.5 Palmitate

Normalized Resistance Non-stimulated cells 0.0 0 100 200

Norm_Time (min)

PP-ICL1 5 B PP-W PP-W6 PP-W7 8 2.0 PP-W PP-W11 1.5 NT(1-13) NP-ICL1 1.0 PP-ICL1-SCR Palmitate 0.5 Non-stimulated cells Normalized Resistance 0.0 0 100 200

Norm_Time (min)

PP-ICL1 2.0 PP-W10 PP-W14

1.5 NT(1-13) NP-ICL1 PP-ICL1-SCR 1.0 Palmitate Non-stimulated cells Normalized Resistance

0 100 200

Norm_Time (min)

Figure 14: Electric cell-substrate impedance sensing assays exhibiting the global morphological changes in response to treatment with pepducins derived from the ICL1 of hNTS1 Each curve represents the normalized resistance to the passage of an alternative current (4000 Hz) versus the time (3- hour) performed in a monolayer of CHO cells stably expressing hNTS1. Compounds were tested at a single concentration of 10 μM. The compound application was set as the “0” time-point. The resistance was normalized to the measurement preceding the injection of each compound. Each data set represents the mean ± S.E.M. of three independent experiments, performed in duplicate. According to the different behaviors described by the normalized resistance, compounds were categorized into three groups A, B, and C.

Table 2: Maximal response of pepducins derived from the ICL1 of hNTS1 in the electric cell-substrate impedance sensing assay Maximal response Compound Mean S.E.M. PP-W5 1.191 0.007 PP-W6 1.204 0.008 PP-W7 1.132 0.009 PP-W8 1.164 0.009 PP-W9 1.618 0.009 PP-W10 1.154 0.003 PP-W11 1.080 0.007 PP-W12 1.399 0.009 PP-W13 1.729 0.011 PP-W14 1.039 0.004 PP-ICL1 1.132 0.007 NT (1-13) 1.183 0.002 NP-ICL1 1.018 0.0004 PP-ICL1-SCR 1.021 0.0005 Palmitate 1.009 0.0001 Non-stimulated cells 1.029 0.0003

3.3 Signaling profiles of pepducins derived from the ICL1 of hNTS1

3.3.1 Signaling pathways associated to the Gα13 subunit BRET assays have been previously used to evaluate the specific interactions between hNTS1 and the heterotrimeric G proteins (Besserer-Offroy et al., 2017; Brouillette et al., 2020). Accordingly, we decided to confirm the capacity of our pepducins to activate G proteins signaling pathway by using BRET biosensors. The decrease in the fluorescence values due to the detachment of the pair Gα13-RlucII and Gγ1-GFP10 were used to monitor the specific engagement of this heterotrimeric G proteins. To choose the appropriate time for the BRET readout, a BRET signal time course was initially performed at a single pepducin concentration (data not shown). These kinetic assays revealed an optimal time of 30 minutes, in which the BRET signal get stabilized, and that corresponds to the processes that occur in the cell membrane before the pepducins start modulating the activity of the receptor. Figure 14 shows the percentage of activation of the Gα13 pathway 30 minutes after incubation with pepducin concentrations ranging from 10-7 to 10-4 M (at half-log

concentration intervals). The efficacy and potency to promote the engagement of the Gα13 subunit vary considerably depending on the position chosen to introduce the Trp in the ICL1 sequence. As expected, the negative control compounds (NP-ICL1, PP-ICL1-SCR, and the palmitate) do not induce any activation of the Gα13 pathway. Table 3 shows the efficacy and EC50 values obtained from the concentration-response curves of the most active pepducins of the series. PP-W13 and PP-W5 stood out for being the compounds displaying the better efficacy values in the pepducin series, which were superior to the NT efficacy. However, NT displays a potency value of 17.34 nM which is superior to those obtained with any of our compounds. Besides, PP-W13 and PP-W5 exceed the values of efficacy and potency fulfilled by the positive control PP-ICL1 which was 94.62 % and 8.75µM. Taking together, these results revealed a preferential augmentation in the potency values when the replacement is made in the residues close to the N-terminus and C-terminus of the peptide.

NT(1-13) α PP-W5 NT(1-13) G 13 dissociation Gα13 dissociation PP-W6 11 150 150 PP-W 7 PP-W PP-W12 100 10 100 PP-W PP-W13 8 PP-W 14 PP-W 50 50 9 PP-W PP-ICL1

%of activation PP-ICL1 0 %of activation 0 NP-ICL1 NS -11 -10 -9 -8 -7 -6 -5 -4 NP-ICL1 NS -11 -10 -9 -8 -7 -6 -5 -4 Palmitate (normalized NT(1-13)) to (normalized NT(1-13)) to -50 log[compound], (M) Palmitate -50 log[compound], (M) PP-ICL1-SCR PP-ICL1-SCR

Figure 15: Gα13 subunit engagement induced by pepducins derived from the ICL1 of hNTS1 BRET2 assay presenting the effect of increasing concentration of pepducins on the BRET between Gα13-RlucII and Gγ1-GFP10 after stimulation with coelenterazine 400A in HEK cells expressing hNTS1. Measurements were performed 30 minutes after pepducin stimulation. BRET2 ratios after stimulation with NT (1-13) at 1 μM were established as 100% of pathway activation in the normalization, whereas BRET2 ratios for non-treated cells were set as 0 % activation. Data represent the mean ± S.E.M. of two independent experiments, tested each time in duplicate.

Table 3: Efficacy and potency of pepducins derived from the ICL1 of hNTS1 to engage the Gα13 subunit Efficacy Potency Compound Emax (%) S.E.M. (%) EC50 (µM) S.E.M. (µM) PP-W5 116.0 4.870 2.0 0.467 PP-W6 30.45 9.836 12.35 1.94 PP-W9 50.18 9.480 14.93 9.68 PP-W11 76.83 4.184 13.89 3.04 PP-W12 80.91 1.546 12.53 0.88 PP-W13 132.6 4.192 5.42 0.78 PP-ICL1 94.62 3.370 8.75 1.25 NT (1-13) 92.82 3.153 1.73 (nM) 0.42 (nM)

3.3.2 Signaling pathways linked to the Gαq subunit

Historically, the hNTS1 has been linked to signal transduction cascades coupled to the Gαq subunit (Najimi et al., 2002). Consequently, we decided to test the effects of our pepducins on HEK cells transfected with the BRET biosensor Gαq–RlucII and Gγ1-GFP10. Figure 15 shows the percentage of activation of the Gαq pathway in response to increasing concentration of pepducins ranging from 10-7 to 10-4 M (at half-log concentration intervals). Table 4 represents the efficacy and the EC50 values obtained from the concentration- response curves of the most active pepducins of the series. NT displayed an efficacy of 102.6 % and EC50 value of 1.49 nM which were the best values reported for any of the tested compounds. Among the pepducins, PP-W5 and PP-W13 showed the highest efficacy and EC50 values. Their potency and efficacy values are superior to the positive control PP-ICL1 for which no curve was obtained at the range of concentrations evaluated. It is worth mentioning that apart from the compounds PP-W5, PP-W6, PP-W11, and PP-W13, the other pepducins exhibit signaling profiles similar to those of NP-ICL1, PP-ICL1-SCR and palmitate.

The Gαq subunit engagement in response to pepducins treatment was lower than the previously evaluated pathway. The comparison of the activation of both canonical G protein pathways revealed a biased mechanism of agonist allosteric modulation (Figure 15).

NT(1-13) α G q dissociation 5 Gαq dissociation PP-W NT(1-13) 6 PP-W 11 150 150 PP-W 7 PP-W PP-W12 100 10 100 PP-W PP-W13 PP-W8 PP-W14 50 9 50 PP-W PP-ICL1 PP-ICL1 %of activation 0 %of activation 0 NP-ICL1 NS -11 -10 -9 -8 -7 -6 -5 -4 NP-ICL1 NS -11 -10 -9 -8 -7 -6 -5 -4 Palmitate (normalized NT(1-13)) to Palmitate (normalized NT(1-13)) to -50 log[compound], (M) -50 log[compound], (M) PP-ICL1-SCR PP-ICL1-SCR

Figure 16: Gαq subunit engagement induced by pepducins derived from the ICL1 of hNTS1 BRET2 assay presenting the effect of increasing concentration of pepducins on the BRET between Gαq-RlucII and Gγ1-GFP10 after stimulation with coelenterazine 400A in HEK cells expressing hNTS1. Measurements were performed 30 minutes after pepducin stimulation. BRET2 ratios after stimulation with NT (1-13) at 1 μM were established as 100% of pathway activation in the normalization, whereas BRET2 ratios for non-treated cells were set as 0 % activation. Data represent the mean ± S.E.M. of two independent experiments, tested each time in duplicate.

Table 4: Efficacy and potency of pepducins derived from the ICL1 of hNTS1 to engage the Gαq subunit Efficacy Potency Compound Emax (%) S.E.M. (%) EC50 (µM) S.E.M. (µM) PP-W5 59.10 3.859 5.59 1.52 PP-W6 43.71 8.886 29.99 16.1 PP-W11 37.78 3.941 19.1 6.78 PP-W12 21.13 4.431 19.23 12.8 PP-W13 54.06 5.130 12.88 4.2 NT (1-13) 102.6 2.749 1.49(nM) 0.27 (nM)

3.3.3 Non-canonical G protein signaling pathways The hNTS1 induce the recruitment of β-arrestins 1 and 2 after being activated by NT (Besserer-Offroy et al., 2017). Therefore, we decided to use the BRET biosensors hNTS1- GFP10 and RlucII-β-arrestins 1 or 2 to assess whether or not our pepducins at the same range of concentration mentioned above can induce the recruitment of β-arrestins 1 or 2 (Figure 16). Interestingly, none of our pepducins seem to contribute to the recruitment of neither β-arrestins 1 or 2. Pepducins exhibit similar profiles to those exhibited by the negative controls. Even at the highest concentrations tested the percent of recruitment was not enough to build a concentration-response curve. PP-W9 showed the highest efficacy in the test of recruitment of β-arrestin 1, and its value was inferior to 40% at 100 mM.

Likewise, in the case of the recruitment of β-arrestin 2, the highest percent of activity was performed by PP-W5 and was less than 50% of recruitment at the higher concentration used. It is worth to mention that the preferential activation of canonical G protein- dependent pathways instead of the recruitment of β-arrestins corroborates a biased mechanism of activation.

NT(1-13) β-arrestin 1 recruitment PP-W5 β-arrestin 1 recruitment NT(1-13) 11 150 PP-W6 150 PP-W 12 PP-W7 PP-W 100 100 13 PP-W8 PP-W 14 50 PP-W9 50 PP-W PP-ICL1 PP-W10 %of activation 0 %of activation 0 NP-ICL1 NS -11 -10 -9 -8 -7 -6 -5 -4 PP-ICL1 NS -11 -10 -9 -8 -7 -6 -5 -4 Palmitate (normalized NT(1-13)) to NP-ICL1 (normalized NT(1-13)) to -50 log[compound], (M) -50 log[compound], (M) PP-ICL1-SCR Palmitate PP-ICL1-SCR

NT(1-13) PP-W5 β-arrestin 2 recruitment β-arrestin 2 recruitment NT(1-13) PP-W6 150 150 PP-W11 PP-W7 12 8 PP-W 100 PP-W 100 PP-W13 PP-W9 50 50 PP-W14 PP-W10 PP-ICL1

%of activation PP-ICL1 0 %of activation 0 NP-ICL1 NS -11 -10 -9 -8 -7 -6 -5 -4 NP-ICL1 NS -11 -10 -9 -8 -7 -6 -5 -4

(normalized NT(1-13)) to Palmitate (normalized NT(1-13)) to -50 log[compound], (M) Palmitate -50 log[compound], (M) PP-ICL1-SCR PP-ICL1-SCR

Figure 17: β-arrestin 1 and 2 recruitment induced by pepducins derived from the ICL1 of hNTS1 BRET2 assay presenting the effect of increasing concentration of pepducins on the BRET between hNTS1-GFP10 and RlucII-β-arrestin 1 or 2 after stimulation with coelenterazine 400A in HEK cells expressing hNTS1. Measurements were performed 30 minutes after pepducin stimulation. BRET2 ratios after stimulation with NT (1-13) at 1 μM were established as 100% of pathway activation in the normalization, whereas BRET2 ratios for non-treated cells were set as 0 % activation. Data represent the mean ± S.E.M. of two independent experiments, tested each time in duplicate.

3.4 Effect of pepducins on NT binding to NTS1 ECIS and BRET experiments demonstrated that pepducins were able to cause phenotypic cellular changes and to engage some G proteins signaling pathway in the absence of the orthostheric ligand. However, the effect of our pepducins on the NT binding to the hNTS1 has not yet been tested. The effect of the most potent pepducins (PP-W5, PP-W11, and PP- W13) on the NT bound to the orthosteric site of hNTS1 was evaluated using a competitive radioligand binding assay. Figure 17 shows the effect of increasing concentration of pepducins, NT (8-13) and our controls on the displacement of the radiolabeled [125I]-NT bound to hNTS1. As Table 5 shows, PP-W5, PP-W11, and PP-W13 displaced the radiolabeled

125 [ I]-NT attached to the receptor binding site with IC50 of 3.92± 0.55, 7.96 ± 1.02, 12.02 ±

1.79 µM, respectively. These IC50 were higher than those shown by PP-ICL1 which was 21.43 ± 4.36 µM. As controls, NP-ICL1, the PP-ICL1-SCR, and palmitate failed to displace radiolabeled NT from the hNTS1. Taking in account all those results the three selected pepducins seem to displace the radiolabeled NT from hNTS1 by an allosteric mechanism.

Binding hNTS1

150 NT(8-13) PP-ICL1 100 Palmitate

-NT bound-NT NP-ICL1 3 PP-ICL1-SCR 50 PP-W5 I]-Tyr PP-W11 125 0 PP-W13 NS -12 -11 -10 -9 -8 -7 -6 -5 -4

%of [ Log[Analogs], M -50

Figure 18: Displacement curves of [125I] Neurotensin on hNTS1 by NT (8-13) and pepducins. Concentration-response curves exhibiting the displacement of radiolabeled [125I]-NT binding in response to increasing concentrations of NT (8-13), PP-W5, PP-W11, PP-W13, NP-ICL1, PP-ICL1-SCR, PP-ICL1 and palmitate performed on hNTS1-expressing CHO cell membranes. Values were normalized according to NT (8-13); values for cells treated with 10 μM of NT (8-13) were set as 0 % [125I]-NT specific binding, and those for non-treated cells were set as 100 % [125I]-NT specific binding. Each data set represents the mean ± S.E.M. of two independent experiments, tested each time in triplicate.

Table 5: NT (8-13) and pepducins binding affinity on hNTS1. IC50 was derived from the resulting dose-response curves Compound IC50 (µM) S.E.M. (µM) PP-W5 3.92 0.55 PP-W11 7.96 1.02 PP-W13 12.02 1.79 Palmitate < 100 - NP-ICL1 < 100 - PP-ICL1-SCR < 100 - PP-ICL1 21.43 4.36 NT (1-13) 1.82 0.19

3.5 In vivo physiological effects of pepducins 3.5.1 In vivo physiological effects, analgesic effects Considering that our pepducins were able to engage G protein signaling pathways and displace radiolabeled NT from hNTS1, we decided to assess their physiological effects in Sprague-Dawley rats. Specifically, NT is able to produce a potent analgesia in various pain models and this effect is exerted through activation of the NTS1 and NTS2 receptors (Clineschmidt and McGuffin, 1977). The antinociceptive properties of pepducins were then assessed in an acute pain model using the tail-flick test (Figure 18), which reflects pain sensitivity or analgesia. The latency time to flick the rat’s tail to a noxious thermal stimulus (light beam) was measured following i.t. injection of pepducins. The longer the latency time to flick the tail, the stronger the analgesic effect. Pepducins were screened at an equimolar dose of 275 nmol/kg corresponding to that of PP-ICL1 which was previously found by our group to induce an analgesic effect (Brouillette et al., 2020). As seen in Figure 18A, the injection of 275 nmol/kg of PP-W5 leads to a significant analgesic effect with a magnitude similar to PP-ICL1 whereas PP-W13 did not significantly increase the tail-flick latency. Interestingly, PP-W11 induced a drastic and prolonged antinociceptive effect lasting 60 min and reaching the maximal effect (cut-off of the apparatus, threshold of 10 sec) during 30 minutes from 10 to 40 min post-i.t. injection. No changes in tail-flick latencies were observed following the injection of the vehicle or the scrambled peptide at the same dose. At a lower dose of 100 nmol/kg, only PP-W11 was able to induce an analgesic effect among

all pepducins and the effect was still strong 10 min-post injection nearly reaching the cutoff of the apparatus (Figure 18B).

A B *** *** **** **** **** 10 * * 10

*** 8 8 *** **** * **** **** * * Vehicle (10% DMSO, 20% PEG) *** * 6 * * PP-ICL1 6 * * * * * *** PP-ICL1-SCR * * * ** 4 PP-W5 4 * PP-W11

13 time (sec) Latency Latency time (sec) Latency 2 PP-W 2

0 0 0 10 20 30 40 50 60 0 10 20 30 40 50 60 Time (min) Time (min)

Figure 19: Analgesic effect of acute intrathecal injection of SCR peptide, PP-ICL1, PP-W5, PP-W11, PP-W13 at 275 nmol/kg (A) and 100 nmol/kg (B) on tail-flick latencies in Sprague-Dawley rats Results are expressed as mean ± S.E.M. (N=5/group). Comparisons between the vehicle and the pepducins at equimolar doses were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Dunnett's correction. *p<0.05, **p<0.01, ****p<0.0001.

As PP-W11 showed the most promising antinociceptive effect, this pepducin was subject to a dose-response curve (Figure 19). Figure 19A shows the analgesic effect of PP-W11 at three doses. Note that the injection of PP-W11 at the lowest dose of 50 nmol/kg still resulted in a significant increase of the tail-flick latency. The maximal possible effect (% MPE) was observed at 10 min post-injection when the antinociceptive effect was maximal for each dose (Figure 19B). % MPE reached 45% for the lowest dose and 100% for the highest dose,

11 which corresponds to the machine’s cut-off. Based on the %MPE, PP-W displayed an ED50 of 54.81 nmol/kg. However, PP-W11 exhibited the best efficacy with 100 % MPE while NT hardly reaches 50 % at the highest doses (Figure 19C) (Sarret et al., 2005).

To assess whether the PP-W11-induced analgesic effect was mediated by NTS1 activation, we co-administered PP-W11 and the selective NTS1 antagonist, SR48692. When injected with the NTS1 antagonist, the antinociceptive effect of PP-W11 was reversed. These results demonstrate that the analgesic effect of this pepducin is dependent on NTS1 activation (Figure 19D).

Figure 20: Analgesic effect of acute intrathecal injection of increasing doses of PP-W11 on tail-flick latencies in Sprague-Dawley rats (A) time-course of PP-W11 analgesic effect at doses ranging from 50 to 275 nmol/kg, (B) % maximal possible effect calculated at 10 min post-injection of the pepducin where the antinociceptive response was maximal for each dose, (C) dose-effect of the compound and (D) antagonizing effect with SR48692 (NTS1 antagonist). Results are expressed as mean ± S.E.M. (N=5/group). SR48692 was administered at a 5x equimolar dose of PP-W11. For the time-courses of the effects, comparisons between the vehicle and the pepducin at different doses (A) or between the vehicle and the compounds at equimolar doses (D) were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Dunnett's correction. For the % maximal possible effect at 10 min, comparisons were performed using one-way analyses of variance for repeated measures followed by multiple comparisons using Dunnett’s correction between the vehicle and the pepducin at different doses. Nonlinear regression using three parameters was used to determine the dose-effect relationship. *p<0.05, ***p<0.001, ****p<0.0001.

3.5.2 In vivo physiological effects, hypothermic effects In addition to the previous mentioned analgesic effect, NT is also responsible for other physiological effects including hypothermia and hypotension. We first measured pepducins’ effect on body temperature using a rectal probe (Figure 20A and B). The transient hypothermia observed following i.t. administration of the vehicle, the scrambled peptide, or any of the pepducins result from the short anesthesia with isoflurane allow. Following i.t. injection of 275 nmol/kg of pepducins, PP-W11 induced a significant hypothermia of - 1°C as compared to vehicle from 20 to 60 minutes post-injection (Figure 20A). At an equimolar dose, PP-W5 significantly decreased body temperature at 30 and 60 min after administration while PP-W13 had no effect. It is important to notice that PP-ICL1 had no effect on body temperature when compared with vehicle injection. When decreasing the injected dose to 100 nmol/kg, only PP-W11 was once again able to significantly decrease the body temperature by -1°C resulting in a small and long-lasting hypothermia (Figure 20B).

A B Time (min) Time (min) 0 10 20 30 40 50 60 0 10 20 30 40 50 60 0.0 0.0 Vehicle (10% DMSO, 20% PEG) -0.5 PP-ICL1 -0.5 PP-ICL1-SCR -1.0 -1.0 **** * 5 **** * PP-W **** **** **** * **** * **** 11 -1.5 * PP-W -1.5 **** PP-W13 -2.0 -2.0 DeltaBody Temperature (°C) DeltaBody Temperature (°C)

Figure 21: Variation of body temperature monitored after intrathecal injection of pepducins Delta body temperature monitored after intrathecal injection of vehicle, PP-ICL1, PP-W5, PP-W11 and PP-W13 at 275 (A) and 100 nmol/kg (B). Results are expressed as mean ± S.E.M. (N=5/group). Comparisons between the vehicle and the pepducins were performed using two-way analyses of variance for repeated measures followed by multiple comparisons using Dunnett's correction. *p<0.05, ****p<0.0001.

3.5.3 In vivo physiological effects, hypotensive effects Finally, we monitored the mean arterial blood pressure after an i.v. administration of 55 nmol/kg of pepducins using direct and continuous intracarotid measurements (Figure 21). NT was previously shown to lead to a sharp drop in blood pressure at 0.1 mg/kg i.v. (0.12 µmol/kg). This strong hypotensive action is triphasic with a first short drop of blood pressure (about -25 mmHg) rapidly followed by a swift return to baseline level before a sustained hypotension (third phase) (Chartier 2021, Fanelli 2015). As previously observed, PP-ICL1 drastically decreases blood pressure following i.v. administration lasting at least 1000 sec. Our new pepducins PP-W5 and PP-W13 displayed lower hypotensive effects as compared to PP-ICL1 when used at an equimolar dose. Surprisingly, PP-W11 did not produce any hypotension at this dose. As expected, no change in blood pressure was observed following i.v. injection of the scrambled peptide.

Time (s) 30 500 1000 20 10 0 NT(8-13) -10 PP-ICL1-SCR -20 PP-ICL1 -30 PP-W5 -40 PP-W11 -50 PP-W13 Delta MABP (mmHg) -60 -70 Figure 22: Delta mean arterial blood pressure Delta mean arterial blood pressure (MABP) monitored after intravenous injection of NT (8-13) at 123 nmol/kg and PP-ICL1-SCR, PP-ICL1, PP-W5, PP-W11 and PP-W13 at 55 nmol/kg. Results are expressed as mean ± SEM (N=5/group).

DISCUSSION 4.1 Synthesis of pepducins derived from the ICL1 of hNTS1 The vast majority of the synthesized pepducins to date are based on the sequences of the ICL2 and ICL3 of their targeted receptors (O’Callaghan et al., 2012). Interestingly, these compounds seem to allosterically stabilize the receptor’s conformation in a way that contributes to a closer contact between the intracellular signaling machinery and the receptor’s ICL2 and ICL3 (Wess, 1997). The discovery that pepducins derived from the ICL1 favors the interaction between GPCRs and the heterotrimeric G proteins provided new elements to guide the synthesis of new pepducins derived from this intracellular loop (Oldham et al., 2008). In the case of the hNTS1, most of the ligands that have been developed interact orthosterically with the receptor’s binding site (Eiselt et al., 2019). Therefore, their mechanisms of action are different from that of pepducins. Accordingly, the synthesis of pepducins targeting the hNTS1 was one of the initial aims of our research group. Several SAR studies were performed with this sort of molecules derived from the ICL1 of hNTS1. It should be mentioned that ICL1 was chosen due to the high degree of sequence identity in rats and humans (Uniprot website, Ref: P30989), which makes possible to perform our in vitro assays on the human subtype and in vivo experiment in rats. These first studies consisted of the substitution of each ICL1 amino acid residue by opposite stereogenic analogs as well as an alanine residue. Those substitutions aimed to obtain insights into the importance of the stereochemistry of the chiral center and the relevance of the amino acid side chain for the activity of the molecule (non-published results). Besides, a series of pepducins was also synthesized by removing progressively the last two C- terminal residues of the ICL1 of hNTS1 sequence to determine the minimal amino acids sequence required to maintain a biological effect. These truncated pepducins induced a partial activation of signaling pathways dependent on the Gαq and Gα13 subunit at 10 µM. PP-ICL1 whose sequence contains all the amino acids present in the ICL1 (sequence presented in Table 1 in page 51) displayed a marked analgesic effect when administered intrathecally in acute, tonic, and chronic pain models (Brouillette et al., 2020). Therefore, we decided to explore the effect of introducing a hydrophobic residue into the structure of

PP-ICL1. As a result of these substitutions, we obtained a library of 10 compounds, as presented in Table 1. Since there was no information about the pepducins’ residues involved in the interaction with the allosteric site placed in the intracellular surface of hNTS1, all the positions were modified. The decision to choose the tryptophane as a substitute was based on its capacity to mediate hydrophobic effects which is known to enhances protein-protein interaction (Bissantz et al., 2010), which represented, in this case, an increase in the interaction pepducin-hNTS1. To my knowledge, this is the first time that this strategy has been applied to the pepducins approach.

4.2 Cell response measured through a label-free phenotypical assay ECIS was chosen to evaluate our compounds' ability to induce a biological response expressed in terms of total mass distribution. ECIS has previously been used to evaluate the global cell phenotypical response induced by orthosteric ligands such as NT, NN, and hNTS1 antagonist (Besserer-Offroy et al., 2017). Similarly, ECIS have been used to characterize the phenotypical cellular effects induced by a pepducins library derived from the ICL1 of hNTS1 generated by progressive removal of the last two amino acids near the C-terminal (Brouillette et al., 2020). Accordingly, the morphological responses resulting from the activation of hNTS1 by orthosteric, as well as allosteric ligands have been well characterized. These assays demonstrated that pepducins could modulate hNTS1 in a manner that produced effects distinct from those of NT (8-13). Furthermore, they informed us that, pepducin-induced activity is triggered when the cells are stimulated at high concentrations range (10-100 µM). In that sense, we used this technique to test whether the tryptophan introduced into the PP-ICL1 sequence represents an improvement in their biological potential. Consequently, each of the synthesized compounds was analyzed at the lowest concentration from which PP-ICL1 started showing a particular biological effect in previous experiments (Brouillette et al., 2020).

The maximal value reached by the variation of the normalized electrical resistance over time was used to monitor and quantify the whole cell integrated response and this way to evaluate the effect of the substitution in the compound activity. PP-W5, PP-W6, PP-W9, PP-

W12 and PP-W13 have shown maximum responses superior to the 1.18-fold increase displayed by NT (1-13) when it was evaluated at the single concentration of 1000 nM as shown in Table 2. They exhibit a maximal response of 1.191±0.007, 1.204±0.008, 1.618±0.009, 1.399±0.008, and 1.729±0.01, respectively. These pepducins also induced a greater global response than those of PP-ICL1 and its truncated derivates evaluated at a 100 µM concentration (Brouillette 2019, unpublished results). These results suggest an improvement in the biological potential because of the introduction of tryptophan into the sequence of pepducins derived from the ICL1 of the hNTS1. On the other hand, the behavior of the global induced response over time described different curves as compared to that of NT which suggests a difference in the biological activity (Besserer-Offroy et al., 2017). In the ICL1 sequence, there is a core made up of the following amino acid residues (Ser5- Leu6-Gln7) whose sequence is repeated twice. No significant change in the cellular response was observed followed by the substitution of any of those homologs’ residues in the second triads. These results may suggest that the different cellular responses obtained could be due to the position where tryptophan is introduced in the ICL1 chain. We also identified a pattern characterized by the higher values of normalized resistance when the substitutions were made closer to the pepducin’s C-terminus and N-terminus.

4.3 Signaling pathways associated to G proteins and β-arrestins 1 and 2 Impedance assays were useful to assess the global responses induced by our pepducin series. Nonetheless, it did not provide any insight about which signaling pathways were involved in those morphological changes. Accordingly, we decided to specifically evaluate the engagement of heterotrimeric G proteins and the recruitment of beta arrestins 1 and 2 by using BRET biosensors. The capacity of pepducins to induce an active conformational state of the receptor favorable to coupling G protein subunits has been mainly tested indirectly (L. Covic et al., 2002; Lidija Covic et al., 2002). Most of the evidence collected comes from the measurement of second messengers associated with the engagement of G proteins (Wettschureck and Offermanns, 2005). The direct determination of the interaction between hNTS1 and G proteins has been previously evaluated. Besserer-Offroy et al were the first to report these kinds of interactions by using BRETs biosensors. This research

confirmed that the Gαq, Gαi1, GαoA, and Gα13 protein signaling pathways as well as the recruitment of β-arrestins 1 and 2 are engaged after hNTS1 activation. Moreover, this approach always involved the use of endogenous ligands like NT, NT (8-13), NN, and hNTS1 antagonists. Therefore, the modulation was always mediated by the interaction of the ligand with the orthosteric site of hNTS1 (Besserer-Offroy et al., 2017). hNTS1 has also been targeted by cell penetrating lipopeptides based in the ICL1 of the hNTS1 that may allosterically modulate the receptor activity by inducing the engagement of the Gαq and

Gα13 protein signaling pathways (Brouillette et al., 2020).

In our study, pepducins have been designed to reach the intracellular surface of hNTS1. Their possible mechanism of action is based on their probable interaction with an allosteric site of hNTS1 placed in the inner cell surface. The use of BRET biosensors for Gα13 (Gα13-

RlucII) and Gγ1-GFP10, for Gαq (Gαq-RlucII) and Gγ1-GFP10, and hNTS1-GFP10 and RlucII-β- arrestin 1 or 2 for the β-arrestins 1 and 2 allowed us to evaluate the effects of our pepducins on four different signaling pathways. The BRET results indicated that our series of pepducins favors the coupling of heterotrimeric G proteins to the hNTS1. Specifically, the best efficacy and potency values were observed for the BRET biosensor targeting the Gα13 signaling pathway. Among all the pepducins tested, PP-W5 and PP-W13 stood out for displaying efficacy values even superior to that of the NT, which confirmed that the introduction of tryptophan as a substitute residue in these two positions can enhance the activation of G proteins. PP-W11 and PP-W12 also showed better efficacy values than the rest of the synthesized pepducins, however they never exceeded the efficacy and potency of NT and PP-ICL1. It is worth mentioning that the NT and PP-ICL1 displayed potencies in the range of nanomolar and micromolar, respectively. These values agreed with what was previously reported in the literature (Besserer-Offroy et al., 2017; Brouillette et al., 2020). The

5 pepducins were also capable to activate the pathway dependent on the Gαq subunit. PP-W PP-W13, PP-W11 and PP-W6 displayed the best efficacy in the series (Table 3). However, the potency and efficacy values were slightly lower than for Gα13 pathway. Moreover, none of the pepducins exceeded the maximum NT efficacy value as in the previous pathway.

Pepducins PP-W7, PP-W8, PP-W10, PP-W12 and PP-W14 showed no activation. On the other hand, β-arrestin 1 and 2 were not activated for any of the pepducin. Pepducins induced the engagement of the G protein pathway without recruiting β-arrestins 1 and 2 suggesting the existence of a biased regulatory mechanism.

Regarding the nature and position of the substitution, there seems to be a general pattern leading to larger efficacies and potencies and therefore, a better engagement of G proteins’ subunit when the substituted amino acid is placed at both extremes of the peptide sequence (N-terminus and/or at the C-terminus). Indeed, in the middle of the ICL1 sequence, there is a core of three amino acids (Ser5-Leu6-Gln7) which repeats twice. The substitution of the homolog's residues like the Leu9 and Gln10 in the second core influenced neither the hNTS1 activation nor the potency's values. These results could suggest that the tryptophan position in the ICL1 could contribute to the increase of the signaling pathway associated with G proteins.

4.4 Effect of pepducins on NT binding to NTS1 BRET assays have revealed that pepducins can interact with the hNTS1. Specifically, BRET demonstrated the preferential engagement of some heterotrimeric G proteins in a total absence of the endogenous natural ligand (allosteric modulation). Previous experiments performed by Covic et al, with human platelets expressing PAR1, demonstrated that the treatment with the competitive inhibitor BMS-200661 does not suppress the physiological effect triggered by pepducins. These results suggested that pepducins interact with a site different from the receptor’s binding site and that they accomplish the agonist or antagonist allosteric effect without affecting the binding of the native peptide (L. Covic et al., 2002). Similar results were obtained by using the pepducins ICL1-9, ICL3-8 and ICL3-9 at concentration of 3, 10 and 30 µM. These pepducins did not displaced the radiolabeled orthosteric ligand [125I]-iodocyanopindolol in HEK cells overexpressing FLAG-β2AR (Carr et al., 2014). In contrast, Brouillette et al, reported a displacement of the radiolabeled [125I]- NT after treatment with pepducins derived from the hNTS1 (Brouillette et al., 2020). In this study, we have demonstrated that the introduction of the tryptophan in PP-W5, PP-W11, and PP-W13 seems to enhance the displacement of the radiolabeled [125I]-NT bound to the

hNTS1 orthosteric site. This effect could be due to an increase in the interaction between these pepducins and hNTS1 in an allosteric binding site placed in the receptor’s intracellular surface. The non-palmitoylated peptide (NP-ICL1) does not act by the previous described mechanism of action of pepducins since it lacks the hydrophobic tail. This molecule does not displace the radiolabeled [125I]-NT, by acting on the orthosteric binding site which confirmed the importance of the peptide moiety in the pepducin mechanism of action and the possible modulation of the activity from an intracellular location. Finally, the use of the scrambled peptide demonstrated that the displacement of the radiolabeled ligand was sequence dependent.

4.5 In vivo physiological effects of pepducins 4.5.1 In vivo physiological effects of pepducins: Analgesic effects hNTS1 mediates physiological responses associated with hypotension, hypothermia, and analgesia (Boules et al., 2013). Besides, the central administration of NT has been associated with higher analgesic effects than those observed with morphine at equimolar dose (Clineschmidt et al., 1979). Similarly, the antinociceptive effect of compounds targeting the orthosteric site of hNTS1 has been previously described (Eiselt et al., 2019). In addition, Brouillette et al evaluated the ability of a family of truncated pepducins derived from ICL1 of hNTS1 to allosterically induces an analgesic effect after intrathecal administration. This test was performed in an acute pain model or tail-flick test and the compounds were evaluated in agonist mode which means without the co-administration of NT (Brouillette et al., 2020).

In this study, the substitution of specific residues in the structure of pepducins derived from the ICL1 of hNTS1 was found to be effective in increasing global cellular responses. Similarly, the introduction of tryptophane residue favored the activation of the signaling pathway dependent on Gα13 and Gαq subunits. Consequently, we also decided to evaluate the analgesic potential of this family of pepducins at 275 nmol/kg, which was the dose from which PP-ICL1 was shown to exert an analgesic activity (Brouillette et al., 2020). Accordingly, the analgesic efficacy of each compound was evaluated by comparing the time of apparition of a spinal reflex in response to the application of radiant heat on the rat’s tail. 68

The intrathecal administration of PP-W5 and PP-W11 resulted in a significant increase in the latency time compared to the negative controls and to PP-W13, which displayed a moderate analgesic effect. It should be mentioned that PP-W5 induced analgesic responses like those reported by PP-ICL1 (Brouillette et al., 2020). However, the effects of PP-W5 seem to last slightly longer over time. On the other hand, the introduction of the tryptophan residue in position 11 was characterized by an immediate antinociceptive response. This response is greater than all analgesic values reported by any pepducin synthesized so far. Besides, it is characterized by a maximum effect that lasts over time for approximately 30 minutes. PP- W11 equally excelled among the other pepducins for inducing an appreciable analgesic response at a dose almost 2 times lower than those tested previously (Brouillette et al., 2020). Accordingly, the injection of PP-W11 showed a dose-response antinociceptive behavior which reached almost 50% of the maximum analgesic effect even at the lowest concentration evaluated. On the other hand, the % MPE obtained from the dose-response

11 curve allowed the determination of the ED50. PP-W reported a value of ED50 of 54.81 nmol/kg, which is 15 times higher than that of the NT, which was of 1 nmol/rat or 3-4 nmol/kg (Sarret et al., 2005). Regarding, morphine whose ED50 was of 1.3 µg/rat or 4

11 nmol/rat or 3.4-4.6 nmol/kg, the ED50 of PP-W was 4 times higher (Makuch et al., 2013). However, PP-W11 exhibited better efficacy with 100 % MPE while NT barely reached 50 % at the highest dose (Sarret et al., 2005). The lower potency displayed by pepducins when we tested their analgesic effects may be explained by their allosteric mechanism of action regarding orthosteric ligands. Our research team previously reported the binding of a macrocyclic NT(8-13) analog to the orthosteric site of hNTS1 producing a potent analgesic activity with an ED50 of 4.63 μg/kg (5.68 nmol/kg) using the same pain model (Sousbie et al., 2018).

In addition, the use of the specific antagonist SR48692 corroborated that the antinociceptive response triggered by PP-W11 is the result of the specific activation of hNTS1. Besides, It also confirmed the proposed mechanism of action of pepducins which require the mandatory presence of a targeted receptor for the triggering of any physiological response (L. Covic et al., 2002).

4.5.2 In vivo physiological effects of pepducins: Hypotensive effects The link between the neurotensinergic system and the cardiovascular system has been widely demonstrated. The hNTS1 receptor plays an important role in this relationship since the binding of its endogenous ligand can trigger a vascular effect (Boules et al., 2013). The hNTS1r has been targeted by numerous drugs to modulate the hypotensive effects. Most of these drugs have been designed to act on the orthosteric site of hNTS1 (Eiselt et al., 2019). However, Brouillette et al have described a family of pepducins that interacts with an allosteric site probably located on the intracellular surface of the hNTS1. Specifically, one of the members of the family of compounds derived from the ICL1 of hNTS1 induced a pronounced vascular response (Brouillette et al., 2020).

Our series of pepducins are derived from the same structural motif as those described by Brouillette et al. Therefore, the possibility of inducing any vascular response remained high. Accordingly, the blood pressure was measured with the help of an intracarotid cannula connected to a pressure transducer followed by the administration of our compounds to rats. NT revealed an immediate triphasic hypotensive effect, characterized by a first phase with a short drop (about -25 mmHg) rapidly followed by a swift return to baseline level (second phase) before a sustained depression (third phase). This observation coincides with previously reported behaviors not only reported by NT but also with NT analogs, such as JMV2007 and PD149163 (Eiselt et al., 2019; Fanelli et al., 2015). Interestingly, the introduction of the tryptophan residue into the ICL1 sequence at position 11 does not seem to promote any vascular effect at an equimolar dose to that of PP-ICL1. Specifically, PP-W11 that produces a strong analgesic effect, was not effective to induce variation in blood pressure. In addition, the hypotensive effects provoked by PP-W5 and PP-W13 were very weak compared to those shown by PP-ICL1. As expected, PP-ICL1 reported a marked hypotensive response that matched the results previously reported by Brouillette et al. (Brouillette et al., 2020).

4.5.3 In vivo physiological effects of pepducins: Hypothermic effects The interaction between hNTS1 and NT results in the variation of the body temperature (Boules et al., 2013). In this regard, analogs of NT (8-13) have been designed to interact with the active site of hNTS1. These molecules have shown a considerable hypothermic effect by causing a decrease in body temperature by several degrees. Specifically, the hNTS1 agonist PD149163 markedly reduces the body temperature by 3 degrees followed by its administration at a dose of 10 nmol (25 g/kg). However, so far, there is no information about which signaling pathways are engaged in this physiological effect (Eiselt et al., 2019). On the other hand, the administration of compounds that target hNTS1 at sites other than the orthosteric site has shown a moderate hypothermic effect (Brouillette et al., 2020). Interestingly, PP-ICL1, our control peptide derived from ICL1 of hNTS1, does not cause any variation in body temperature. Its behavior was relatively similar to that of the negative controls used (PP-SCB-ICL1 and DMSO) (Brouillette et al., 2020). In contrast, the compounds obtained by introducing the tryptophane at the positions 5 (pepducin PP-W5) displayed a hypothermic effect that lasted for the 60 minutes of the test. PP-W11 which previously had shown a potent analgesic effect was also associated with a decrease in the body temperature by at least one degree. However, the dose of 275 nmol/kg at which P-W11 was tested was higher than the 10-20 nmol (25 µg/kg) dose at which analogs of NT (8-13) acting on the orthosteric site trigger a certain physiological effect (Eiselt et al., 2019).

4.5.4 Further insight into the tryptophane derivatives PP-ICL1 was the parental compound and best hit because of its marked analgesic and hypotensive effects in a library of pepducins based on the ICL1 of hNTS1. The SAR study presented here aimed to use the hydrophobic effect caused by the insertion of tryptophans in the PP-ICL1 sequence to enhance the interaction with hNTS1 in order to promote better physiological responses. The key residues in the interaction between PP-ICL1 and hNTS1 are not yet known. Therefore, all the amino acid positions in the ICL1 were explored. Tryptophane substitutions were able to abolish the undesirable hypotensive effect induced by PP-ICL1. Besides, the modification in tree punctual position pepducins PP-W5, PP-W11, and PP-W13 triggered an analgesic profile superior to the parental pepducin. The results

obtained in this thesis slightly suggest that analgesia and hypotension might not be induced by the same signaling pathways, since our tryptophan derivatives seem to trigger analgesia reducing the hypotensive effect. Nonetheless, analgesia and hypothermia seem to be linked since PP-W11 displaying the better analgesic values provoked a reduction in body temperature Table 6. At this point, the hypothetical character of the last statements needs to be validated in further researches, since the signaling pathway associated with analgesia, hypotension, and hypothermia are not yet established.

Table 6: Physiologic effect of pepducins in vivo. The amounts of addition or subtraction symbols are associated with the magnitude of the physiological effect.

Physiological Effects Compound Analgesia Hypotension Hypothermia PP-ICL1 + + - PP-W5 ++ - - PP-W11 +++ - + PP-W13 + - -

CONCLUSIONS The goal of this research was to evaluate the effect of increasing the hydrophobic properties of a previously characterized analgesic molecule (PP-ICL1) on its pharmacological potential. Consequently, our research included the synthesis of a library of pepducins based on the sequence of PP-ICL1, where each amino acid position was substituted by a tryptophan. The insertion of tryptophan into position 9, 12, and 13 of the PP-ICL1 was associated with greater global cellular responses that could be related to an increase in the biological activity. The results obtained, also point to an enhancement in the engagement of the Gαq and Gα13 protein-dependent signaling pathways over β-arrestins revealing a possible biased allosteric modulation. The competitive radioligand binding assay confirmed that this allosteric modulation affects the NT binding to the orthosteric binding site of NTS1. PP-W11 stands out for being one of the most potent compounds characterized in vitro. This pepducin also demonstrated a marked improvement in the analgesic effects regarding the parental pepducin PP-ICL1. Taking together these results confirmed that enhancing selectively the hydrophobic potential of PP-ICL1 could constitute a valid approach in the refinement of these pepducin properties.

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