Peptidic opioid receptor antagonists and uses thereof

Abstract

The present invention relates to antagonistic peptides specifically binding the µ-opioid receptor (µOR). Said antagonists are mimetic peptides of a single domain antibody ligand having binding affinity to the µOR. The invention furthermore provides pharmaceutical compositions comprising such antagonistic peptides, as well as the use of such antagonistic peptides in methods for treating diseases.

Description

[0001] PEPTIDIC OPIOID RECEPTOR ANTAGONISTS AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to antagonistic peptides specifically binding the p-opioid receptor (pOR). Said antagonists are mimetic peptides of a single domain antibody ligand having binding affinity to the pOR. The invention furthermore provides pharmaceutical compositions comprising such antagonistic peptides, as well as the use of such antagonistic peptides in methods for treating diseases.

[0004] BACKGROUND

[0005] G protein-coupled receptors (GPCRs) represent key therapeutic targets due to their central roles in cellular signaling and control over a plethora of physiological processes. Developing new ligands that bind a given GPCR with high selectivity remains a significant challenge in drug discovery (1-3). Small molecule ligands have historically dominated the landscape of GPCR-targeted drugs but recently biologies, including antibodies and nanobodies (Nbs), have emerged as an alternative class of ligands that offer distinct advantages and hold promise for therapeutic developments (4, 5). Nbs are single domain antibody fragments derived from heavy chain-only antibodies, which naturally occur in camelids and cartilaginous fish, and are characterized by a small size, high antigen binding affinity, and binding loops that can access deep cavities on target proteins (6). Nbs can show enhanced selectivity over small molecules due to their ability to interact with unique and extended epitope surfaces. Over the last decade, Nbs that bind GPCRs on their intracellular side have served as innovative research tools to uncover GPCR signal transduction mechanisms (7, 8). For example, Nbs were used as crystallization chaperones or as fiducial markers in high resolution structural studies (9-72). Conformation-selective Nbs were also repurposed into biosensors to report GPCR activity in living cells (13, 14). Only recently, several Nbs that bind GPCRs as extracellular ligands and thereby modulate receptor function have been described (15-20). Generating knowledge on GPCR-targeting Nbs is key to unlocking their potential as both versatile research tools and therapeutic compounds.

[0006] Opioid receptors (ORs) are prototypical members of the rhodopsin-like GPCR family and function in pain modulation and analgesia (21, 22). The OR family comprises four major receptor subtypes, including the pOR, 5OR, KOR, and nociceptin-OR (NOPR), with the pOR representing the prime therapeutic target for pain relief. Approved drugs that target pORs are diverse small molecule compounds, including the widely used analgesics morphine and fentanyl. The ligand repertoire has recently been expanded through structure-based molecular docking, rational design, and high-throughput screening, delivering new OR ligands with distinct pharmacological profiles, including biased agonism, receptor subtype selectivity, and pharmacokinetic properties (23-25).

[0007] Opioid receptor-targeting ligands that activate the receptor (agonists) are important analgesics yet produce significant toxicity and have high abuse potential. Therefore, ligands that antagonize the receptor (antagonists) are of clinical relevance due to their ability to reverse opioid-induced side effects during pain treatment and after drug abuse, and represent approved treatment options for the reversal of respiratory depression and opioid- induced constipation. Currently available pOR antagonists, such as naloxone and naltrexone, are centrally acting antagonists frequently used in drug overdose. Peripherally restricted OR antagonists, including naloxegol or methyl-naltrexone, are versions of naloxone or naltrexone that contain chemical modifications, which reduce their passage across the blood-brain barrier (BBB). They reverse the gastrointestinal effects of opioid agonists but preserve the CNS-mediated analgesia and are used in the treatment of opioid- induced constipation. Both types of pOR antagonists have limitations: centrally acting opioid antagonists often have a very short in vivo half-life (naloxone <1 h), which means that the effects may not last as long as the duration of opioids whose effects they are intended to reverse, and they are not useful in rendering ineffective the administration of exogenous opioids for extended durations. The peripherally acting antagonists lack pOR selectivity and their remaining partial BBB passage attenuates the analgesic effects of opioids, rendering clinical dosing difficult.

[0008] The discovery of new modulators of pOR function, in particular antagonists, remains a pressing necessity in medicine, such as for developing compounds that can reduce or reverse the deleterious opioid side effects (26). Until now, no antibody or single domain antibody ligand for ORs has been characterized in-depth, representing a hurdle in exploiting the unique features of biologies to effectively target ORs.

[0009] SUMMARY OF THE INVENTION

[0010] The present invention provides highly selective p-opioid receptor (pOR)-targeting antagonistic peptides. These antagonists are derived from a study of the cryoEM structure of the pOR bound to an extracellular single domain antibody ligand (termed ‘NbE’) (67, 9). The antagonists of the invention are therefore mimetic peptides of the NbE ligand, in particular of the CDR3 loop of NbE. The antagonists of the invention present novel properties based on NbE’s unique pharmacological characteristics (such as high selectivity, slow off-rate, possibly long in vivo half-life), and have the potential to outperform currently available pOR antagonists.

[0011] In one aspect, the invention provides an antagonist of p-opioid receptor (pOR), wherein the antagonist is a mimetic peptide of the CDR3 loop of a single domain antibody comprising a sequence of SEQ ID NO: 1 , wherein the CDR3 loop comprises a sequence of SEQ ID NO: 2.

[0012] In another aspect, the invention provides a pharmaceutical composition comprising a therapeutically effective amount of the antagonist according to the invention and at least one of a pharmaceutically acceptable carrier, adjuvant or diluent.

[0013] In a further aspect, the invention provides the use of the antagonist according to the invention or the pharmaceutical composition according to the invention to antagonize pOR signaling activity.

[0014] In a further aspect, the invention provides the antagonist or the pharmaceutical composition according to the invention for use in a method of treating a medical condition, preferably wherein the medical condition is an opioid receptor-related medical condition, more preferably a pOR-related medical condition. In some embodiments, the invention provides a method of treating a medical condition, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of the antagonist or the pharmaceutical composition according to the invention. In some embodiments, the invention provides the use of the antagonist or the pharmaceutical composition according to the invention for the manufacture of a medicament for the therapeutic treatment of an opioid receptor-related medical condition, more preferably a pOR-related medical condition.

[0015] BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 : Single domain antibody NbE binds the extracellular side of the pOR and acts as an antagonist, (a) Confocal images of HEK293 cells expressing FLAG-pOR, 5OR, KOR, or NOPR (labeled with anti-FLAG M1-AF647) and incubated with 1 pM purified AF488-labeled NbE. Scale bar, 10 pm. (b) FACS-based quantification of AF488-NbE binding to HEK293 cells expressing murine pOR, 5OR, KOR, or NOPR at different NbE concentrations (cells gated for similar receptor surface levels), N = 3, mean ± SEM. (c) FACS-based quantification of AF488-NbE binding (1 pM) to pOR-expressing HEK293 cells pretreated with naloxone at different concentrations. N = 3, mean ± SEM. (d) Maximum cAMP response in HEK293 stably expressing pOR, stimulated with 2.5 pM forskolin (FSK, norm, to 100%), treated with increasing concentrations of DAMGO (N = 5) or NbE (N = 3), mean ± SEM. (e) Maximum cAMP response in HEK293 stably expressing pOR, stimulated with 2.5 pM FSK (norm, to 100%), treated with 10 nM DAMGO (N = 4) or 30 nM morphine (N = 2) (corresponds to agonists’ EC50, respectively) and pre-incubated with increasing concentrations of NbE, mean ± SEM. In all panels, N indicates the number of independent experiments.

[0017] Figure 2: Architecture of the NbE-pOR complex, (a) Cryo-EM map (left) and ribbon representation (right) of the NbE-pOR-Fab module complex, with the murine pOR, NbE, heavy chain (NabFab_H) and light chain (NabFab_L) of the Fab fragment and anti-Fab single domain antibody (FabNb) indicated, (b) The NbE-pOR complex as ribbon representation, with a focus on the NbE-pOR interface. NbE deeply inserts its CDR3 - hairpin loop into the orthosteric binding pocket. For reasons of clarity the NabFab module is omitted. CDR1 , 2, and 3 are indicated.

[0018] Figure 3: The NbE-pOR interaction interface, (a) Overview of the NbE-pOR complex. Lower dashed square indicates the close-up views shown in b, and upper dashed square indicates the close-up views shown in c and d. (b) Close-up of the orthosteric binding pocket, with interface residues shown as sticks. NbE residues F107 (left panel) and Y106 (right panel) are centered. Interface residues shown as sticks, (c) Top view of the pOR ligand binding pocket with CDR3NbEdeeply inserted. Interactions between ECL2 of the pOR and CDR3 of NbE are highlighted. Interface residues are shown as sticks and hydrogen bonds and salt bridges are indicated as black-and-grey dashed lines and black-and-white dashed lines, respectively, (d) Interaction interface between ECL3 of the pOR and CDR3 of NbE. Interface residues are shown as sticks and salt bridges are indicated as black-and- grey dashed lines, (e) The tip-forming residues Y106, F107, and Y108 (shown as sticks) of CDR3 are binding to distinct hydrophobic patches inside the orthosteric ligand binding pocket (shown as surface representation), (f) Binding mode superposition of NbE and the small molecule antagonists p-FNA (PDB: 4DKL) and alvimopan (PDB: 7LIL4). NbE’s Y108 stacks against pOR’s L219ECL2, which is a unique ligand-receptor interaction. Alvimopan inserts a phenyl group in a binding cleft not occupied by NbE or p-FNA. (g) Binding mode superposition of NbE and opioid peptide agonists p-endorphin (PDB: 8F7Q) or endomorphin (PDB: 8F7R). Y108NbEuniquely recognises L219ECL2, whereas a phenol group of endomorphin binds to a similar site also recognised by alvimopan (f) or fentanyl (h). (h) Binding mode superposition of NbE and the small molecule agonists fentanyl (PDB: 8EF5) or morphine (PDB: 8EF6).

[0019] Figure 4: ECL regions confer NbE binding selectivity, (a) Superposition of the inactive state of the NbE-bound pOR, inactive bOR (PDB: 4EJ4), and inactive KOR (PDB: 4DJH) with ECL2 and ECL3 indicated, (b) Binding interface of different ECL2 regions (murine pOR, murine bOR, and human KOR) with NbE. For comparison, the bOR and KOR have been superimposed onto the pOR. Q212ECL2is unique to the pOR. (c) Binding interface of TM7 and ECL3 regions from the pOR, bOR, and KOR with NbE. For comparison, the bOR and KOR have been superimposed onto the TM7 and ECL3 regions of the pOR. Most interface residues differ between the different receptor subtypes. In (b) and (c), for superimposed residues, top one corresponds to pOR, middle one corresponds to bOR and bottom one corresponds to KOR, hydrogen bonds and salt bridges are indicated as black-and-grey dashed lines and black-and-white dashed lines, respectively, (d) NbE-binding to the cells expressing wild-type (wt) pOR and ECL mutants (gated for similar receptor surface levels using anti-FLAG M1-AF647). NbE binding to wt pOR was normalized to 100%. pOR-KRQL- A mutant: K209, R211 , Q212 and L219 substituted by alanine, E310A mutant: E310 substituted by alanine. pOR-KRQL-A & E310A: both sets of mutations combined. N = 5, mean ± SEM. **P = 0.0012 (ECL2), 0.0047 (ECL3), 0.0010 (ECL2&3) by ordinary one-way ANOVA. (e) NbE binding to cells expressing bOR mutants (gated for similar receptor surface levels using anti-FLAG M1-AF647). NbE-binding to wt pOR was normalized to 100%. The three bOR mutants include bOR L300W735, a triple bORD193Q’M199T’L300Wmutant, and a bOR mutant with the entire ECL3 and distal parts of TM7 substituted by pOR residues (5OR287-300-M°R-306-318).5OR wt: N = 5, bOR mutants: N = 3, mean ± SEM. **P = 0.0011 , ****P = <0.0001 , n.s. = 0.8998 by ordinary one-way ANOVA. In all panels, N indicates the number of independent experiments.

[0020] Figure 5: Design and affinity measurements of linear peptides, (a) Ribbon representation of the CDR3 region (comprising SEQ ID NO: 2) of NbE when bound to pOR (left) from which linear peptides SBL-NbE-01 to -05 and SBL-NbE-12 (SEQ ID NOs: 4 to 9, respectively) were designed (right). SBL-NbE-01 (SEQ ID NO: 4) comprises the tip-forming residues SYFY, peptides SBL-NbE-02 to -05 and SBL-NbE-12 are designed by incremental addition of an N- and C-terminal residues each, (b) HTRF competition binding assay profiles of naloxone, NbE, and peptides SBL-NbE-05 (SEQ ID NO: 8) and SBL-NbE-12 (SEQ ID NO: 9). Peptide SBL-NbE-05 has an N-terminus acetylation and a C-terminus amidation. Binding to HEK293 cells stably expressing SNAP-pOR, labeled with SNAP-Lumi4-Tb and 3 nM red-labeled naltrexone derivative. All data normalized to cells without red-labeled naltrexone; N > 3, mean ± SEM.

[0021] Figure 6: Representative cyclic peptides of the invention, (a) Sequences of tested cyclic peptides SBL-NbE-21 (SEQ ID NO: 10), SBL-NbE-14 (SEQ ID NO: 11), SBL-NbE-22 (SEQ ID NO: 12), SBL-NbE-23 (SEQ ID NO: 13), SBL-NbE-26 (SEQ ID NO: 14) and SBL- NbE-27 (SEQ ID NO: 15). The “c” and square brackets indicate the cyclization through the terminal positions of the included residues, (b) Chemical structure of the cyclic peptides SBL-NbE-26 and -27 with anticipated hydrogen bonding network shown as dashed lines.

[0022] Figure 7: Functional characterization of cyclic peptides of the invention, (a)-(b) Affinity measurements of SBL-NbE-21 / -14 / -22 / -23 (in (a)) and SBL-NbE-26 and -27 (in (b)) using HTRF competition assays. Naloxone and NbE serve as references. N > 3, mean ± SEM. (c) IC50 values of the peptides measured in HTRF assays, and the inhibitory constant Ki, determined using the Cheng-Prusoff equation (Ki= IC50 / (1+ [L] / Kd)), with [L] (concentration of red labeled ligand) = 3 nM, and Kd of red labeled ligand = 1.3 nM (determined in saturation binding assay), (d) Quantification of the concentration dependent reversal of DAMGO-driven pOR signaling by the linear peptides SBL-NbE-05 and -12, and the cyclic peptides SBL-NbE-26 I -27. N > 3, mean ± SEM. (e) Reversal of DPDPE-driven inhibition of cAMP accumulation. DPDPE-driven 5OR signaling is not affected by the peptides SBL-NbE-05, -26, or -27. N = 3, mean ± SEM.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art. For the purpose of the present invention, all references cited herein are incorporated by reference in their entireties.

[0025] Antagonists of the p-opioid receptor (pOR) of the invention

[0026] In a first aspect, the invention relates to an antagonist of the p-opioid receptor (pOR). The antagonist of the pOR described herein is a peptide, in particular a mimetic peptide of the CDR3 loop of single domain antibody NbE, wherein NbE comprises sequence SEQ ID NO: 1 . Accordingly, the terms antagonist and mimetic peptide will be used interchangeably. More particularly, the antagonist of the invention is a mimetic peptide of the CDR3 loop of NbE, wherein said CDR3 loop of NbE comprises sequence SEQ ID NO: 2.

[0027] As used herein, the p-opioid receptor (pOR) refers to one of the four types of opioid receptors (OR) belonging to the superfamily of GPCRs. The other three opioid receptors are 5 (delta) OR, K (kappa) OR, and nociceptin-OR (NOPR). The amino acid sequences (and the nucleotide sequences of the cDNAs which encode them) of the opioid receptors are readily available, for example by reference to GenBank (http: / / www.ncbi.nlm.nih.gov / entrez). Preferably, the amino acid sequence of murine pOR, human pOR, human bOR, human KOR, human NOPR, murine bOR, murine KOR and murine NOPR comprises SEQ ID NOs: 18 to 25 of the present disclosure, respectively.

[0028] The term antagonist as used e.g. in “pOR antagonist peptide” refers to the fact that the antagonist binds the pOR without stimulating any activity. An “antagonist” is also known as a “blocker” because of its ability to prevent binding of other ligands and, therefore, block agonist-induced signaling activity. By contrast, an “agonist” refers to a ligand that, by binding a receptor protein, increases the receptor’s signaling activity.

[0029] According to a preferred embodiment, the antagonist of the invention is directed against and / or specifically binds to pOR. The nature of the pOR is not critical to the invention and can be from any organism including a fungus (including yeast), nematode, virus, insect, plant, bird (e.g. chicken, turkey), reptile or mammal (e.g., a mouse, rat, rabbit, hamster, gerbil, dog, cat, goat, pig, cow, horse, whale, monkey, camelid, or human). Preferably, the pOR is of mammalian origin, even more preferably of human origin.

[0030] In some embodiments, the antagonist of the invention is directed against and / or specifically binds to the mouse pOR comprising SEQ ID NO: 18. In preferred embodiments, the antagonist of the invention is directed against and / or specifically binds to the human pOR comprising SEQ ID NO: 19.

[0031] A prerequisite of the antagonist of the invention is its capability to specifically bind to the pOR. In some embodiments, the antagonist of the invention is directed against and / or specifically binds to the orthosteric pocket of the pOR. Amino acids that forms the tip of the CDR3 region (102YSGSYFYKSE111) of NbE are mainly responsible for mediating the selective binding to the pOR. In a more specific embodiment, the binding agent directed against and / or specifically binding to the pOR is capable of displacing an orthosteric ligand bound to the orthosteric binding pocket of pOR or prevents binding of an orthosteric ligand to the pOR. Orthosteric ligands of the pOR include for example naloxone and the endogenous peptides p-endorphin and endomorphin.

[0032] In some embodiments, the antagonist of the invention is directed against and / or specifically binds the pOR by interacting with residues comprised within pOR’s transmembrane (TM) helices 3, 5, 6, and / or 7. Preferably, the secondary structure elements of pOR are defined as in Table 1 :

[0033] Table 1

[0034] *ICL = intracellular loop; ECL = extracellular loop; TM = transmembrane helix

[0035] In some embodiments, the antagonist of the invention is directed against and / or specifically binds the pOR by interacting with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 residues selected from the group consisting of: D147, Y148, M151 , K209, R211 , Q212, L219, F221 , E229, L232, K233, V236, W293, I296, H297, V300, K303, E310, W318, I322 and Y226, with reference to SEQ ID NO: 18. In preferred embodiments, the antagonist of the invention is directed against and / or specifically binds the pOR by interacting with 1 , 2, 3, 4, 5 or 6 residues selected from the group consisting of: D147, K209, R211 , Q212, L219 and W318, with reference to SEQ ID NO:18. In further preferred embodiments, the antagonist of the invention is directed against and / or specifically binds the pOR by interacting with 1 , 2 or 3 residues selected from the group consisting of: D147, L219 and W318, with reference to SEQ ID NO: 18.

[0036] In some embodiments, the antagonist of the invention is directed against and / or specifically binds the pOR by interacting with 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 residues selected from the group consisting of: D149, Y150, M153, K211 , R213, Q214, L221 , F223, E231 , L234, K235, V238, W295, I298, H299, V302, K305, E312, W320, I324 and Y228, with reference to SEQ ID NO: 19. In preferred embodiments, the antagonist of the invention is directed against and / or specifically binds the pOR by interacting with 1 , 2, 3, 4, 5 or 6 residues selected from the group consisting of: D149, K211 , R213, Q214, L221 and W320, with reference to SEQ ID NO:19. In further preferred embodiments, the antagonist of the invention is directed against and / or specifically binds the pOR by interacting with 1 , 2 or 3 residues selected from the group consisting of: D149, L221 and W320, with reference to SEQ ID NO: 19.

[0037] In some embodiments, the antagonist of the invention specifically binds the pOR with an inhibitory constant (Ki) of about 10'5M or less, about 10'6M or less, about 10'7M or less, about 10'8M or less, or about 10'9M or less. In some embodiments, the antagonist of the invention does not substantially bind to any one of or any combination of the b-OR, the K- OR, and the NOPR-OR. Thus in some embodiments, the antagonist of the invention binds to any one of or any combination of the b-OR, the K-OR, and the NOPR-OR with a respective dissociation constant (KD) of about 10'4M or more, or about 10'3M or more, each selected independently.

[0038] Linear peptide antagonists of the invention

[0039] In some embodiments, the antagonist of the invention is a mimetic peptide of the CDR3 loop of NbE, wherein said CDR3 loop of NbE comprises sequence SEQ ID NO: 2.

[0040] In some embodiments, the antagonist of the invention comprises a sequence consisting of (1) a fragment of SEQ ID NO: 2 comprising SEQ ID NO: 3, or (2) a sequence derived from the sequence defined in (1) by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids. In more particular embodiments, said fragment of SEQ ID NO: 2 comprising SEQ ID NO: 3 comprises 11 to 19, 11 to 18, 11 to 17, 11 to 16, 11 to 15, or 11 to 14 amino acids.

[0041] In some embodiments, the antagonist of the invention comprises a sequence selected from (1) SEQ ID NOs: 4 to 9 or (2) a sequence derived from any one of SEQ ID NOs: 4 to 9 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids.

[0042] In some embodiments, the antagonist of the invention comprises a sequence selected from (1) SEQ ID NOs: 8 to 9 or (2) a sequence derived from any one of SEQ ID NOs: 8 to 9 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids. In some embodiments, the antagonist of the invention comprises or consists of SEQ ID NO: 8, wherein the sequence is acetylated in N-terminus and amidated in C-terminus. In some embodiments, the antagonist of the invention comprises or consists of SEQ ID NO: 9.

[0043] Cyclic peptide antagonists of the invention

[0044] In some embodiments, the antagonist of the invention is a cyclic peptide derived by cyclization of a linear mimetic peptide described herein.

[0045] The invention encompasses such cyclic peptides, which are cyclized via side chain- to-side chain cyclization, head-to-side chain cyclization, head-to-tail cyclization, and / or side chain-to-tail cyclization, which can be accomplished through amide formation between the backbone amine and carboxylic acid of two terminal amino acids, amide formation between the side chains and / or backbone termini of two amino acids (lactam), and other functionalities such as the formation of thioether, disulfide, alkene and 1 ,4- or 1 ,5- substituted 1 ,2,3-triazoles linkages. Such cyclization methods are known by the skilled person in the art. Preferably, the antagonistic cyclic peptides of the invention are obtained following the procedure described in Example 1.

[0046] Cyclization tethers can be formed by: (i) Lactam bond formation between a free amine and carboxylic acid (obtained through deprotection of orthogonal protecting groups) either on the side chains and / or backbone termini of amino acids, (ii) Cu(l)-catalyzed or Ru(ll)- catalyzed azide-alkyne cycloaddition (i.e. CuAAC or RuAAC) of introduced alkyne- containing amino acids (examples are, but not limited to, D- or L-propargylglycine and homopropargylglycine) and azide-containing amino acids (examples are, but not limited to, D- or L-azidolysine, azidoornithine, azidohomoalanine and azidoalanine) to give 1 ,4- or 1 ,5- substituted 1 ,2,3-triazoles linkages; (iii) Disulfide bond formation between introduced thiol- containing amino acids (examples are, but not limited to, D- or L-cysteine and homocysteine), (iv) Thioether bond formation between introduced thiol-containing amino acids through use of a bivalent linker by a substitution reaction, (v) Formation of an olefin functionality through ring-closing metathesis (RCM) through use of terminal alkene-bearing amino acid derivatives. Additionally, non-covalent cyclization can be envisioned through addition of terminal interacting residues, such as two Trp residues (Trp zipper) or Trp and alkylated lysine residues (including, but not limited to, shorter analogues of ornithine and aminohomoalanine). In some embodiments, the antagonist of the invention is a cyclic peptide derived by side chain-to-side chain cyclization of a linear mimetic peptide described herein. In more particular embodiments, the antagonist of the invention is a cyclic peptide comprising a sequence selected from (1) SEQ ID NOs: 10 to 13, or (2) a sequence derived from any one of SEQ ID NOs: 10 to 13 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids. In specific embodiments, the antagonist of the invention comprises or consists of a sequence selected from:

[0047] (SBL-NbE-23, SEQ ID NO: 13) wherein Azk, Pra and Aha correspond to L-azidolysine, L-propargylglycine and L- azidohomoalanine respectively.

[0048] In some embodiments, the antagonist of the invention is a cyclic peptide derived by head-to-tail cyclization of a linear mimetic peptide described herein.

[0049] In some embodiments, the antagonist of the invention is a cyclic peptide comprising a p-turn inducing scaffold. The nature and position of the p-turn-inducing scaffold is preferably chosen to create a conformational bias towards a p-hairpin structure, such as shown by the CDR3 conformation of NbE when bound to pOR in Fig. 5a. In preferred embodiments, the p-turn inducing scaffold is a D-Pro-L-Pro dipeptide or an analogue thereof, including D-Pro-TfmOxa (2-trifluoromethyloxazolidine-2-carboxylic acid), D-Pro- Gly, Aib-Gly and Asn-Gly. In some embodiments, the antagonist of the invention is a cyclic peptide according to formula I, or a sequence derived from formula I by substitution or chemical modification of one or more amino acids: - - - - - -1- 2-3-4-5- - -6-7-8-9-10- - - - - -

[0050] (formula I), wherein

[0051] Xi is D or is absent

[0052] X2 is Y or is absent,

[0053] X3 is W or is absent,

[0054] X4 is G, or is absent,

[0055] X5 is K or is absent, dP corresponds to a D-Pro,

[0056] Xe is Y or is absent,

[0057] X7 is C or is absent,

[0058] Xs is N or is absent,

[0059] X9 is F or is absent, and

[0060] X10 is K or is absent; wherein either X5 and Xe, only, are absent; or X4, X5, Xe, and X7, only, are absent; or X3, X4, X5, Xe, X7, and Xs, only, are absent; or X2, X3, X4, X5, Xe, X7, Xs and X9, only, are absent; or all of Xi to X10 are absent.

[0061] In some embodiments, X5 and Xe, only, are absent. In some embodiments X4, X5, Xe, and X7, only, are absent. In some embodiments X3, X4, X5, Xe, X7, and Xs, only, are absent. In some embodiments X2, X3, X4, X5, Xe, X7, Xs and X9, only, are absent. In some embodiments, all of Xi to X10 are absent. In some embodiments, the antagonist of the invention derives from formula I by substitution or chemical modification of 1 , 2, 3, 4, or 5 amino acids , In some embodiments, the antagonist of the invention derives from formula I by substitution or chemical modification of the aromatic amino acids F and / or Y. Chemical modification of F or Y encompasses (i) modifications which conformationally rigidity the amino acid side chain angles through use of covalent or non-covalent steric constraints, or (ii) substitution of their aromatic groups through introduction of halogen (F, Cl, Br, I, CF3), alkyl or aryl groups at the ortho, meta or / and para positions in case of F, at the ortho or / and meta positions in case of Y, or / and by substitution of the phenolic OH of Y by alkyl or aryl groups. In some embodiments, the antagonist of the invention is a cyclic peptide according to formula II, or a sequence derived from formula II by substitution or chemical modification of one or more amino acids: - - - - - - -1-2- - -3-4- - - - - - -1(formula ||) wherein

[0062] Xi is D or is absent,

[0063] X2 is Y or is absent, dP corresponds to a D-Pro,

[0064] X3 is F or is absent, and

[0065] X4 is K or is absent, wherein either X2 and X3, only, are absent, or all of Xi to X4 are absent.

[0066] In some embodiments, X2 and X3, only, are absent. In some embodiments, all of Xi to X4 are absent. In some embodiments, the antagonist of the invention derives from formula II by substitution or chemical modification of 1 , 2, 3, 4, or 5 amino acids. In some embodiments, the antagonist of the invention derives from formula II by substitution or chemical modification of the aromatic amino acids F and / or Y. Chemical modification of F or Y encompasses (i) modifications which conformationally rigidity the amino acid side chain angles through use of covalent or non-covalent steric constraints, or (ii) substitution of their aromatic groups through introduction of halogen (F, Cl, Br, I, CF3), alkyl or aryl groups at the ortho, meta or / and para positions in case of F, at the ortho or / and meta positions in case of Y, or / and by substitution of the phenolic OH of Y by alkyl or aryl groups.

[0067] Formula I and formula II correspond to SEQ ID NOs: 16 and 17 respectively.

[0068] In more particular embodiments, the antagonist of the invention is a cyclic peptide comprising a sequence selected from (1) SEQ ID NOs: 14 and 15 or (2) a sequence derived from any one of SEQ ID NOs: 14 and 15 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids.

[0069] In some embodiments, the antagonist of the invention is a cyclic peptide comprising a sequence selected from (1) SEQ ID NOs: 14, 15 and 27 or (2) a sequence derived from any one of SEQ ID NOs: 14, 15 and 27 by substitution or chemical modification of one or more amino acids, preferably of 1 , 2, 3, 4 or 5 amino acids. In some embodiments, the antagonist of the invention is a cyclic peptide comprising a sequence selected from (1) SEQ ID NOs: 14, 15, and 26-33 or (2) a sequence derived from any one of SEQ ID NOs: 14, 15, and 26-33 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids.

[0070] In some embodiments, the antagonist of the invention is a cyclic peptide comprising a sequence selected from (1) SEQ ID NOs: 14, 15, and 27-33 or (2) a sequence derived from any one of SEQ ID NOs: 14, 15, and 26-33 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids.

[0071] In some embodiments, the antagonist of the invention is a cyclic peptide comprising a sequence selected from (1) SEQ ID NOs: 26-33 or (2) a sequence derived from any one of SEQ ID NOs: 26-33 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids.

[0072] In some embodiments, the antagonist of the invention is a cyclic peptide comprising a sequence selected from (1) SEQ ID NOs: 27-33 or (2) a sequence derived from any one of SEQ ID NOs: 27-33 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids.

[0073] In some embodiments, the antagonist of the invention is a cyclic peptide comprising a sequence selected from (1) SEQ ID NOs: 15 and 27 or (2) a sequence derived from any one of SEQ ID NOs: 15 and 27 by substitution or chemical modification of 1 , 2, 3, 4 or 5 amino acids.

[0074] In specific embodiments, the antagonist of the invention comprises or consists of a sequence selected from: and

[0075] (SBL-NbE-26, SEQ ID NO: 14)

[0076] (SBL-NbE-27, SEQ ID NO: 15) wherein dP is D-Pro. In specific embodiments, the antagonist of the invention comprises or consists of a sequence selected from: and

[0077] (SBL-NbE-27, SEQ ID NO: 15) Atc-Y-K-S-E-Y-D-Y-dP-P-F-K-Y-Y-S-G-S-Y

[0078] (SBL-NbE-29, SEQ ID NO: 27) wherein dP is D-Pro and Ate is:

[0079] In specific embodiments, the antagonist of the invention comprises or consists of:

[0080] (SBL-NbE-29, SEQ ID NO: 27) wherein dP is D-Pro and Ate is:

[0081] A "substitution", as used herein, results from the replacement of one amino acid by a different amino acid as compared to an amino acid sequence of a parental protein or peptide. It is understood that a protein or peptide may have conservative amino acid substitutions which have substantially no effect on the activity of said protein or peptide. By conservative substitutions is intended combinations such as Gly, Ala; Vai, lie, Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; Cys, Met; and Phe, Tyr, Trp. A substitution as used herein is preferably a conservative substitution. A substitution as used herein also encompasses substitution of an L-isomer to a D-isomer (or vice-versa).

[0082] Chemical modifications as used in “chemical modification of an amino acid” is not particularly limited and includes any chemical modification of the amino acid, so long as the modified amino acid and / or resulting peptide comprising the modified amino acid is an equivalent in terms of effect and exhibits characteristics similar to those of the parental amino acid and / or peptide comprising the modified amino acid. Chemical modifications encompass incorporation of chemical groups that induce steric and / or conformational constraints via non-covalent interactions or covalent bonds. Such chemical groups can have structure-inducing or conformation-stabilizing roles. Additionally, chemical modifications involve substitution of the amino acid side chain, including the introduction of halogen (F, Cl, Br, I, CF3), alkyl or aryl groups at the ortho, meta or / and para aromatic positions in case of F, at the ortho or / and meta aromatic positions in case of Y, or / and by substitution of the phenolic OH of Y by alkyl or aryl groups.

[0083] Compositions

[0084] In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of an antagonist of the invention and at least one of a pharmaceutically acceptable carrier, adjuvant or diluent. The invention also relates to uses and methods of treatment using the pharmaceutical compositions disclosed herein. The methods and uses encompassed by the present invention are described in more detail below.

[0085] The pharmaceutical compositions described herein may be prepared using methods known in the art. Standard pharmaceutical carriers include a phosphate buffered saline solution, water, emulsions such as an oil / water or water / oil emulsion, and various types of wetting agents.

[0086] The pharmaceutical compositions may comprise any other pharmaceutically acceptable ingredients, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, colouring agents, desiccants, detergents, diluents, disinfectants, dis integrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavour enhancers, flavouring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity-increasing agents, water-absorbing agents, water-miscible cosolvents, water softeners, or wetting agents. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0087] In general, a diluent, adjuvant or carrier can be selected based upon the mode and route of administration, and standard pharmaceutical practice.

[0088] Therapeutic uses and methods

[0089] As outlined above, the present invention further relates to the first medical use of the antagonists of the invention.

[0090] Accordingly, in another aspect, the invention relates to the use of the antagonist or the pharmaceutical composition of the invention for the treatment of a medical condition. For that purpose, the antagonist or the pharmaceutical composition of the invention is administered to a subject in need thereof, in a therapeutically effective amount.

[0091] In one embodiment, the invention relates to the antagonist or the pharmaceutical composition of the invention for use in the treatment of a medical condition.

[0092] In one embodiment, the invention relates to a method of treating a medical condition, the method comprising the step of administering to a subject in need thereof a therapeutically effective amount of the antagonist or the pharmaceutical composition of the invention.

[0093] In one embodiment, the invention relates to the antagonist or the pharmaceutical composition of the invention for use in a method of reversing opioid-induced side effects during pain treatment and / or after opioid drug abuse or overdose. In particular embodiments, the invention relates to the antagonist or the pharmaceutical composition of the invention for use in a method of reversing respiratory depression and / or opioid-induced constipation.

[0094] In the context of the invention, the terms “medical condition”, “disease” and “disorder” are used interchangeably and include but are not limited to an opioid receptor-related medical condition, in particular a p-opioid receptor (pOR)-related medical condition. In preferred embodiments, said medical condition is selected among respiratory depression, constipation sedation, dizziness, nausea, vomiting, physical dependence to opioid, and tolerance to opioid.

[0095] The administration of the antagonists or pharmaceutical compositions of the invention may include topical administration, oral administration, and parenteral administration. In some embodiments, said subject or patient is a mammal. In preferred embodiments, the subject or patient is a human.

[0096] In one embodiment, the invention relates to the use of the antagonist or pharmaceutical composition of the invention, for manufacturing of a medicament.

[0097] In another aspect, the invention relates to the use of the antagonist or the pharmaceutical composition of the invention to antagonize opioid receptor signaling activity, in particular p-opioid receptor (pOR) signaling activity.

[0098] As used herein, "treatment" or "treating" is an approach for obtaining beneficial or desired results including clinical results. For purposes of this invention, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, diminishing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the worsening of the disease), preventing or delaying the spread of the disease, preventing or delaying the recurrence of the disease, delaying or slowing the progression of the disease, ameliorating the disease state, providing a remission (partial or total) of the disease, decreasing the dose of one or more other medications required to treat the disease, and / or prolonging survival. The use of the present invention contemplates any one or more of these aspects of treatment. Definitions

[0099] Unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry described herein are those well-known and commonly used in the art.

[0100] The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms unless otherwise noted. If aspects of the invention are described as "comprising" a feature, embodiments also are contemplated "consisting of or "consisting essentially of the feature. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illustrate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise stated. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0101] The term “about” as used herein is equivalent to ± 10% of a given numerical value, unless otherwise stated.

[0102] As used herein, the term “peptide” refers to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. Accordingly, the term “peptide” used in the present disclosure encompasses “peptidomimetics” defined as small protein-like chains designed to mimic a peptide. Typically, the term “amino acid” will refer to “proteinogenic amino acid”, i.e. those amino acids that are naturally present in proteins. In general, the amino acids are in the L isomeric form, but D amino acids can also be comprised in the peptides of the invention (in which case the D isoform is specified), as well as any non-natural amino acid.

[0103] The term "affinity", as used herein, refers to the degree to which a ligand binds to an antigen on a target receptor so as to shift the equilibrium of receptor and ligand toward the presence of a complex formed by the binding of the ligand to the receptor. The dissociation constant (Kd) is commonly used to describe the affinity between the ligand (e.g. the peptide described herein) and the target (e.g. the pOR). Other ways of describing the affinity between a ligand and its target protein are the association constant (Ka), the inhibitory constant (Ki), or indirectly by evaluating the potency of ligands by measuring the half maximal inhibitory concentration (IC50) or half maximal effective concentration (EC50).

[0104] As used herein, the terms "complementarity determining region" or "CDR" within the context of immunoglobulin single variable domains, in particular single domain antibodies, refers to variable regions containing the amino acid sequences capable of specifically binding to antigenic targets. These CDR regions account for the basic specificity of the single domain antibody for a particular antigenic determinant structure. Such regions are also referred to as "hypervariable regions". Single domain antibodies generally comprise a single amino acid chain that can be considered to comprise 4 “framework sequences or regions” or FRs and 3 “complementary determining regions” or CDRs, each non-contiguous with the others (termed CDR1 , CDR2, CDR3).

[0105] The invention will be further illustrated in view of the following examples.

[0106] EXAMPLE 1

[0107] The disclosure of Yu, Kumar, et al. "Structural basis of p-opioid receptor-targeting by a nanobody antagonist." bioRxiv (2023) is incorporated herein by reference in its entirety (67).

[0108] 1.1 NbE binds the extracellular side of uOR and is an antagonist

[0109] We first tested whether the single domain antibody NbE, which was part of a single domain antibody library previously generated against the pOR (9), binds to the extracellular side of pOR in the surface of living cells. We covalently conjugated purified NbE (SEQ ID NO: 1) with Alexa Fluor 488 and incubated OR-expressing HEK293 cells with the fluorescently labeled NbE. Confocal microscopy analyses showed a pronounced NbE signal at the plasma membrane of cells expressing mouse pOR (comprising SEQ ID NO: 18) or human pOR (comprising SEQ ID NO: 19), while cells expressing the closely related mouse bOR (comprising SEQ ID NO: 23) or human bOR (comprising SEQ ID NO: 20), mouse KOR (comprising SEQ ID NO: 24), or mouse NOPR (comprising SEQ ID NO: 25) were not labeled by NbE (Fig. 1a). Selective binding of NbE to pOR-expressing cells was also detected by flow cytometry analyses after adding NbE at increasing concentrations (Fig. 1 b). bOR-, KOR-, or NOPR-expressing cells gated for similar receptor surface levels did not exhibit NbE binding above control cells (Fig. 1 b). Since the NbE staining intensity of pOR expressing cells did not plateau at the highest NbE concentration tested (10 pM), we turned to grating-coupled interferometry (GCI) to determine the NbE binding strength. We immobilized biotinylated AVI-tagged NbE on a streptavidin coated biosensor surface and perfused it with buffer containing purified murine pOR at different concentrations. From the binding curves we extracted the affinity of the NbE-pOR interaction (KD=56 nM) and the kinetic parameters (ka=2.8*103M'1s'1and kd=1.6*10'4s-1), which revealed a slow NbE off- rate.

[0110] Next, we investigated if NbE binds competitively with orthosteric pOR ligands. We incubated pOR-expressing HEK293 cells with increasing concentrations of naloxone, subsequently added fluorescent NbE, and quantified the NbE signal by flow cytometry. NbE binding decreased in a naloxone concentration-dependent manner and was entirely abolished at high naloxone concentrations (Fig. 1c). The data suggested that the NbE binding site on the pOR potentially overlaps with the orthosteric ligand binding pocket, and thus we tested if NbE modulates pOR activity. We first assessed whether NbE behaves as an agonist by measuring pOR-mediated inhibition of cyclic AMP (cAMP) accumulation in living cells, a readout of Gi-driven OR signaling. Application of NbE did not activate pOR even at high concentrations, in contrast to the peptide agonist DAMGO (Fig. 1d). We then tested whether NbE acts as a pOR antagonist and reduces the signaling effects of opioid peptides and small molecule opioid drugs. Indeed, pre-incubation of pOR-expressing cells with NbE caused a concentration-dependent decrease in DAMGO- and morphine-induced Gi signaling (Fig. 1e). At high concentrations, NbE fully blocked DAMGO and morphine- driven pOR inhibition of cAMP production.

[0111] Taken together, NbE specifically binds to the extracellular side of the pOR and competes with orthosteric opioid ligands. Furthermore, NbE acts as an antagonist when added to cells as an extracellular ligand.

[0112] 1.2 Cryo-EM structure determination of the NbE-pOR complex

[0113] Advancement of structural biology techniques have led to the determination of several pOR structures in complex with either antagonists (77, 27), partial agonists (25, 28), or full agonists, including the endogenous peptides p-endorphin and endomorphin (9, 28-30). All ligands bind to the orthosteric ligand binding pocket that is on the extracellular side and largely solvent exposed. To identify the precise molecular binding mode of NbE, we determined the structure of the NbE-pOR complex using cryo-EM. Initial structure determination was precluded due to the absence of large extracellular features of the complex. The surrounding detergent micelle additionally impaired accurate particle projection alignments, resulting in unsuccessful 3D reconstruction. To provide extra features for accurate particle alignment, we incubated the purified NbE-pOR complex with a Fab module consisting of a single domain antibody-binding Fab fragment (NabFab) and an anti-Fab single domain antibody, recently developed as a fiducial marker (31). The resulting stable and homogenous NbE-pOR-Fab module complex was purified by size exclusion chromatography. Cryo-EM analyses enabled us to determine the structure of the NbE-pOR-Fab module complex at a global resolution of 3.1 A.

[0114] The overall structure of the NbE-pOR complex bound to the Fab module is shown in Fig. 2a. The NabFab attaches to the NbE scaffold at a site distal to the CDR loops. NabFab binding to NbE is ensured by introducing three point mutations in NbE’s scaffold, thereby assimilating NbE to the scaffold of Tc-Nb4, the original target antigen of NabFab (31). Introducing the scaffold mutations did not significantly alter NbE binding (Ki) to pOR in cell membranes, as measured in homogeneous time-resolved fluorescence (HTRF) Tag-lite ligand competition binding assays (not shown). The pre-assembled NbE-NabFab complex showed a modest reduction in the Ki (not shown), possibly due to the larger ligand size and slight steric clashes between the membrane surrounding pOR and the NbE-NabFab module, as suggested by the EM density map (Fig. 2a). The Fab fragment binds in a single and rigid conformation to NbE, enabling high-resolution structure determination of the NbE- pOR complex. For reasons of clarity, the Fab module is omitted in all subsequent figures (Fig. 2b).

[0115] When bound to NbE, the pOR adopts an inactive conformation, which closely resembles the structures of the pOR bound to the morphinan antagonist p-funaltrexamine (P-FNA) or alvimopan (11, 27). The average root mean square deviation (RMSD) for all Caatoms of pOR between the NbE-pOR complex and the pOR complexes bound to antagonists is 0.78 A, whereas the RMSD between NbE-pOR and agonist-bound structures is on average 1.72 A. In particular, transmembrane helix 6 (TM6), which is displaced by roughly 10 A in the activated state (9, 29), superimposes well between the NbE-pOR and the p-FNA- or alvimopan-bound inactive pOR structures. Moreover, a conserved core triad consisting of the amino acids I155340, P244550, and F289644(Ballesteros- Weinstein numbering scheme (32)), which lies below the ligand binding pocket and propagates structural rearrangements involved in receptor activation (9), superimposes well with the pOR in the inactive form (not shown). Structural differences in the ligand binding pocket between agonists, partial agonists, and antagonists are relatively subtle (33, 34), however, binding of NbE to the pOR induces several unique conformational changes in the orthosteric binding pocket and the extracellular loops. 1.3 Unique interaction profile of NbE with the inactive uOR

[0116] Binding of NbE to the pOR is mediated through its complementarity-determining regions (CDRs) CDR1 and CDR3, with the main interaction interface being formed between the p-hairpin loop of CDR3 and pOR’s TM helices 3, 5, 6, and 7 (Fig. 3a-d). CDR3 deeply inserts into the orthosteric ligand binding pocket with the three aromatic residues Y106NbE, F107NbEand Y108NbEforming the tip of the loop (Fig. 3b and 3e).

[0117] Similar to previously determined pOR structures bound to the ligands DAMGO (29), BU72 (9), p-FNA (27), alvimopan (11), PZM21 , and FH210 (25), as well as p-endorphin and endomorphin (30), H297652is positioned closely to a phenol hydroxyl group of the NbE ligand (Fig. 3b). While binding of BU72 and p-FNA is mediated through a hydrogen bond network that involves two water molecules (9, 27), in the NbE-pOR complex, H297652and Y106NbEare directly forming a hydrogen bond (Fig. 3b). In addition, Y106NbEis surrounded by mainly hydrophobic residues, including Y148333, M151336, V236542, F237543, I296651, and V300655(Fig. 3b). F107NbE, the second tip-forming aromatic residue, inserts itself into a neighboring hydrophobic cavity (Fig. 3b and 3e) and interacts with the key residues I143330, Y148333, M151336, W293648, I322739and Y326743of pOR (Fig. 3b). Interestingly, the aspartate D147332, present in many family A GPCRs, and crucial for the recognition of DAMGO, p-FNA, BU72, PZM21 , and endogenous peptides by forming a salt bridge with an amine group of the ligand, is rotated compared to all other pOR structures (not shown). Instead of forming a salt bridge with the ligand, D147332stacks in this conformation onto F107NbE, representing a novel ligand-receptor interaction mode (Fig. 3b). The third aromatic residue Y108NbEis situated at the periphery of the orthosteric binding pocket (Fig. 3c). The aromatic ring of the phenol group stacks against L219ECL2, part of ECL2, whereas the hydroxyl group forms a hydrogen bond with K233539(Fig. 3b and 3c). K233539had previously been identified as the side chain to which the morphinan ligand p-FNA is covalently attached (27, 35). Another notable difference in the ligand binding pocket involves W318735that is uniquely positioned, likely due to steric constraints when inserting the large NbE ligand into the narrow ligand binding pocket. This specific rotamer conformation has only been observed when the pentameric peptide ligand DAMGO is bound to the pOR, however, in the NbE-pOR structure W318735is shifted by several A. Because of the unusual positioning of W318735and its rotamer conformation, K303658that often stacks onto W318735is displaced by several A, hydrogen bonding with S103NbEof NbE instead (Fig. 3d). The three tip-forming aromatic residues of NbE are recognized by neighboring but distinct binding cavities in the orthosteric binding pocket (Fig. 3e). Here, Y106NbEand F107NbEare binding to sites usually occupied by small molecule ligands (Fig. 3f-h), whereas Y108NbEstacks onto L219 of ECL2, thereby stabilizing the CDR3 p-hairpin loop in its conformation. Binding of ligands to this peripheral binding site has not yet been observed in other pOR structures. Conversely, the ligands alvimopan (antagonist), endomorphin, and fentanyl (agonists) are inserting aromatic functional groups into another binding cavity, which is not occupied by NbE (Fig. 3f-h).

[0118] To probe if the single domain antibody ligand undergoes conformational rearrangements upon pOR binding, we crystallized NbE in its unbound form. The crystal contained three NbE molecules per asymmetric unit with all molecules virtually identical to each other, including the CDRs. A superposition between the unbound and bound NbE structures revealed that the CDR1 region and the CDR3 p-hairpin loop undergoes a conformational shift when comparing the free and the pOR-bound state, while the single domain antibody scaffold is nearly identical (not shown). The conformational differences suggest that CDR1 and CDR3 exhibit intrinsic flexibility, which could potentially be important for conformational selection of the inactive pOR and high-affinity binding of NbE.

[0119] In summary, NbE shows a unique interaction profile with the pOR. In particular, D147332, L219ECL2, and W318735of the pOR engage with NbE in a so far undetected ligand binding mode that can offer new possibilities for structure-guided drug design.

[0120] 1.4 Extracellular loops confer NbE binding selectivity

[0121] To provide a rationale for NbE’s selectivity for pOR over other OR family members (Fig. 1a and 1 b), we focused our structural analyses on the extracellular loops, which represent the most variable regions in the OR family. In particular, ECL2 shows variations in sequence and length between the receptors (Fig. 4a), providing a possible explanation for the observed pOR selectivity given the specific interactions of NbE with ECL2 and ECL3 (Fig. 3c and 3d). K209ECL2, R211ECL2and Q212ECL2interact with E111NbE, Y102NbE, and T35NbErespectively, thereby contributing to high-affinity binding (Fig. 4b). Importantly, Q212ECL2is unique to the pOR. The centered L219ECL2of ECL2 stacks onto Y108NbEof CDR3 (Fig. 3b and 3c). K303658, also unique to pOR, forms a specific hydrogen bond with S103NbEthat likely stabilizes ECL3 in a conformation that allows several nonspecific side chain-main chain interactions between pOR and NbE, with E310ECL3being at ECL3’s center (Fig. 4c). Binding of NbE to pOR induces clear shifts for ECL2, parts of TM6, ECL3 and TM7 compared to the other two antagonist-bound pOR structures (not shown).

[0122] To probe the relevance of pOR’s ECLs in NbE binding, we first substituted ECL2 residues K209ECL2, R211ECL2, Q212ECL2and L219ECL2(‘KRQL’ motif) by alanine and tested NbE binding to the pOR mutant by flow cytometry, gating for cells with similar surface expression. Compared to wild-type pOR, the KRQL-A mutant showed 50% reduced NbE binding, indicating a lower affinity for the ECL2 mutant (Fig. 4d). Exchanging E310ECL3in ECL3 with alanine also significantly reduced NbE binding (Fig. 4d). Combining both the ECL2 and ECL3 mutations did not lead to further reduction in NbE binding, highlighting the central role of the orthosteric pocket interactions (Fig. 4d). All mutants retained comparable responses to DAMGO (not shown).

[0123] Next, we reasoned that assimilating the bOR to the pOR, based on the uncovered interaction interfaces, might transform the bOR from a non-binder to a NbE-binder. Given the established role of the amino acid position 7.35 in opioid receptor subtype selectivity (36, 37), we first mutated bOR’s L300735in the orthosteric pocket to the corresponding pOR residue W318735(bORL300Wmutant). We then also converted the bOR residues D193ECL2(corresponds to pOR Q212ECL2) and M199ECL2(corresponds to pOR T218ECL2) into glutamine and threonine residues respectively (Fig. 4b, c). T218ECL2of pOR forms an intramolecular hydrogen bond with the guanidinium group of R211ECL2. As a consequence of this loop stabilization, K209ECL2and R211ECL2of the pOR are ideally positioned to create the aforementioned hydrogen bond and salt bridge network with Y102NbEand E111NbEof NbE (Fig. 4b). Because T218ECL2is unique to the pOR, we speculated that mutating bOR M199ECL2into threonine might stabilize the ECL2 of the bOR in a conformation that favors NbE binding. We quantified fluorescent NbE binding to cells expressing pOR (control), wildtype bOR, or bOR mutants by flow cytometry in cells gated for similar receptor levels. As expected, only non-specific background NbE binding was detected for cells expressing wildtype bOR (Fig. 4e). The NbE signal was not increased for cells expressing bORL300W. However, strong NbE binding was observed when cells expressed the triple mutant bORD193Q’M199T’L300W(Fig. 4e). The results show that mutating two residues in the ECL25OR, combined with the orthosteric L300W mutation, can convert the non-binding bOR into a strong NbE binder. To test the role of ECL3, we next substituted the entire ECL3 as well as residues of the connecting a-helix 7 of the bOR (residues 287-300, with respect to SEQ ID NO: 23) with residues of the pOR (residues 306-318, with respect to SEQ ID NO: 18). The substitution led to a moderate but significant increase in NbE binding (Fig. 4e), indicating that pOR’s ECL3 region partially contributes to NbE binding.

[0124] In sum, the mutational and gain-of-function studies identify ECL2 and ECL3 as important contributors to NbE binding and receptor subtype selectivity.

[0125] 1.5 Constrained peptide mimetics of NbE’s CDR3 efficiently antagonize uOR

[0126] The centering of key contacts on a single CDR makes NbE’s CDR3 a promising starting point for the design of ligands that downsize the single domain antibody towards smaller peptides based on the antigen-binding paratope (38). First, we designed and synthesized a library of linear peptides of increasing length, with the shortest peptide based on the four residues105SYFY108that compose the p-turn segment at the tip of NbE’s CDR3 (Fig. 5a). We systematically extended each peptide by one N- and one C-terminal residue of the CDR3NbE(SEQ ID NO: 2) with the longest linear peptide spanning 14 residues (Fig. 5a), as detailed below:

[0127] SBL-NbE-01 (SEQ ID NO: 4),

[0128] SBL-NbE-02 (SEQ ID NO: 5),

[0129] SBL-NbE-03 (SEQ ID NO: 6),

[0130] SBL-NbE-04 (SEQ ID NO: 7),

[0131] SBL-NbE-05 (SEQ ID NO: 8), and

[0132] SBL-NbE-12 (SEQ ID NO: 9).

[0133] Peptides SBL-NbE-01 I -02 I -03 I -04 I -05 are further acetylated in N-terminal and amidated in C-terminal. Peptide SBL-NbE-12 is an unacetylated C-terminal carboxylic acid for improved solubility.

[0134] Peptide binding to pOR was assessed using the HTRF Tag-lite binding assay, which relies on fluorescence resonance energy transfer (FRET) between SNAP-tagged pOR labeled with terbium cryptate as FRET donor, and the red fluorescent opioid ligand naltrexone, serving as acceptor, with a decrease in FRET indicating competitive binding of a test compound. Binding of the control ligand naloxone and of NbE was readily detected with Ki values of 2.4 nM and 20 nM respectively (Fig. 5a and Fig. 7c). In the Tag-lite binding assay, significant binding of SBL-NbE-05 (SEQ ID NO: 8) and SBL-NbE-12 (SEQ ID NO: 9) was detected at Ki values of 1.2 pM and 8.7 pM, respectively, while the shorter peptides 1-4 showed little to no binding (Fig. 5a and Fig. 7c). Linear peptides are intrinsically flexible molecules able to adopt multiple conformations, which commonly deviate from ordered secondary structures, such as p- hairpins. This was confirmed by circular dichroism (CD), showing predominantly random coil spectra for SBL-NbE-01 to -05 and SBL-NbE-12 in water and in the a-helix stabilizing solvent TFE (2,2,2-trifluoroethanol) (not shown).

[0135] To mimic the more rigid CDR3 p-hairpin structure observed in NbE, a series of cyclic peptides was designed using several parallel strategies including side chain-to-side chain macrocyclizations, as well as head-to-tail main chain cyclization. Sequences of resulting representative peptides are shown in Fig. 6a and are detailed below:

[0136] SBL-NbE-21 (SEQ ID NO: 10), side chain-to-side chain cyclized (Azk to Pra link),

[0137] SBL-NbE-14 (SEQ ID NO: 11), side chain-to-side chain cyclized (Azk to Pra link),

[0138] SBL-NbE-22 (SEQ ID NO: 12), side chain-to-side chain cyclized (Azk to Pra link),

[0139] SBL-NbE-23 (SEQ ID NO: 13), side chain-to-side chain cyclized (Aha to Pra link),

[0140] SBL-NbE-26 (SEQ ID NO: 14), head-to-tail cyclized and SBL-NbE-27 (SEQ ID NO: 15), head-to-tail cyclized.

[0141] SBL-NbE-26 and SBL-NbE-27 where obtained by grafting sequences corresponding to linear peptides SBL-NbE-05 and SBL-NbE-12, respectively, on the p-turn-inducing D- Pro-L-Pro dipeptide (39) and are shown in Fig. 6b. The position of the p-turn-inducing scaffold in these sequences was chosen to create a conformational bias for the observed hydrogen bonds in the NbE CDR3 p-hairpin structure, comparable to the conformational rigidity imposed by the single domain antibody framework (Fig. 5a). CD measurements confirmed the beneficial effect of cyclization, as typical spectra for a p-hairpin structure were observed for cyclic peptide 8 (not shown).

[0142] Tag-lite binding assay results are shown in Fig. 7a for cyclic peptides SBL-NbE-21 1 - 14 I -22 I -23 and in Fig. 7b for cyclic peptides SBL-NbE-26 and SBL-NbE-27. Cyclic peptides SBL-NbE-21 / -14 / -22 I -23 bind the pOR at varying affinity and Ki values in the nM-to-low pM range, with SBL-NbE-14 and -22 exhibiting improved affinity over the linear peptides (Fig. 5b). Both cyclic peptides SBL-NbE-26 and -27, which have an increased tendency to adopt a p-hairpin structure compared to the linear peptides, bind the pOR with high affinity (Fig. 7b) and Ki values of 244 nM and 39 nM, respectively, which closely approaches the parental NbE affinity (Ki 20 nM). All IC50 and Ki values are shown in Fig. 7c.

[0143] Importantly, this is the first single domain antibody CDR3 peptidomimetic that recapitulates the binding properties of the parental single domain antibody. Previous work on such mimetics already delivered proof-of-concept, but never with equivalent binding properties (40, 41).

[0144] Next, we tested whether the peptide mimetics retain the pOR-selective antagonism observed for NbE. Peptides SBL-NbE-051 -121 -261 -27 caused a concentration-dependent decrease in DAMGO-driven pOR signaling, with cyclic peptides SBL-NbE-26 and -27 fully reversing pOR-mediated inhibition of cAMP production at high concentrations (Fig. 7d). Furthermore, DPDPE-driven 5OR signaling was not antagonized by the peptides, showing that the CDR3 analogues retain receptor subtype selectivity (Fig. 7e).

[0145] Taken together, the extensive interaction interface between the CDR3 of NbE and the pOR allowed the design of downsized linear and cyclic peptide mimetics that retain high affinity pOR binding and antagonism.

[0146] 1.6 Material and Methods

[0147] Mammalian cell culture conditions and plasmids

[0148] HEK293 cells (CRL-1573, ATCC, female) were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco), supplemented with 10% fetal bovine serum (FBS, Thermo Fisher). HEK293 cells stably expressing N-terminally signal sequence FLAG (ssf)-tagged murine pOR (HEK293-pOR) were cultured in the presence of 250 pg / ml Geneticin (Gibco) (14). A clonal HEK293 cell line stably expressing N-terminally signal sequence (ss) SNAP-tagged murine pOR was generated by cloning a ‘GAG promoter, ssSNAP-pOR, RGK promoter, PuroR’ cassette into a piggyBac transposon plasmid (Addgene #84239), co-transfection of the plasmid with piggyBac transposase, followed by puromycin selection and isolating single cell clones by FACS. For transient DNA expression, Lipofectamine 2000 (Invitrogen) was used according to the manufacturer's instructions. Published plasmids used in this study: Murine ssf-SOR, ssf-pOR, ssf-KOR, and ssf-NOPR in pcDNA3.1 , NbE-6xHis in pXAP100. Plasmids generated in this study: mNbE (N45K, Q117K, Q120P)-6xHis in pXAP100, Mutant versions of murine ssfMOR and ssfDOR, and human ssf-pOR in pcDNA3.1. Plasmids were generated by In-Fusion cloning or QuikChange Site-Directed Mutagenesis (Agilent). NbE purification and labeling

[0149] Escherichia coliMMKQ cells were transformed with NbE (originally named ‘Nb35’ (9)) cloned into the pXAP100 plasmid, containing an N-terminal pelB signal sequence for periplasmic expression and C-terminal 6xHis-tag for purification. A single colony was grown in Terrific Broth media (1.2% w / v Tryptone, 2.4% w / v Yeast Extract, 0.6% Glycerol, 0.1 % Glucose, 2mM MgCI2) at 37°C with 50 pg / mL ampicillin to an optical density (OD) at 600 nm of 0.7, followed by induction with 1 mM Isopropyl p-D-1 -thiogalactopyranoside (IPTG, Biosolve Chimie, Catalog number 0010006204) and incubated overnight at 37°C at 180 rpm shaking. Cell pellets were resuspended in TES buffer (0.2M Tris, 5mM EDTA, 0.5M sucrose, pH 8.0) equivalent to 5% of bacterial culture volume, and kept on a shaker at 4°C for at least 60 min. Cells underwent an osmotic shock by adding double the volume of TES diluted 1 :4 with H2O and kept on the 4°C shaker for 45 minutes. Cells were pelleted and discarded, and NbE contained in periplasmic lysate purified by Ni2+-affinity (HisPur Ni NTA Resin; Thermo Fisher Scientific, Catalog number 88221) and eluted by low pH by adding Acetate Buffer (50 mM NaAc,1M NaCI, pH4.5-4.7) and immediately adding 1 M Tris (pH 7.5) to the eluates. NbE was further purified by size-exclusion chromatography (Superdex 75, Cytiva) and purity assessed by SDS-PAGE gel electrophoresis and gel staining with Coomassie blue (Bio-Rad, Catalog number 1610400). NbE was covalently conjugated at primary amines of lysines with Alexa Fluor 488 (Invitrogen, Catalog number A10235). For conjugation, 1 mg NbE was incubated with AF488 for 1 hour at room temperature (RT) and NbE-AF488 separated from free dye using gel filtration. The concentration and degree of labeling was determined with a NanoDrop spectrophotometer (Thermo Scientific) and NbE- AF488 subsequently used in fluorescence microscopy- and flow cytometry-based binding assays.

[0150] Flow cytometry-based binding assay

[0151] HEK293 cells were transiently transfected with ssf-tagged murine pOR, bOR, KOR, and NOPR or with pOR and bOR mutants. 24 h post transfection, cells were detached with PBS- EDTA and resuspended in ice-cold assay buffer (PBS, 1 mM Ca2+, 0.5 mM Mg2+) at 8 million live cells / ml. Cells were incubated for 30 min at RT with NbE-AF488 at various concentrations and 10 minutes with AF647-conjugated anti-FLAG M1 antibody (Sigma- Aldrich, F-3040). Cells were washed twice with PBS and resuspended in FACS buffer (PBS, 1 mM Ca2+, 0.5 mM Mg2+, BSA 0.5%). Flow cytometry was performed using a Beckman Coulter CytoFLEX Flow Cytometer. Data were analyzed with FlowJoTM v10 software and cells were gated for i) singlets, ii) living cells, and iii) comparable receptor expression using the FLAG-AF647 signal. To probe competitive binding with naloxone, HEK293-pOR cells were seeded at 5 x 10*4 live cells / cm2density. Cells were detached and resuspended in ice-cold assay buffer. Cells were then pre-treated with different concentrations of naloxone (Sigma-Aldrich, N7758) for 5 minutes, followed by addition of 1 pM NbE-AF488 for 30 minutes. Cells were washed twice with PBS, resuspended in FACS buffer, and analyzed using the Beckman Coulter CytoFLEX Flow Cytometer. Data were analyzed with FlowJoTM v10 software and cells were gated for singlets and further gated for similar receptor expression levels. Individual conditions were normalized to the maximum AF488 signal from the cells treated with 10 pM NbE-AF488. The analyzed data was plotted using GraphPad Prism Version 9.2 software.

[0152] Luminescence-based cAMP assay

[0153] HEK293-pOR cells were transfected with a plasmid encoding pGloSensor-20F cAMP reporter (Promega). Cells were harvested 24 h post transfection and resuspended at a 1.5x10A6 live cells / ml in assay media (DMEM without phenol red or FluoroBrite DMEM, 250 pg / ml luciferin). 100 pl cell suspension was plated into each well of a clear bottom 96-well plate, and equilibrated for 60 min at 37°C. To probe the reversal of 10 nM DAMGO (Sigma- Aldrich, E7384) or 30 nM morphine (morphine HCI, Sintetica) (concentrations correspond to their respective EC50 values in the assay, agonist stock in H2O, diluted in assay media), cells were pre-incubated for 15 min with different concentrations of NbE. Before addition of ligands, sequential luminescence images were collected to obtain baseline luminescence values using the FDSS / pCELL kinetic plate imager (Hamamatsu) with an integrated simultaneous dispensing head and simultaneous detection across the plate. Cells were then treated with ligands and 2.5 pM Forskolin (FSK, Sigma-Aldrich F6886), followed by continuous luminescence imaging for 10 min, using two technical replicates per condition. Luminescence signals from cells not stimulated with FSK were considered as background and subtracted from all experimental conditions. The difference in luminescence signal between the baseline (average luminescence before addition of drugs) and the maximum signal in each condition was determined, and cells treated with 2.5 pM FSK (no agonist or NbE) normalized to 100%. To illustrate data on peptide-driven reversal of the DAMGO or DPDPE effect, the inhibition mediated by 10 nM DAMGO or 1 nM DPDPE in the absence of peptides was normalized as 100% pOR or 100% 5OR signaling. GraphPad Prism Version 9.2 software was used for data analysis, statistical tests and graph plotting.

[0154] Confocal microscopy-based live cell binding assay HEK293 cells were seeded on poly-L-lysine-coated 35-mm Cellvis glass-bottomed dishes (I BL, 220.110.022) and, after 24 hours, transfected with mouse ssf-pOR, -5OR, -KOR, or - NOPR, or human ssf-pOR or ssf-SOR (0.8 g DNA) using 3 l of Lipofectamine 2000. 16 to 24 h post-transfection, cells were incubated at 37°C with 1 pM NbE-AF488 for 30 minutes and Alexa Fluor™ 647 conjugated (Invitrogen, Catalog number A20173) M1 antibody for 10 min in HBS imaging solution (Hepes-buffered saline with 135 mM NaCI, 5 mM KCI, 0.4 mM MgCI2,1.8 mM CaCI2, 20 mM Hepes, 1 mM d-glucose, 1 % FBS, adjusted to pH 7.4). Cells were subsequently washed 2x with PBS and imaged with a spinning disk confocal microscope (Nipkow, Zeiss) using Plan Apo 63x / 1.4 Oil DICIII objective in a temperature and CO2-controlled environment (37°C, 5% CO2).

[0155] Flow cytometry-based internalization assay

[0156] HEK293 cells were seeded onto 12 well-plate and transiently transfected with ssf-tagged murine wt pOR or pOR mutants. 24 hours post transfection, HEK cells were treated with 10 pM DAMGO or vehicle control for 25 min. Cells were immediately washed with ice-cold PBS, and surface receptors labeled for 10 min with anti-FLAG M1-647 at 4°C. Cells were then resuspended in 500 pl FACS buffer (PBS, 1 mM Ca2+, 0.5 mM Mg2+, BSA 0.5%). Cells were analyzed by flow cytometry (Beckman Coulter Cytoflex). Data was analyzed with FlowJoTM v10 software and cells were gated for singlets and further gated for the receptorexpressing population. The area under curve (AUC) was calculated as a measure of the receptor level from the histogram of the gated population using formula, AUC = Total sum of counts * Width of the histogram bin; where the width of the bin was calculated using Sturges' Formula. The receptor levels of the vehicle control (no DAMGO) was normalized to 100 % for each condition and each experimental replicate.

[0157] Tag-lite HTRF binding assay

[0158] HEK293 cells stably expressing SNAP-pOR were labeled with 100 nM Tag-lite SNAP- Lumi4-Tb (Revvity Catalog number SSNPTBC). The labeled cells were resuspended at a concentration of 1x106 cells / ml. 10 pl of the resuspension was plated in each well of low volume 96-well plates or 384-well plates to obtain 10'000 cells per well. 5 pl of labeled antagonist (Revvity Catalog number L0005RED) was added to each well, resulting in a final concentration of 3 nM, followed by addition of different concentrations of the test compound, and incubation for 3 h at room temperature, protected from light. Each experimental condition was performed in technical triplicates. Upon reaching equilibrium after 3 h, the FRET signal at 620 nm and 660 nm was read using a SpectraMax Paradigm Multi-Mode Microplate Reader (Molecular Devices). The ratio of the acceptor and donor emission signals (HTRF ratio signal) for each individual well was calculated. HTRF signals were plotted against concentrations. All conditions were normalized to the maximum HTRF ratio from the wells only containing labeled ligand L0005RED. The signal from 100 nM naloxone condition was considered as background. GraphPad Prism Version 9.2 software was used for data analysis, statistical tests and graph plotting.

[0159] Grating coupled interferometry assay

[0160] Grating Coupled Interferometry (GCI) experiments were conducted on a Creoptix WAVE delta system using 4PCP chips (Creoptix AG). The chips were conditioned with 100 mM sodium borate (pH 9.5) and 1 M NaCI (Xantec). Neutravidin (100 pg / pl in 10 mM sodium acetate, pH 5.0) was immobilized on the chip surface using standard amine coupling. This included 420 s of surface activation with a 1 :1 mix of 400 mM EDC and 100 mM NHS (Xantec), 420 s neutravidin injection, 420 s BSA (0.5%) injection, and a final 420 s surface passivation with 1 M ethanolamine at pH 8.0. Subsequently, biotin-NbE was captured at three concentrations: 100 pg / pl, 20 pg / pl, and 1 pg / pl, yielding surface masses of approximately 3700, 3500, and 2300 pg / mm2, respectively. Any remaining neutravidin sites were filled with 100 pg / pl biotin-BSA. All preparation steps were performed at 10 pl / min flow rate. The analyte, containing purified pOR or the TCR-CD3 complex (negative control), was injected in a 1 :2 dilution series from 7.8 nM to 1000 nM at 45 pl / min. The running buffer contained 25 mM HEPES (pH 7.5), 100 mM NaCI, 0.01% LMNG, and 0.001% CHS. Blank injections and a reference channel were used for double referencing, and a 0-2% dimethylsulfoxide (DMSO) calibration curve was employed for bulk refractive index correction.

[0161] Data were analyzed with Creoptix WAVEcontrol software, with corrections made for X and Y offset, DMSO calibration, and double referencing. A 1 :1 Langmuir binding model with bulk correction was used for all experiments. Consistent results were observed across the three NbE concentrations.

[0162] Purification of the anti-Fab single domain antibody

[0163] A fragment encoding the anti-Fab single domain antibody (37) was synthesized by GeneArt (Thermo Fisher Scientific) and subsequently cloned into the pET28a vector downstream of a 6xHis tag. The resulting fusion proteins were expressed in Escherichia coli BL21 (DE3) at 18°C overnight. Protein expression was induced with 0.5 mM Isopropyl B-D-1- thiogalactopyranoside (IPTG) at an OD Aeoo of 0.6. The fusion proteins were first purified in batch by a Ni2+-affinity step (HisPur Ni-NTA Resin; Thermo Fisher Scientific), before being further purified by size-exclusion chromatography (Superdex 75 Increase 10 / 300 GL). The final protein buffer solution contained 25 mM Tris-HCI (pH 7.5) and 150 mM NaCI (no reducing agents). Monomeric anti-Fab single domain antibody fractions were pooled, concentrated to ~3.8 mg / ml, flash-frozen in liquid nitrogen, and stored at -70°C.

[0164] Purification of the NabFab

[0165] The NabFab plasmid was kindly provided by the group of Prof. Kaspar Locher, ETH Zurich (31). Chemically competent C43 Escherichia coli cells were used for protein expression. Four liters of TB autoinduction media (Terrific Broth containing 0.4% glycerol, 0.01 % glucose, 0.02% lactose, 1.25 mM MgSC>4 and 100 pg / ml ampicillin) were inoculated with overnight cultures from single colonies and incubated for 6 h at 37 °C, before shifting to 30°C for expression overnight at 180 rpm shaking. Cells were resuspended in lysis buffer containing 50 mM Tris-HCI pH 7.5, 200 mM NaCI, protease inhibitor cocktail tablets (PIC) (complete EDTA-free, Roche Diagnostics) and 5 units / ml supernuclease (Novagen) and lysed by sonication. The lysate was incubated in a water bath at 65°C for 40 min and spinned down at 20,000x g for 30 min. The filtered supernatant was loaded on a HiTrap™ Protein L column (Cytiva), pre-equilibrated with 50 mM Tris-HCI (pH 7.5) and 500 mM NaCI. The protein was eluted using 0.1 M acetic acid and immediately loaded onto a Hitrap SP HP (Cytiva) column pre-equilibrated with buffer A (50 mM sodium acetate, pH 5.0). After a washing step, the NabFab was eluted using a salt gradient with buffer B (50 mM sodium acetate, 2 M NaCI, pH 5.0). The final protein buffer contained 25 mM Tris-HCI pH 7.5, 150 mM NaCI, the protein was concentrated to 7.0 mg / ml, flash-frozen and stored at -70°C.

[0166] Purification of / JOR

[0167] DNA coding for the full-length murine pOR was subcloned into the pF1 vector. The resulting construct comprises an N-terminal signal sequence, a Flag epitope tag and a C-terminal HRV-3C protease cleavage site followed by a Strep II tag and 8 x His tag. pOR was expressed in Sf9 cells using baculoviruses in the presence of 10 pM naloxone. Typically, 20 ml of P3 viruses were used to infect 500 ml of Sf9 cells. Cells were infected at a cell density of roughly 3.0 x 106cells / ml, incubated for 48 h at 27°C at 110 rev / min and harvested by centrifugation at a cell viability of 80-85%. The purification of pOR has been previously described (27). All purification steps were performed at 4°C. Briefly, Sf9 cell pellets were lysed using hypotonic buffer containing 20 mM HEPES pH 7.5, 5 mM MgCh, PIC, 5 units / ml supernuclease, 10 pM naloxone (Sigma-Aldrich) and 2 mg / ml iodoacetamide (Sigma-Aldrich), and gently stirred for 1 h. Cell membranes were separated by ultracentrifugation at 100,000x g for 40 minutes and resuspended in solubilization buffer made of 25 mM HEPES pH 7.5, 500 mM NaCI, 1% LMNG, 0.1 % CHS, PIC, 10 pM naloxone and 2 mg / ml iodoacetamide using a Dounce homogenizer. The solution was gently stirred for 4 h. The insoluble debris was removed by ultracentrifugation at 100,000x g for 1 h. The supernatant (supplemented with 20 mM imidazole) was incubated for 2-3 hours with HisPur Ni-NTA Resin and extensively washed with wash buffer A (25 mM HEPES pH 7.5, 500 mM NaCI, 0.1% LMNG, 0.01% CHS, 1 pM naloxone, 10 mM imidazole), followed by buffer B (25 mM HEPES pH 7.5, 500 mM NaCI, 0.1 % LMNG, 0.01 % CHS, 1 pM naloxone, 10 mM MgCh, 10 mM ATP). Proteins were eluted using an imidazole gradient and subsequently loaded onto a 5 ml StrepTactin Superflow Cartridge (Qiagen) at a flow rate of 0.8 ml / min. The column was washed with wash buffer C (25 mM HEPES pH 7.5, 500 mM NaCI, 0.05% LMNG, 0.005% CHS) to remove contaminations and residual naloxone. pOR was eluted using 2.5 mM desthiobiotin. The pOR was further purified by size exclusion chromatography. A Superose 6 Increase 10 / 300 GL column (GE Healthcare Life Sciences) was pre-equilibrated with 20 mM HEPES pH 7.5, 100 mM NaCI, and 0.001% LMNG / 0.0001% CHS. Fractions containing pOR were concentrated, flash-frozen and stored at -80°C.

[0168] NbE- OR-Fab module complex assembly

[0169] To assemble the NbE-pOR-Fab module complex, freshly prepared pOR was incubated with NbE (containing framework mutations N45K, Q117K, Q120P to enable NabFab binding (37)), NabFab and anti-Fab single domain antibody in a molar ratio of 1 :2:2.5:3 overnight, gently mixed at 4°C. A final size-exclusion step using a Superose 6 Increase 10 / 300 GL column was conducted. Fractions containing the complex were pooled, concentrated to around 2.8 mg / ml and immediately used for EM grid preparation. The assembled complex was analyzed by application to a Superose 6 Increase 5 / 150 column.

[0170] Cryo-EM sample preparation and data collection

[0171] 3 pl of freshly purified NbE-pOR-Fab module complex were applied onto holey gold grids (Quantifoil UltrAuFoil R1.2 / 1.3, 300 mesh), front blotted for 3-4 seconds with 1 mm additional movement (95% humidity at 15°C) before being plunged into liquid ethane using an EM GP2 automatic plunge freezer (Leica). The cryo-EM data sets of NbE-pOR-Fab module complex were acquired on a Thermo Scientific Talos Arctica Cryo-TEM at an accelerating voltage of 200 kV. A total of 5’938 movies were recorded using a Falcon III direct electron detector at a nominal magnification of 150,000x, resulting in a pixel size of 0.9759 A. Data were collected using EPU (Thermo Fisher Scientific) with one image per hole, a defocus range of -0.6 to -2.0 pm and a total electron dose of 40e7A2distributed over 44 frames per acquisition. Data acquisition was monitored on-the-fly pre-processing using CryoSPARC v.3.3.1 (53).

[0172] Cryo-EM image processing

[0173] All data were processed using CryoSPARC v.3.3.1 and RELION 3.1 (53, 54). First, raw movies were aligned and dose weighted using patch-based motion correction (55). Contrast transfer function (CTF) parameters were estimated by patch-based CTF estimation (53). Only micrographs with a CTF fit better than 4.0 A resolution were selected for further processing, resulting in a set of 5’686 micrographs. Particles were initially picked using a blob picker with a minimum and maximum diameter of 70 A and 200 A, respectively. The resulting 2D classes were fed into the Topaz particle-picking pipeline to increase the number and accuracy of picked particles. After several rounds of 2D classification (and removal of duplicated particles) 704’786 particles were selected. Ab initio reconstruction with multiple classes was followed by heterogeneous refinement. The best class contained 509,809 particles. 3D classification helped to further clean up the particle set. 3D refinement using non-uniform refinement and CTF refinement (global and local CTF refinement) resulted in a reconstruction of 3.1 A resolution. To further improve the density of the pOR, refined particles were imported into RELION 3.1 for 3D classification without alignment (K=4 and T=12), using a soft mask on the seven transmembrane helices of the pOR. The best class showing high resolution features was selected (445,766 particles) and particles were re-imported to CryoSPARC for non-uniform refinement followed by local refinement with a soft mask around the NbE-pOR. As a result, the density for the NbE-pOR interface was significantly improved. All maps were sharpened with deepEMhancer (56). All resolution estimations were derived from Fourier shell correlation (FSC) calculations between reconstructions from two independently refined half-sets, and reported resolutions are based on the FSC = 0.143 criterion. Local resolution estimations are obtained by ResMap (57).

[0174] Cryo-EM model building and refinement

[0175] The crystal structure of inactive pOR bound to a covalent antagonist (PDB: 4DKL) was used as an initial reference (27). Similarly, the crystal structure of the NbE (PDB: 8V8K) was used as an initial model. For NabFab and anti-Fab single domain antibody, the cryo-EM structure of VcNorM complex was used as initial models (37). All initial models were fitted into cryo- EM maps using Chimera X (58), then manually built in Coot (59), iteratively and real-space refined using PHENIX (60). Model validation was performed with MolProbity (67). Structural figures were generated in Chimera X (58). Crystallization and data collection

[0176] Purified NbE was concentrated to ~30 mg / mL for crystallization. Crystals were grown using the hanging drop vapor diffusion method at 16°C in a temperature-controlled incubator. The best NbE crystals grew in 1 M succinic acid pH 7.0, 1-2% polyethylene glycol 2000 monomethyl ether (PEG 2000 MME) and 0.1 M Hepes, pH 7.0, reaching their final size within 3 days. Crystals of NbE were cryoprotected using the well solution supplemented with 25% glycerol and flash frozen in liquid nitrogen. Diffraction data was collected at the Advanced Photon Source on beamline 23-IDD on a Pilatus 6M detector. The final dataset was collected from a single crystal of NbE, with reflections extending to 2.85 A resolution.

[0177] Crystallographic data processing and model refinement

[0178] Reflection data was integrated using XDS (62), and scaled and merged using Aimless as part of the CCP4 suite (63). Initial phases were obtained using molecular replacement in Phaser (64), using a homology model from SwissModel (65) yielding three copies of NbE in the asymmetric unit. Iterative rounds of manual model building and automated refinement were carried out in Coot (66) and Phenix (60), respectively. The final model was refined to a Rfree of 0.331 with favorable geometry (96% Ramachandran favored, 3% allowed, and 0.3% outliers). Of the three chains in the asymmetric unit, one (chain A) is characterized by exemplary density for the resolution and was used as an initial model for building of the NbE-pOR-Fab complex.

[0179] Synthesis of linear peptides SBL-NbE-01, -02, -03, -04, -05 and -12

[0180] Peptides were synthesized using Fmoc-based solid phase peptide synthesis (SPPS) on a microwave assisted peptide synthesizer (CEM Liberty Lite). The synthesis was performed on 0.1 mmol scale using preloaded Wang resin or Rink Amide resin depending on the desired C-terminal end of the peptide, being a carboxylic acid or carboxamide. Fmoc deprotection was performed at 90°C for 3 min using a solution of 20% 4-methylpiperidine in / V, / V-dimethylformamide (DMF) during the entire synthesis. Each coupling was done using 5 equivalents of Fmoc protected amino acid, with 0.5 M / V, / V'-diisopropylcarbodiimide (DIC) and 1 M Oxyma as coupling reagents. N-terminal acetylation has been done manually using 10 equivalents of acetic anhydride and 5 equivalents of N,N-diisopropylethylamine (DIPEA) during 1 h in DMF. After completion of the sequence, the resin was washed several times with dichloromethane (DCM), followed by the cleavage using a cocktail solution consisting of 90 % trifluoroacetic acid (TFA), 5 % triisopropylsilane and 5 % distilled water during 4 h. After freeze-drying, crude peptides were obtained and purified using preparative HPLC. More specifically, a Gilson HPLC system, equipped with Gilson 322 pumps and a Supelco Discovery BIO Wide Pore C18 column (25 cm x 21.2 mm, 10 pm), was used. Crude peptides were dissolved in DMSO and purified using H2O-MeCN-0.1% TFA as mobile phase. Finally, fractions were collected, and the accompanying purities were assessed by analytical RP- HPLC, after which the pure fractions were combined and lyophilized to obtain the final purified peptide as a powder (TFA salt) with a high purity (> 95 %).

[0181] Synthesis of side chain-to-side chain cyclized peptides SBL-NbE-21, -14, -22, -23

[0182] Peptides were manually synthesized using Fmoc / tBu-based SPPS as linear variants using polypropylene syringes equipped with a polyethene frit. The synthesis was performed on a 0.10-0.20 mmol scale using a 2-chlorotrityl chloride resin. Between every step, the resin was washed with DMF (3 times) and DCM (3 times). Loading of the resin (2 equiv) was performed overnight with the appropriate Fmoc / tBu-protected amino acid (1 equiv) and DI PEA (2 equiv) in dry DCM. Fmoc deprotections were performed using a solution of 20% 4-methylpiperidine in DMF for 5 and 15 min. Amino acid couplings were carried out using 4 equiv of protected amino acid, 4 equiv of / V, / V, / V( / V-tetramethyl-O-(1 H-benzotriazol-1- yl)uronium hexafluorophosphate (HBTLI) and 8 equiv of DIPEA in DMF for 1 to 3 h. After final Fmoc removal, the peptides were cleaved from the resin as linear peptides using a cocktail solution consisting of 90% trifluoroacetic acid (TFA), 5% triisopropylsilane and 5% water during 4 h. After freeze-drying, crude peptides were obtained and purified using preparative HPLC as described above for the linear peptides SBL-NbE-01 , -02, -03, -04, - 05 and -12 using a ReproSil 100 C18 column (25 cm x 20 mm, 5 pm). Afterwards, the purified linear peptides were cyclized (0.005-0.015 mmol scales) using CuSO4-5H2O (3-5 equiv), sodium ascorbate (3-5 equiv) and NH4HCO3 (10 equiv) in H2O:MeCN 9:1 (0.4-0.5 mM) for 1 hour. After freeze-drying, crude peptides were obtained and purified by preparative HPLC as described above using a ReproSil 100 C18 column (25 cm x 20 mm, 5 pm).

[0183] Synthesis of head-to-tail cyclized peptides SBL-NbE-26 and -27

[0184] Peptides were manually synthesized using Fmoc / tBu-based SPPS as linear variants using polypropylene syringes equipped with a polyethene frit. The synthesis was performed on a 0.15 mmol scale using a 2-chlorotrityl chloride resin. Between every step, the resin was washed with DMF (3 times) and DCM (3 times). Loading of the resin (2 equiv) was performed overnight with the appropriate Fmoc / tBu-protected amino acid (1 equiv) and DI PEA (2 equiv) in dry DCM. Fmoc deprotections were performed using a solution of 20% 4-methylpiperidine in DMF for 5 and 15 min. Amino acid couplings were carried out using 4 equiv of protected amino acid, 4 equiv of / V, / V, / V( / V-tetramethyl)-O-(1 H-benzotriazol-1- yl)uronium hexafluorophosphate (HBTLI) and 8 equiv of DIPEA in DMF for 1 to 3 h. After final Fmoc removal, the peptides were cleaved from the resin under their side chain protected forms using a solution of 20% hexafluoro-2-propanol in DCM for 90 minutes. After evaporation under reduced pressure, a powder was obtained which was directly used for head-to-tail cyclization in solution. Cyclization of the linear, side-chain protected peptides was directly performed on the crude peptides (0.05-0.1 mmol scales) using PyOxim (5 equiv), Oxyma Pure (1 equiv) and DIPEA (12 equiv) in DCM or DMF (0.5-1 mM). After 21 h the solvent was removed under reduced pressure, followed by removal of the side chain protecting groups using a mixture consisting of 90% trifluoroacetic acid, 5% triisopropylsilane and 5% ultrapure water during 90 minutes. After evaporation under reduced pressure, the peptides were precipitated and washed with cold diethyl ether. After freeze-drying, crude peptides were obtained and purified by preparative HPLC as described above for the linear peptides using a ReproSil 100 C18 column (25 cm x 20 mm, 5 pm).

[0185] Circular dichroism of linear and cyclic peptides

[0186] Circular dichroism spectra were recorded on an Applied Photophysics ChiraScanTM-plus spectrometer (ChiraScan v.4.5.1848.0) at 20°C with nitrogen gas purging at 4 L / min. Measurements were performed in a quartz cuvette with a path length of 1 mm containing 100 pL of the peptide solutions. The spectral range of 300-180 nm was sampled with a 1 nm step size, 1 nm bandwidth and 1 s collection time per step. The background was subtracted for all spectra. Raw data was obtained in ellipticity 0 (mdeg) and converted towards the mean residue molar ellipticity [0] considering the number of amide bonds.

[0187] EXAMPLE 2

[0188] 2.1 Additional series of cyclic peptide variants

[0189] A further series of cyclic peptide variants based on SBL-NbE-27 (SEQ ID NO: 15) described in Example 1 were designed, synthetized and characterized.

[0190] The side chain dihedral angles (chi; / ) in combinations with the backbone angles are critical for molecular recognition. Considering the amino acid Ca-Cb bond, the side chain of this amino acid can adopt three low-energy staggered conformations: gauche(-), gauche(+) and anti (also termed trans). The orientation of the side chain relative to the peptide backbone differs greatly between these three conformers. The energy difference and barrier between these conformations is low, so that generally all three conformations are accessible in solution. However, during the interaction of the cyclic peptide with its receptor, each of the critical side chains will adopt one of these low-energy conformations, thereby creating a unique pharmacophore.

[0191] As such, the chi / / torsion angles can be restricted by van der Waals forces, which can be introduced by substituents at the p-carbon. Alternatively, the rotation around the Ca-Cp (but also the subsequent Cp-Cy, etc.) bond can be restricted by incorporation of the side chain into various ring structures.

[0192] A single p -methyl substitution in aromatic amino acids does not constrain significantly the (%1 , / 2) space. Generally, there is a small energy preference of the gauche(-) rotamer for the erythro (e) isomer (2S,3S) and of the anti rotamer for the threo (t) isomer (2S,3R), which can be rationalized on the basis of steric interactions in the different rotamers.

[0193] Considering that the side chain of aromatic amino acids can also adopt three low- energy conformations around the Ca-Cp bond, cyclic analogues of residues such as phenylalanine and tyrosine were introduced. In contrast to a non-covalent introduction of conformational bias, such as the one of p-methyl residues, the conformational preferences of cyclic residues will be stronger. For example, the 1 ,2,3,4-tetrahydroisoquinoline-3- carboxylic acid or Tic residue to limit the conformations to gauche(-) or gauche(+), excluding the anti. According to the same reasoning, linking the aromatic side chain to the alpha carbon, resulting in 1-amino-tetralin-1-carboxylic acid (Ate), limits / 1 to the gauche(-) and anti conformations, and excludes gauche(+). These two types of constrained amino acids therefore have complementary side chain constraints, which makes them very useful for studying the conformational preferences during receptor interaction. Of note, more of these complementary residues exist.

[0194] In view of the above, and because the tip of the CDR3 loop was protruding the pOR binding cleft, conformational constraints at the level of residues 106 and 107 of the CDR3 loop, a tyrosine and phenylalanine, respectively were inserted. Looking at the cryo-EM structure, both residues’ side chains adopted a gauche(-) conformation. Hence, the following unnatural residues forcing the side chains in that conformation were inserted into SBL-NbE-27, to replace the parental Phe (corresponding to position 1 in SEQ ID NO: 15) or Tyr (corresponding to position 18 in SEQ ID NO: 15): Modified residues introduced at Phe (corresponding to position 1 in SEQ ID NO: 15): Modified residues introduced at Tyr (corresponding to position 18 in SEQ ID NO: 15):

[0195] Of note, the hydroxy-aminotetralin amino acid Hat induces the same constraint as the Ate residue, and p-MePhe building block was used as a mixture of threo and erythro, which leads to two peaks (peak 1 and 2 in Table 2) in HPLC chromatograms. These were separated by means of preparative HPLC, but without assigning the exact identity to each peak.

[0196] The resulting variants are summarized in Table 2 (parental SBL-NbE-27 is also shown for reference):

[0197] Table 2

[0198] 2.2 Binding characteristics

[0199] Tag-lite binding assay as described in Example 1 were performed for the above cyclic peptides SBL-NbE-281 -291 -301 -31 I -32 I -331 -341 -35. Parental SBL-NbE-27 was also tested, next to Naloxone and a non-binding control peptide. The resulting IC50 and Ki values are shown in Table 3.

[0200] Table 3

[0201] REFERENCES

[0202] 1. R. Santos, et al. Nat. Rev. Drug Discov. 16, 19-34 (2017). 2. K. Sriram, et al. Mol. Pharmacol. 93, 251-258 (2018).

[0203] 3. A. S. Hauser, et al. Nat. Rev. Drug Discov. 16, 829-842 (2017).

[0204] 4. C. J. Hutchings, et al. Nat. Rev. Drug Discov. 16, 661 (2017).

[0205] 5. T. Laeremans, et al. Front Mol Biosci 9, 863099 (2022).

[0206] 6. I. Jovcevska, et al. BioDrugs 34, 11-26 (2020).

[0207] 7. A. Manglik, et al. Annu. Rev. Pharmacol. Toxicol. 57, 19-37 (2017).

[0208] 8. R. Heukers, et al. Curr. Opin. Cell Biol. 57, 115-122 (2019).

[0209] 9. W. Huang, et al. Nature 524, 315-321 (2015).

[0210] 10. S. G. F. Rasmussen, et al. Nature 469, 175-180 (2011).

[0211] 11. M. J. Robertson, et al. Nat. Struct. Mol. Biol. 29, 1188-1195 (2022).

[0212] 12. A. C. Kruse, et al. Nature 504, 101-106 (2013).

[0213] 13. R. Irannejad, et al. Nature 495, 534-538 (2013).

[0214] 14. M. Stoeber, et al. Neuron 98, 963-976. e5 (2018).

[0215] 15. C. McMahon, et al. Proceedings of the National Academy of Sciences 117, 20284- 20291 (2020).

[0216] 16. P. Scholler, et al. Nat. Commun. 8, 1967 (2017).

[0217] 17. Y. Ma, et al. Sci Adv G, eaax7379 (2020).

[0218] 18. A. Wu, et al. Nat. Commun. 14, 5209 (2023).

[0219] 19. M. A. Skiba, et al. Nat. Chem. Biol., doi: 10.1038 / s41589-024-01620-6 (2024).

[0220] 20. R. R. Schlimgen, et al. Nat. Commun. 15, 4611 (2024).

[0221] 21. G. Corder, et al. Annu. Rev. Neurosci. 41 , 453-473 (2018).

[0222] 22. B. L. Kieffer, et al. Neuropharmacology 56 Suppl 1 , 205-212 (2009).

[0223] 23. A. Manglik, et al. Nature 537, 185-190 (2016).

[0224] 24. A. Faouzi, et al. Nature 613, 767-774 (2023).

[0225] 25. H. Wang, et al. Angew. Chem. Int. Ed Engl. 61 , e202200269 (2022).

[0226] 26. N. D. Volkow, et al. N. Engl. J. Med. 377, 391-394 (2017).

[0227] 27. A. Manglik, et al. Nature 485, 321-326 (2012).

[0228] 28. Y. Zhuang, et al. Cell 185, 4361-4375.e19 (2022).

[0229] 29. A. Koehl, et al. Nature 558, 547-552 (2018).

[0230] 30. Y. Wang, et al. Cell 186, 413-427.e17 (2023).

[0231] 31. J. S. Bloch, et al. Proc. Natl. Acad. Sci. U. S. A. 118 (2021).

[0232] 32. J. A. Ballesteros, et al. Methods in Neurosciences, S. C. Sealfon, Ed. (Academic Press, 1995)vol. 25, pp. 366-428.

[0233] 33. A. Manglik, Biol. Psychiatry 87 , 6-14 (2020).

[0234] 34. Z. Li, J. Liu, F. Dong, N. Chang, R. Huang, M. Xia, T. A. Patterson, H. Hong, Int. J. Mol. Sci. 24 (2023). 35. C. Chen, et al. J. Biol. Chem. 271 , 21422-21429 (1996).

[0235] 36. C. Marie-Pepin, et al. J. Biol. Chem. 272, 9260-9267 (1997).

[0236] 37. S. Granier, et al. Nature 485, 400-404 (2012).

[0237] 38. K. Van Holsbeeck, et al. Bioorg. Chem. 119, 105563 (2022).

[0238] 39. D. Obrecht, et al. [Preprint] (2012). https: / / doi.Org / 10.1016 / j.ddtec.2011.07.006.

[0239] 40. K. Van Holsbeeck, et al. Angew. Chem. Int. Ed Engl. 62, e202219095 (2023).

[0240] 41. C. Martin, et al. [Preprint] (2017). https: / / doi.org / 10.1002 / chem.201701321.

[0241] 42. J. C. Zamora, et al. Pharmacol. Res. Perspect. 9, e00887 (2021).

[0242] 43. D. R. Maguire, et al. Neuropsychopharmacology 45, 1986-1993 (2020).

[0243] 44. L. R. Gerak, et al. J. Pharmacol. Exp. Ther. 371 , 507-516 (2019).

[0244] 45. Y. Toyoda, et al. Nat. Commun. 14, 1-13 (2023).

[0245] 46. C. Hong, et al. Nat. Commun. 12, 815 (2021).

[0246] 47. C. T. Dooley, et al. Proc. Natl. Acad. Sci. U. S. A. 90, 10811-10815 (1993).

[0247] 48. P. W. Schiller, et al. Life Sci. 73, 691-698 (2003).

[0248] 49. L. C. Purington, et al. J. Med. Chem. 52, 7724-7731 (2009).

[0249] 50-52. NA.

[0250] 53. A. Punjani, et al. Nat. Methods 14, 290-296 (2017).

[0251] 54. J. Zivanov, et al. Elife 1 (2018).

[0252] 55. S. Q. Zheng, et al. Nat. Methods 14, 331-332 (2017).

[0253] 56. R. Sanchez-Garcia, et al. Commun Biol 4, 874 (2021).

[0254] 57. A. Kucukelbir, et al. Nat. Methods 11 , 63-65 (2014).

[0255] 58. E. C. Meng, et al. Protein Sci. 32, e4792 (2023).

[0256] 59. A. Casahal, et al. Protein Sci. 29, 1069-1078 (2020).

[0257] 60. D. Liebschner, et al. Acta Crystallogr D Struct Biol 75, 861-877 (2019).

[0258] 61. C. J. Williams, et al. Protein Sci. 27, 293-315 (2018).

[0259] 62. W. Kabsch, XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125-132 (2010).

[0260] 63. J. Agirre, et al. Acta Crystallogr D Struct Biol 79, 449-461 (2023).

[0261] 64. A. J. McCoy, Acta Crystallogr. D Biol. Crystallogr. 63, 32-41 (2007).

[0262] 65. T. Schwede, et al. Nucleic Acids Res. 31 , 3381-3385 (2003).

[0263] 66. P. Emsley, et al. Acta Crystallogr. D Biol. Crystallogr. 60, 2126-2132 (2004).

[0264] 67. Yu, Jun, et al. "Structural basis of p-opioid receptor-targeting by a nanobody antagonist." bioRxiv (2023).

Claims

45CLAIMS1. An antagonist of p-opioid receptor (pOR), wherein the antagonist is a mimetic peptide of the CDR3 loop of a single domain antibody comprising a sequence of SEQ ID NO: 1 , wherein the CDR3 loop comprises a sequence of SEQ ID NO: 2.

2. The antagonist according to claim 1 , wherein the antagonist comprises a sequence consisting of (1) a fragment of SEQ ID NO: 2 comprising SEQ ID NO: 3, or (2) a sequence derived from the sequence defined in (1) by substitution or chemical modification of one or more amino acids.

3. The antagonist according to any one of claims 1 to 2, wherein the antagonist is a cyclic peptide.

4. The antagonist according to claim 3, wherein the antagonist comprises a sequence selected from (1) SEQ ID NOs: 10 to 13 or (2) a sequence derived from any one of SEQ ID NOs: 10 to 13 by substitution or chemical modification of one or more amino acids.

5. The antagonist according to claim 3, wherein the cyclization is a head-to-tail cyclization.

6. The antagonist according to claim 3 or 5, wherein the peptide comprises a p-turn inducing scaffold.

7. The antagonist according to claim 6, wherein the p-turn inducing scaffold is a D- Pro-L-Pro dipeptide or an analogue thereof.

8. The antagonist according to any one of claims 5 to 7, wherein the peptide has a sequence of formula I:- - - - - -1-2-3- 4-5- - -6-7-8-9-1£r- - - - - -(formula I), whereinXi is D or is absentX2 is Y or is absent,46X3 is W or is absent,X4 is G, or is absent,X5 is K or is absent, dP corresponds to a D-Pro,Xe is Y or is absent,X7 is C or is absent,Xs is N or is absent,X9 is F or is absent, andX10 is K or is absent, wherein either X5 and Xe, only, are absent; or X4, X5, Xe, and X7, only, are absent; or X3, X4, X5, Xe, X7, and Xs, only, are absent; or X2, X3, X4, X5, Xe, X7, Xs and X9, only, are absent; or all of Xi to X10 are absent, or a sequence derived from formula I by substitution or chemical modification of one or more amino acids.

9. The antagonist according to any one of claims 5 to 8, wherein the peptide has a sequence of formula II:(formula II), whereinXi is D or is absent,X2 is Y or is absent, dP corresponds to a D-Pro,X3 is F or is absent, andX4 is K or is absent, wherein either X2 and X3, only, are absent, or all of Xi to X4 are absent , or a sequence derived from formula II by substitution or chemical modification of one or more amino acids.

10. The antagonist according to any one of claims 5 to 9, wherein the antagonist comprises a sequence selected from (1) SEQ ID NOs: 14, 15 and 27 or (2) a sequence derived from any one of SEQ ID NOs: 14, 15 and 27 by substitution or chemical modification of one or more amino acids.4711. The antagonist according to any one of claims 1 to 10, wherein the antagonist has a specific binding affinity for the p-opioid receptor (pOR).

12. A pharmaceutical composition comprising a therapeutically effective amount of the antagonist according to any one of claims 1-11 and at least one of a pharmaceutically acceptable carrier, adjuvant or diluent.

13. Use of the antagonist according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 to antagonize pOR signaling activity.

14. The antagonist according to any one of claims 1 to 11 or the pharmaceutical composition according to claim 12 for use in a method of treating a medical condition, preferably wherein the medical condition is an opioid receptor-related medical condition, more preferably a pOR-related medical condition.

15. The antagonist or the pharmaceutical composition for use according to claim 14, wherein the medical condition is selected among respiratory depression, constipation sedation, dizziness, nausea, vomiting, physical dependence to opioid, and tolerance to opioid.

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