Vasopressin-2 receptor antagonistic peptides and uses thereof

Abstract

The present invention relates to a peptide comprising a sequence having 80% or greater amino acid identity to the amino acid sequence of SEQ ID NO. 1: RPSX1CNLPVKPGPCX2 GFFSAFYYSQKX3NKCHSFTYGGCAGNANRFSTX4EK CRRTCX5X6, wherein - X1 represents an amino acid residue F or G, - X2 represents any amino acid residue, except a basic amino acid residue, - X3 represents an amino acid residue T or D, - X4 represents an amino acid residue I, L or E, - (i) X5 represents V and X6 represents G or (ii) X5 represents G and X6 represents V, and - the amino acid located at position 39 is A. The present invention also relates to various diagnostic and therapeutic uses thereof.

Description

Technical Field

[0001] This invention relates to the medical field concerning the treatment of diseases involving the angiotensin-2 receptor. Background Technology

[0002] The type 2 angiotensin receptor (V2R) is a G-protein-coupled receptor encoded by the AVPR2 gene located on chromosome Xq28. Like V1a, V1b, and oxytocin receptors, V2R belongs to the angiotensin-sensitive hormone receptor family. Similar to many GPCRs, V2R induces pleiotropic effects through both G-protein-dependent and G-protein-independent mechanisms.

[0003] V2R is significantly expressed in the distal nephron. Once activated in the collecting duct, the V2R / Gas pathway stimulates intracellular cAMP production, which activates protein kinase A to phosphorylate aquaporin-2, thereby translocating it from intracellular vesicles to the apical membrane via an intracellular calcium-dependent exocytosis mechanism. Furthermore, water can reach chief cells from urine and then into the bloodstream via aquaporin 2 at the apical membrane due to aquaporin 3 and 4 on the basolateral membrane. Poorly circulated V2R is associated with prolonged activation due to the active complex AVP-V2R-β-inhibitor protein. A third V2 pathway leads to ERK1 / 2 activation via metalloproteinase-mediated insulin-like growth factor receptor activation.

[0004] Both loss-of-function and gain-of-function variations of V2R are associated with human diseases. Insufficient arginine-angiotensin (AVP) secretion is associated with hyponatremia, characterized by plasma sodium levels below 135 mmol / L. It is associated with numerous diseases, including chronic heart failure, liver failure, and chronic kidney disease, where it has been shown to be associated with an increased risk of death. Ectopic AVP secretion in lung or prostate cancer can also lead to excessively high circulating AVP levels, resulting in hyponatremia. Furthermore, V2R is also a therapeutic target for autosomal dominant polycystic kidney disease (ADPKD). Many therapeutic strategies targeting V2R-related diseases (including ADPKD) have been developed, but only blocking V2R has demonstrated efficacy in humans (Nagao et al., 2012, Exp Anim, Vol. 61: 477-488).

[0005] Extrarenal, V2R is expressed in endothelial cells, where it is involved in controlling the expression of von Willebrand factor. In the inner ear, V2R is involved in the regulation of lymphatic sac volume and is thought to play a role in Meniere's disease (Eckhard et al., 2012, Mol Aspects Med, Vol. 33: 612-637). In the skeleton, V2R may aid in hemostasis (Tamma et al., 2013, Proc Natl Acad Sci USA, Vol. 110(46): 18644-18649). Neuropeptides, such as AVP, can be overexpressed in neuroendocrine tumors. Furthermore, V2R is ectopically present in human lung, breast, pancreas, colorectal, and gastrointestinal tumors (Pifano et al., 2017, Front Oncol, Vol. 7–Article 11: 1-11).

[0006] V2R insufficiency is associated with many diseases, particularly in humans. However, despite the significant physiological and pathological importance of V2R, the therapeutic repertoire for this receptor is poor. The art primarily utilizes only one antagonist molecule called tolvaptan, a benzozazepine-derived molecule belonging to the lvaptan class, which has high affinity for V2R but moderate selectivity compared to the other three vasopressin-sensitive receptors. Tolvaptan is currently used to treat hyponatremia and ADPKD, but its hepatotoxicity presents numerous challenges.

[0007] Excessive secretion of AVP is a key cause of diseases such as hyponatremia and explains why V2R antagonists are so effective in the following pathologies characterized by euvolemic or hypervolemic hyponatremia: such as SIADH (syndrome of inappropriate antidiuretic hormone secretion), cirrhosis, and congestive heart failure (GIF; Ghali et al., Cardiology, 2008, 111, 147-157).

[0008] Congenital nephrotic diabetes insipidus is associated with V2R inactivation mutations. This leads to polyuria with severe dehydration, especially in children. V2R antagonists (valpromoids) act as drug chaperones, capable of penetrating cells and rescuing the mutated receptor (Morello et al., J. Clin. Investigation, 2000, 105, 887-895).

[0009] By interacting with inhibitory proteins, V2R stimulation leads to activation of signaling pathways involving cAMP and MAP kinases, thus favoring a proliferative response. For example, injection of AVP into rats induced the proliferation of renal tubular epithelial cells, which could be inhibited by V2R antagonists (Alonso et al., Endocrinology, 2009, 150, 239-250). Furthermore, diuretics, such as V2R antagonists, have been able to inhibit the proliferation of renal cancer cells (Bolignano et al., Urol. Oncol., 2010, 28, 642-647) and lung cancer cells (Pequeux et al., Endocr. Relat. Cancer, 2004, 11, 871-885). These results suggest that V2R antagonists are promising therapeutic candidates for various types of cancer.

[0010] Von Weilerbrand factor (VWF) is involved in initial hemostasis. It has been shown that AVP also contains the V2R-specific agonist dDAVP. It increases VWF and factor VIII levels through its interaction with V2R (Kaufmann et al., J. Clin. Invest., 2000, 106, 107-116). Excessive clotting can lead to thrombosis (clots), which can be cured by using V2R antagonists, thereby limiting the secretion of clotting factors.

[0011] The V2R antagonist tolvaptan is also used to treat cirrhosis. Previous meta-analyses have shown that treatment with vaptans increases the risk of total adverse events in patients with cirrhosis due to their well-known hepatotoxicity. (Impact of Vaptans Clinical Outcomes in Cirrhosis Patients: A Meta-Analysis of Randomized Controlled Trials, 2019, Frontiers in Pharmacology).

[0012] The V2R antagonist tolvaptan has been further used to treat heart failure. This large-scale analysis confirmed the efficacy and safety of tolvaptan in a real-world clinical setting. (Tolerability of Tolvaptan in Patients With Heart Failure—Final Results of the Samsca Post-Marketing Surveillance in Heart Failure (SMILE) Study, 2019, Circulation Journal).

[0013] Although valpromide-like drugs have been clearly demonstrated to be effective in treating various conditions, their therapeutic use is limited by some major drawbacks:

[0014] - Valprotans and their metabolites are hepatotoxic. For this reason, their use requires strict patient monitoring, and their long-term administration is limited. Some of them can only be administered intravenously, restricting their use in hospitalized patients.

[0015] -Vaptans have some selectivity for V2R, with their V2 / Vla selectivity index varying from 112 (tolvaptan) to 0.15 (conivatan).

[0016] -Valaptans are antagonists of MAP kinase activation. Therefore, they cannot completely block specific V2R-related signaling pathways. Valaptans have poor solubility in physiological buffers and limited bioavailability (Bernier et al. JASN, 2006, 17, 591).

[0017] The most selective V2R antagonist known in the art is a peptide originally isolated from mamba venom, named mambaquaretin (MQ1). MQ1 is a 57-amino acid peptide with a network of three disulfide bonds forming a Kunitz peptide structure (Ciolek et al., 2017, Proc Natl Acad Sic USA, Vol. 114(27): 7154-7159). MQ1 peptide exhibits high affinity for human V2Rs but no affinity for 156 other GPCRs (including V1aR, V1bR, and OTR) at μM concentrations. MQ1 peptide is a fully competitive antagonist of three pathways associated with interaction with Gas protein, interaction with β-inhibitor protein, and MAP kinase activation (Ciolek et al., 2017, Proc Natl Acad Sic USA, Vol. 114(27): 7154-7159). When administered, MQ1 peptides induce a pure water-promoting effect, i.e., inducing water loss without electrolyte loss. The efficacy of MQ1 in cystic kidney disease has been demonstrated (Ciolek et al., 2017, Proc Natl Acad Sic USA, Vol. 114(27): 7154-7159).

[0018] V2R antagonists are still needed, which are alternatives or improvements to known V2R antagonists, especially for medical purposes. Summary of the Invention

[0019] This invention relates to a peptide comprising a sequence having 80% or greater amino acid identity with the amino acid sequence of SEQ ID NO.1 below:

[0020] RPSX1CNLPVKPGPCX2GFFSAFYYSQKX3NKCHSFTYGGCAGNANRFST X4EKCRRTCX5X6

[0021] in,

[0022] -X1 indicates amino acid residues F or G.

[0023] -X2 represents any amino acid residue, except for basic amino acid residues.

[0024] -X3 indicates that the amino acid residue is T or D.

[0025] -X4 indicates amino acid residues I, L, or E.

[0026] -(i) X5 represents V and X6 represents G or (ii) X5 represents G and X6 represents V, and

[0027] - The amino acid located at position 39 is A.

[0028] In some embodiments of the peptide, X1 represents the amino acid residue G.

[0029] In some embodiments of the peptide, X2 represents an amino acid residue selected from the group consisting of A, D, E, F, G, I, L, N, M, Q, S, T, V, and Y.

[0030] In some embodiments of the peptide, X3 represents amino acid residue D.

[0031] In some embodiments of the peptide, X4 represents amino acid residues E or L.

[0032] In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO.1.

[0033] In some embodiments, the peptide has 80% or greater amino acid identity with peptides selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6. In some embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6. In some embodiments, the peptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO. 2, SEQ ID NO. 3, SEQ ID NO. 4, SEQ ID NO. 5, and SEQ ID NO. 6.

[0034] The peptide can be labeled with a detectable molecular marker.

[0035] The present invention also relates to nucleic acids encoding the above-mentioned peptides, wherein the nucleic acids may be contained in an expression vector.

[0036] The present invention also relates to pharmaceutical compositions comprising peptides as defined above and physiologically acceptable carriers.

[0037] The present invention also relates to the use of the peptides described above as pharmaceutical agents. The present invention further relates to the use of the peptides for treating diseases selected from the group consisting of: pathological diseases characterized by normal-volume or hypovolemic hyponatremia, nephrogenic syndrome due to inappropriate antidiuresis, congenital nephrotic diabetes insipidus, polycystic kidney disease, cancer, thrombosis, Meniere's disease, refractory liver disease, and heart failure.

[0038] The present invention also relates to the in vitro use of the labeled peptides described above for detecting cells expressing angiotensin-2 receptors.

[0039] The present invention also relates to an in vitro method for detecting regulatory abnormalities in the expression of vasopressin-2 receptor on cells, comprising the following steps:

[0040] a) Provide the cells to be tested.

[0041] b) Contact the cells provided in step a) with the labeled peptide according to claim 11.

[0042] c) Measure the expression level of vasopressin-2 receptor in cells.

[0043] d) Compare the expression level value obtained in step c) with the reference expression level value.

[0044] e) Identify abnormalities in the regulation of cellular expression of vasopressin g-2 receptor.

[0045] The present invention also relates to diagnostic reagents comprising the labeled peptides as described above. Attached Figure Description

[0046] Figure 1 Computer predictions of the immunogenicity of the known MQ1 V2R antagonist peptide (also referred to as "U-Da2a") of SEQ ID NO. 7 and the V2R antagonist according to the invention (referred to as "MQ-LEAD") of SEQ ID NO. 6 are shown, respectively.

[0047] The sequences of the two peptides were entered into the NETMHCPAN 3.2 software (www.IEDB.org) by selecting the most common HLA-DR molecules in European and North American populations.

[0048] The NETMHC software allows for the prediction of potential associations between sequences and HLA class II molecules. The selected class II HLA molecules are the most common in European and North American populations. The prediction score is the percentile of each sequence for the first nine amino acids. The lower the score, the stronger the expected association between the sequence and an HLA class II molecule.

[0049] exist Figure 1 In the diagram, each numbered column corresponds to an amino acid residue in each of the 57 amino acid residues in a peptide. Top: Known MQ1 V2R antagonist peptide. Bottom: MQ-LEAD peptide (SEQ ID NO. 6). In the row named “DRBx_nnnn”, DRBx is the name of the HLA-DRB locus, and nnnn is the number of alleles.

[0050] Immunogenicity scores: (i) scores below 10% (black), (ii) 10 to 20% (dark gray), and (iii) 20% to 30% (light gray). In the figure below, bold and underlined areas represent differences in amino acid residues in the MQ-LEAD peptide compared to the MQ1 peptide.

[0051] Figure 2 The diagram shows the V2R binding properties of the V2R antagonist peptide according to the present invention compared to those of known MQ1 peptides. Horizontal axis: Logarithm of the molar concentration of the tested V2R antagonist peptide or MQ1 peptide. Vertical axis: % of bound 3H-AVP (compared to control).

[0052] Figure 3 illustrates the diuretic effect of the V2R antagonist peptide. The tested V2R antagonist peptide was administered in rats (SprageDelay) by intraperitoneal injection at 3 nmol / kg per day (arrow), and diuresis was tracked over time.

[0053] Figure 3A . Horizontal axis: Time period, expressed in hours. Vertical axis: Diuresis, expressed in ml / h / kg. Test conditions: (i) MQ1 peptide [black circle "●", dashed line], (ii) MQ-LEAD [black square "□", solid line] and (iii) MQ K39A [black triangle] (Solid line)

[0054] Figure 3B . Horizontal axis: from left to right: (i) MQ-LEAD [black bar], (ii) MQ1 peptide [dashed bar] and (iii) MQ K39A [blank bar].

[0055] Figure 4 A schematic diagram of the experimental research design is shown.

[0056] Figure 5 The effects of three doses of MQ-LEAD, one dose of tolvaptan, and their mediators on natriuresis were shown in male rats implanted with DDAVP on days 0-2-3-4 and day 0.

[0057] Horizontal axis: Time period, expressed in days. Vertical axis: Blood Na+ concentration, expressed in mM. Test conditions: (i) Hollow circle “○”: Tolvaptan medium, 1% HPMC in sterile water; (ii) Black square “■”: V2R antagonist peptide MQ-LEAD medium, physiological saline; (iii) Hollow square “□”: Tolvaptan; (iv) Inverted triangle (v) MQ-LEAD: 20 μg / kg; (vi) MQ-LEAD: 60 μg / kg; and (vi) MQ-LEAD: 200 μg / kg.

[0058] Figure 6 The inhibition of cAMP production by V2R antagonistic peptides is shown. The horizontal axis represents peptide concentration, expressed as -Log(M). The vertical axis represents the percentage of cAMP production inhibited compared to the control group. (i) Circle "●": MQ1 peptide; (ii) Square "■": MQ K39A peptide; (iii) Triangle MQ-LEAD peptide. Detailed Implementation

[0059] definition

[0060] The term "object" refers to any single subject that requires treatment or is participating in clinical trials, epidemiological studies, or serving as a control, including human and mammalian veterinary patients such as cattle, horses, dogs, and cats. In some preferred embodiments, the object is a human.

[0061] As used herein, the terms "treat" or "treating" cancer refer to the administration of a combination therapy according to the invention to a person suffering from cancer.

[0062] The term "pharmaceutically acceptable" means something that is generally safe, non-toxic, and not biologically or otherwise undesirable in the preparation of pharmaceutical compositions, and includes something acceptable for veterinary and human pharmaceutical use.

[0063] Within the scope of this invention, the “percentage of identity” between two nucleic acid or peptide / protein sequences represents the percentage of identical nucleotide or amino acid residues between the two sequences to be compared after optimal alignment, which is purely statistical and the differences between the two sequences are randomly distributed along their length. Sequence comparison is conventionally performed by comparing sequences after optimized alignment, which can be done segment by segment or by using an “alignment window.” In addition to manual comparison, it can also be performed using the local homology algorithm of Smith and Waterman (1981), the local homology algorithm of Neddleman and Wunsch (1970), the similarity search method of Pearson and Lipman (1988), or by using computer software of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by comparison software BLAST NR or BLAST P).

[0064] The percentage of identity between two sequences is determined by comparing two optimized aligned sequences, where the sequence to be compared may have additions or deletions compared to a reference sequence used for optimized alignment between the two sequences. The percentage of identity is calculated by determining the number of nucleotide or amino acid residues that are the same between the two sequences, preferably two complete sequences, dividing the number of the same positions by the total number of positions in the alignment window, and multiplying the result by 100 to obtain the percentage of identity between the two sequences.

[0065] As used herein, “V2R antagonist activity” refers to the following activities of a substance (e.g., the activity of a peptide): (i) binding to V2R, preferably V2R, with a binding affinity of less than 100 nM; and (ii) inhibiting cAMP production with an IC50 value of less than 1000 nM.

[0066] Detailed disclosure

[0067] This invention relates to a family of vasopressin-2 receptor antagonists that can be used primarily for medical and diagnostic purposes.

[0068] The inventors have identified a family of peptides that share specific structural features and exhibit potent angiotensin-2 antagonists both in vitro and in vivo. These peptides are derived from the known MQ1 peptide (also known as “U-Da2a”) described in PCT application published under No. WO 2014 / 041526.

[0069] The inventors have discovered that several MQ1-derived peptides share the common characteristic of having a lysine (K) residue at amino acid position 39, which inhibits the activity of the human angiotensin-2 receptor (also referred to herein as "V2R") at the nanomolar level. Notably, the disclosed peptide family exhibits significantly higher V2R inhibitory activity than known MQ1 peptides. These peptides have an affinity for V2R several times higher than known MQ1 peptides, for example, an affinity for V2R that is 8 times higher than that of known MQ1 peptides.

[0070] The peptides disclosed in this paper are potent antagonist compounds of the angiotensin-2 receptor, and will be collectively referred to as "V2R antagonist peptides" in this paper.

[0071] As shown in the examples, the V2R antagonistic peptide disclosed herein strongly inhibits cAMP production in V2R-expressing cells under angiotensin activation at concentrations much lower than those of known MQ1 peptides.

[0072] In vivo, the V2R antagonist peptide of this invention actually induces a water-promoting effect. Notably, the V2R antagonist peptide disclosed herein is approximately 500 times more potent than tolvaptan (the most commonly used V2R antagonist compound in treatment) for the treatment of hyponatremia, that is, approximately 500 times more potent than the main V2R antagonist compounds that have been practically used to date in this physiological setting.

[0073] V2R antagonistic peptide

[0074] This invention relates to a peptide comprising a sequence having 80% or greater amino acid identity with the amino acid sequence of SEQ ID NO.1 below:

[0075] RPSX1CNLPVKPGPCX2GFFSAFYYSQKX3NKCHSFTYGGCAGNANRFST X4EKCRRTCX5X6

[0076] in,

[0077] -X1 indicates amino acid residues F or G.

[0078] -X2 represents any amino acid residue, except for basic amino acid residues.

[0079] -X3 indicates that the amino acid residue is T or D.

[0080] -X4 indicates amino acid residues I, L, or E.

[0081] -(i) X5 represents V and X6 represents G or (ii) X5 represents G and X6 represents V, and

[0082] - The amino acid located at position 39 is A.

[0083] As used in this article, “X” n " and "Xn" can be used indiscriminately to represent the same variable amino acid residue.

[0084] As used herein, amino acid residues may be selected from the group consisting of: alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine ​​(C), glutamic acid (E), glutamine (Q), glycine (G), histidine (H), isoleucine (I), leucine (L), lysine (K), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine (V), as well as amino acid analogs, including D-amino acids, β-alanine, γ-aminobutyric acid, δ-aminolevulinic acid, 4-aminobenzoic acid, aminoisobutyric acid, dehydroalanine, cystine, cystathionine, lanethionine, quinone, diaminopimelic acid, ortholeucine, alloleucine, isoserine, N-ethylglycine, N-propylglycine, N-isopropylglycine, N-methylalanine, N-ethylalanine, and isoserine.

[0085] However, the optimal amino acid residues are selected from a group consisting of 20 common amino acid residues.

[0086] All amino acids in the peptides of the present invention may be in D- or L-form, although naturally occurring L-form is preferred.

[0087] Without wishing to be bound by any particular theory, the inventors believe that there is a direct relationship between the high V2R antagonist properties of the peptides disclosed herein and the presence of the alanine residue (A) at amino acid position 39 of SEQ ID NO.1.

[0088] As illustrated in the embodiments herein, the V2R antagonistic peptides disclosed herein are also expected to be endowed with reduced immunogenicity. This reduced immunogenicity makes these peptides suitable for treating a variety of pathologies associated with V2R dysfunction, particularly those requiring repeated administration to the recipient, such as, for example, antidiuretic hormone secretion disorder syndrome or congenital nephrotic diabetes insipidus.

[0089] Furthermore, without wishing to be bound by any particular theory, the inventors believe that the V2R antagonist peptide according to the invention has high selectivity for V2R, as has been shown with known MQ1 peptides. Indeed, the high selectivity of the V2R antagonist peptide for V2R implies that low adverse effects are expected when administered for therapeutic purposes; conversely, for example, the widely used V2R antagonist tolvaptan has been reported to have numerous adverse effects due to its moderate selectivity for V2R.

[0090] The inventors believe that the affinity of the V2R antagonistic peptide disclosed herein is not significantly affected by the identity of amino acid residue X2, unless amino acid residue X2 is composed of basic amino acid residues. In contrast, the presence of basic amino acid residues as amino acid residue X2 negatively affects the peptide's affinity for V2R, leading to an alteration in its binding ability to V2R.

[0091] As used herein, including for amino acid residue X2, basic amino acid residues consist of amino acid residues, which can be conventional or unconventional, i.e., they carry a positive charge at pH 7.0. Basic conventional amino acid residues are selected from the group consisting of K (lysine), R (arginine), and H (histidine).

[0092] Therefore, in a preferred embodiment, X2 represents any amino acid residue other than K, R, and H.

[0093] In a preferred embodiment, X2 represents an amino acid residue selected from the group consisting of: A, D, E, F, G, I, L, N, M, Q, S, T, V, and Y.

[0094] In other preferred embodiments, X2 represents an amino acid residue selected from the group consisting of N, A, E, F, and I.

[0095] In another preferred embodiment, X2 represents amino acid residue N or A.

[0096] Similarly, the identity of the two neutral amino acids X5 and X6 at the C-terminus of the V2R antagonistic peptide disclosed herein should not significantly affect the peptide's ability to bind to V2R.

[0097] In the most preferred embodiment, the present invention relates to a peptide having 80% or greater amino acid identity with the peptide of SEQ ID NO. 1 and containing neither amino acid residue deletions nor amino acid residue additions compared to the peptide of SEQ ID NO. 1. Therefore, most preferably, the V2R antagonistic peptide according to the present invention has a length of 57 amino acids, and its amino acid sequence contains neither amino acid deletions nor amino acid additions compared to SEQ ID NO. 1.

[0098] The peptide having an amino acid sequence that is 80% identical to that of SEQ ID NO.1 is preferably composed of a peptide that differs from the peptide of SEQ ID NO.1 only in that it has one or more amino acid residue substitutions compared to SEQ ID NO.1, and thus the peptide has a length of 57 amino acid residues and contains one or more amino acid substitutions compared to SEQ ID NO.1.

[0099] Peptides whose sequence has 80% amino acid identity with SEQ ID NO.1 may contain up to 11 amino acid substitutions compared to SEQ ID NO.1. This invention covers peptides having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 substitutions of one amino acid residue compared to SEQ ID NO.1.

[0100] This invention therefore covers V2R antagonistic peptides comprising 80% or greater amino acid identity with SEQ ID NO. 1 and including one or more amino acid residues, peptide bonds, N- and / or C-termini modified to a protein, provided that V2R antagonist activity is maintained. These modifications of the peptide, introduced by conventional methods known to those skilled in the art, include, in a non-limiting manner: substitution of the native amino acid with a non-proteinogenic amino acid (D amino acid or amino acid analog); modification of the peptide bond, particularly with a trans or inverse bond or a bond different from a peptide bond; cyclization; and addition of chemical groups to the side chain or one or more ends of the protein, particularly for coupling agents of interest to the V2R peptide antagonists described herein. These modifications can be used to label V2R antagonistic peptides, or alternatively to further enhance their affinity for V2R, their bioavailability, and / or their stability.

[0101] In some embodiments, in the V2R antagonistic peptides disclosed herein, one or more amino acid residues targeted by endopeptides are replaced by their corresponding non-natural D-forms. For example, one or more arginine and / or lysine residues that are targets of trypsin may be replaced by their corresponding non-natural forms.

[0102] In some embodiments, in V2R antagonistic peptides as disclosed herein, one or more disulfide bridges are replaced by non-natural links. Preferably, the non-natural links are reduction-resistant, such as thiazolidinyl linkers. These linkers increase the resistance of the proteins of the present invention to reducing agents present in biological fluids.

[0103] Most preferably, the V2R antagonistic peptide according to the invention (covering peptides comprising one or more amino acid substitutions compared to SEQ ID NO. 1) comprises cysteine ​​residues located at amino acid positions 5 and 55 corresponding to SEQ ID NO. 1.

[0104] Most preferably, the V2R antagonistic peptide according to the invention (covering peptides comprising one or more amino acid substitutions compared to SEQ ID NO. 1) comprises cysteine ​​residues located at amino acid positions 14 and 38 corresponding to SEQ ID NO. 1.

[0105] Most preferably, the V2R antagonistic peptide according to the invention (covering peptides containing one or more amino acid substitutions compared to SEQ ID NO. 1) contains cysteine ​​residues located at amino acid positions 30 and 51 corresponding to SEQ ID NO. 1.

[0106] In some embodiments of the peptide disclosed herein, X1 represents amino acid residue G (glycine).

[0107] In some embodiments of the peptide disclosed herein, X2 represents amino acid residue A (alanine).

[0108] In some embodiments of the peptide disclosed herein, X3 represents amino acid D (aspartic acid).

[0109] In some embodiments of the peptide disclosed herein, X4 represents the amino acid residue E (glutamic acid) or L (leucine).

[0110] The present invention also relates to peptides comprising the amino acid sequence of SEQ ID NO.1, wherein each of X1 to X6 independently has the meaning described in relation to SEQ ID NO.1. The present invention further relates to peptides comprising the amino acid sequence of SEQ ID NO.1, wherein each of X1 to X6 independently has the meaning described in relation to SEQ ID NO.1.

[0111] The present invention also relates to peptides having 80% or greater amino acid identity with the amino acid sequence of SEQ ID NO.1, wherein X1 represents F, X2 represents N, X3 represents T, X4 represents I, and X5 and X6 have the same meaning as for SEQ ID NO.1. Most preferably, X5 represents V and X6 represents G. The present invention also relates to peptides having 80% amino acid identity with the peptide of SEQ ID NO.2, which covers peptides composed of SEQ ID NO.2.

[0112] The present invention also relates to peptides having 80% or greater amino acid identity with the amino acid sequence of SEQ ID NO. 1, wherein X1 represents F, X2 represents N, X3 represents T, X4 represents L, and X5 and X6 have the same meaning as for SEQ ID NO. 1. Most preferably, X5 represents V and X6 represents G. The present invention also relates to peptides having 80% or greater amino acid identity with the peptide of SEQ ID NO. 3. The present invention also relates to peptides consisting of the amino acid sequence of SEQ ID NO. 3.

[0113] The present invention also relates to peptides having 80% or greater amino acid identity with the amino acid sequence of SEQ ID NO. 1, wherein X1 represents F, X2 represents A, X3 represents T, X4 represents L, and X5 and X6 have the same meaning as for SEQ ID NO. 1. Most preferably, X5 represents V and X6 represents G. The present invention also relates to peptides having 80% or greater amino acid identity with the peptide of SEQ ID NO. 4. The present invention also relates to peptides consisting of the amino acid sequence of SEQ ID NO. 4.

[0114] The present invention also relates to peptides having 80% or greater amino acid identity with the amino acid sequence of SEQ ID NO. 1, wherein X1 represents G, X2 represents N, X3 represents D, and X4 represents I, and X5 and X6 have the same meaning as for SEQ ID NO. 1. Most preferably, X5 represents V and X6 represents G. The present invention also relates to peptides having 80% or greater amino acid identity with the peptide of SEQ ID NO. 5. The present invention also relates to peptides consisting of the amino acid sequence of SEQ ID NO. 5.

[0115] The present invention also relates to peptides having 80% or greater amino acid identity with the amino acid sequence of SEQ ID NO. 1, wherein X1 represents G, X2 represents N, X3 represents D, X4 represents E, and X5 and X6 have the same meaning as for SEQ ID NO. 1. Most preferably, X5 represents V and X6 represents G. The present invention also relates to peptides having 80% or greater amino acid identity with the peptide of SEQ ID NO. 6. The present invention also relates to peptides consisting of the amino acid sequence of SEQ ID NO. 6.

[0116] This invention also relates to compounds of formula (I):

[0117] [Nt-EXT]n-L1x-[V2R-AP]-L2y-[Ct-EXT]m(I),

[0118] in:

[0119] -[Nt-EXT] indicates a chemical moiety covalently linked to the N-terminus of a V2R antagonist peptide as disclosed herein.

[0120] -[V2R-AP] represents the V2R antagonistic peptide according to the present invention.

[0121] -[Ct-EXT] indicates a chemical moiety covalently linked to the C-terminus of a V2R antagonistic peptide as disclosed herein, and

[0122] Each of -n and m independently represents an integer equal to 0 or 1.

[0123] Each of L1 and L2 is a linker portion that, in its presence, prevents the inhibition of (i) the interaction between [Nt-EXT] and [V2R-AP] and (ii) the interaction between [Ct-EXT] and [V2R-AP], respectively. More importantly, each of L1 and L2, in its presence, can prevent either [Nt-EXT] or [Ct-EXT] from affecting the binding of the V2R antagonist peptide [V2R-AP] to V2R.

[0124] Each of -x and y independently represents an integer equal to 0 or 1.

[0125] In some preferred embodiments, [Nt-EXT] and / or [Ct-EXT], when present, independently represent a non-protein chemical moiety, such as a non-protein detectable molecule used to label V2R antagonistic peptides, like a radiochemical moiety or a fluorophore-containing chemical moiety, or a stable moiety (e.g., ZZ, DsBa, and DsBb). In some of these embodiments, [Nt-EXT] and / or [Ct-EXT] is an agent that increases the bioavailability of V2R antagonistic peptides as described herein and, in particular, reduces their urinary elimination, such as a polyethylene glycol molecule. In some preferred embodiments, [Nt-EXT] and / or [Ct-EXT], when present, may independently represent a polyalkylene glycol, especially polyethylene glycol. These preferred embodiments cover polyethylene glycol of the formula H-(O-CH2-CH2)nO-, where n is an integer ranging from 5 to 20, advantageously 10 to 15, for example, n consisting of an integer representing 12.

[0126] In some other preferred embodiments, [Nt-EXT] and / or [Ct-EXT], when present, independently represent protein moieties that encompass protein / peptide moieties, including those that allow purification, detection, immobilization, and / or cellular targeting of the proteins of the present invention, and / or increase the affinity of V2R antagonistic peptides for V2R, bioavailability, production in expression systems, and / or stability. These protein moieties may be selected from: (i) labeled moieties, such as fluorescent proteins (GFP and its derivatives, BFP, and YFP), (ii) reporter moieties, such as enzyme labels (luciferase, alkaline phosphatase, glutathione transferase (GST), β-galactosidase), (ii) binding moieties, such as epitope tags (polyHis6, FLAG, HA, myc), DNA-binding domains, hormone-binding domains, poly-lysine tags for immobilization on a support, and (iii) targeting moieties of the compound of formula (I) for specific cell types or cell partitions. Furthermore, the compounds of formula (I) advantageously include linkers (L1, L2) that connect each of [Nt-EXT] and / or [Ct-EXT] to [V2R-AP], said linkers being long enough to avoid inhibiting the interaction between the sequence [V2R-AP] and each of [Nt-EXT] and / or [Ct-EXT] (in the presence of the linker). Linkers L1 and / or L2 may also contain recognition sites for proteases, for example for removing affinity tags and stabilizing moieties from the purified chimeric protein according to the invention.

[0127] In some embodiments, the linker portions L1 and / or L2 may include, for example, recognition sites for proteases, for removing affinity tags and stabilizing portions from compounds of formula (I) according to the invention.

[0128] Furthermore, V2R antagonistic peptides as described herein or compounds of formula (I) of some embodiments can be advantageously modified by methods well known to those skilled in the art to alter their physiological properties and, in particular, to improve their half-life in organisms (glycosylation: HAUBNE R. et al., J. Nucl. Med., 2001, 42, 326-36; conjugation with PEG: KIMTH. et al., Biomaterials, 2002, 23, 2311-7), their solubility (hybridization with albumin: KOEHLER MF. et al., Bioorg. Med. Chem. Lett., 2002, 12, 2883-6), their resistance to proteases (non-natural amino acids (D conformation, e.g.)), and / or their intestinal absorption (Lien et al., TIB, 2003, 21, 556-).

[0129] Synthesis of V2R antagonistic peptides

[0130] The V2R antagonistic peptides described herein can be produced using known cloning techniques or through chemical synthesis.

[0131] For example, DNA encoding a V2R antagonistic peptide is prepared using cloning techniques and inserted into a self-replicating vector to prepare recombinant DNA. The recombinant DNA is introduced into a suitable host, such as *Escherichia coli*, *Bacillus subtilis*, *Actinomyces*, yeast, filamentous fungi, plant cells, insect cells, and animal cells, to obtain transformants. From the culture products of the transformants, the V2R antagonistic peptide as disclosed herein can be obtained. Alternatively, DNA encoding a V2R antagonistic peptide is prepared and used in a decellularized protein synthesis system using cell extracts of wheat germ and *Escherichia coli* to synthesize the peptide according to the invention.

[0132] This invention covers the use of nucleic acids encoding V2R antagonistic peptides as described herein, or recombinant vectors containing said nucleic acids, in an expressible form. Nucleic acids encoding V2R antagonistic peptides in an expressible form refer to nucleic acid molecules that, upon expression in cellular or cell-free systems, produce functional peptides.

[0133] In fact, in embodiments where the compound of formula (I) is composed of a protein, the compound of formula (I) can also be produced as a recombinant protein using the same method as described above.

[0134] Furthermore, conventional chemical synthesis methods for V2R antagonist peptides, such as those disclosed herein, including solid-phase or liquid-phase methods, are used to sequentially link and elongate amino acids via dehydration / condensation. Methods for synthesizing V2R antagonist peptides using chemical synthesis are described in the examples herein.

[0135] Pharmaceutical Composition

[0136] The present invention also relates to the use of the V2R antagonistic peptide described herein or compounds of formula (I) in some embodiments (including the V2R antagonistic peptide) for medical purposes and, in particular, for the treatment of pathological diseases involving the V2R pathway.

[0137] This invention also covers the use of nucleic acids encoding certain protein embodiments as described herein, or recombinant vectors containing said nucleic acids, in an expressible form. Nucleic acids encoding such proteins in an expressible form refer to nucleic acid molecules that, when expressed in cellular or cell-free systems, produce functional proteins, i.e., proteins composed of V2R antagonists.

[0138] Therefore, according to the present invention, the protein, the nucleic acid and / or the recombinant vector may be included in a pharmaceutical composition that also includes a pharmaceutically acceptable carrier.

[0139] The pharmaceutical compositions according to the invention can be formulated for administration via a variety of routes, including but not limited to, enteric (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, subcutaneous, transdermal, intradermal, rectal, vaginal, intraperitoneal, local, mucosal, nasal, buccal, sublingual; and / or inhalation; and / or as oral sprays, nasal sprays and / or aerosols.

[0140] In some embodiments, the pharmaceutical composition according to the invention may be in any suitable form covered by the art, such as an injectable solution or suspension, tablets, coated tablets, capsules, syrups, suppositories, creams, ointments, lotions, etc.

[0141] Pharmaceutical compositions containing V2R antagonistic peptides or selected compounds of formula (I) can be presented as lyophilized powders, wherein the active ingredient is bound to a sugar, such as mannitol. For use, such pharmaceutical compositions are typically reconstituted with an appropriate volume of water or sodium chloride solution. The resulting liquid pharmaceutical composition can then be administered via an appropriate route of administration.

[0142] In a pharmaceutical composition, a V2R antagonistic peptide or a selected compound of formula (I) is combined with one or more pharmaceutically acceptable carriers and optional sustained-release matrix (e.g., a biodegradable polymer) to form a therapeutic composition.

[0143] In some embodiments, the pharmaceutical composition may also contain one or more salts, one or more buffers, and / or one or more preservatives.

[0144] Pharmaceutically acceptable carriers are those that are known to technicians and are routinely used.

[0145] Within the scope of this invention, "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or medium, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting a preventive or therapeutic active agent.

[0146] In some embodiments, suitable pharmaceutically acceptable carriers may be selected from the group consisting of: sugars, such as lactose, glucose, and sucrose; ethylene glycols, such as propylene glycol; polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; buffers, such as magnesium hydroxide and aluminum hydroxide; pyrogen-free water; isotonic saline; Ringer's solution; ethanol; phosphate buffer solution; etc.

[0147] The pharmaceutical compositions according to the invention comprise a therapeutically effective amount of the V2R antagonist peptide described herein, or a compound of formula (I) of selected embodiments, or a nucleic acid or recombinant vector encoding thereof, for example, an amount sufficient to produce a medical benefit to the individual administering it. The effective dosage of the drug depends on the type of composition used, the route of administration, whether the administration is a single or multiple dose, the type of mammal being treated (human or non-human mammal), parameters of the individual's physical characteristics, including age, physical condition, size, weight, concurrent medication, and other factors that a person skilled in the medical field would recognize. Therefore, within the scope of the invention, the effective amount of the active ingredient to be administered, particularly the V2R antagonist peptide or the selected compound of formula (I), can be determined by a physician or a licensed person skilled in the art and can be appropriately adjusted during the course of treatment.

[0148] The V2R antagonist peptide or a selected compound of formula (I) may be administered in an amount ranging from 0.1 to 300 nanomoles of the selected V2R antagonist peptide per kg body weight per administration step, depending on the individual parameters described above. The inventors believe that administration of the V2R antagonist peptide as disclosed herein at amounts below 0.1 nanomoles per kg body weight would not be therapeutically effective. The inventors also believe that administration of the V2R antagonist peptide as disclosed herein at amounts above 300 nanomoles per kg body weight may lead to adverse effects and may result in some toxicity in subjects.

[0149] Indeed, the exact molecular weight (Mw) of each of the disclosed V2R antagonistic peptides is always determinable.

[0150] Assuming the Mw of the V2R antagonist peptide according to the invention is 6500, administering 0.1 nanomoles / kg of it to a subject weighing 80 kg consists of administering 52 ng of the peptide to the subject. Still assuming the Mw of the V2R antagonist peptide according to the invention is 6500, administering 300 nanomoles / kg of it to a subject weighing 80 kg consists of administering 0.156 mg of the peptide to the subject.

[0151] For clarity, in some embodiments where the selected active ingredient comprises a V2R antagonistic peptide according to the invention, the amount of the active ingredient applied to a desired subject is calculated based on the Mw value of the V2R antagonistic peptide contained therein, rather than based on the Mw value of the active ingredient itself. For example, in some embodiments where the selected active ingredient is a compound of formula (I) disclosed herein, the amount of the active ingredient applied to a desired subject is calculated based on the Mw value of the [V2R-AP] portion contained therein, rather than based on the Mw value of the active ingredient itself.

[0152] As used herein, 0.1 nmol / kg or greater of the selected V2R antagonist peptide encompasses 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, or 20 nmol / kg or greater of the selected V2R antagonist peptide.

[0153] As used herein, the amount of 300 nmol / kg or less of the selected V2R antagonist peptide encompasses amounts of 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 150 nmol / kg or less of the selected V2R antagonist peptide.

[0154] Medical use

[0155] This invention relates to the use of the V2R antagonistic peptide described herein or selected compounds of formula (I) described herein in the preparation of pharmaceutical agents. This invention also relates to the use of the V2R antagonistic peptide described herein or selected compounds of formula (I) described herein as pharmaceutical agents.

[0156] The present invention also relates to a method for treating a subject suffering from a pathological disease involving the V2R pathway, comprising the step of administering the V2R antagonistic peptide described herein, or a selected compound of formula (I) described herein, or a nucleic acid or recombinant vector encoding the thereof to the subject.

[0157] The present invention also relates to the use of the V2R antagonistic peptide described herein, or selected compounds of formula (I) described herein, or nucleic acids or recombinant vectors encoding the thereof, in the preparation of medicaments for treating subjects suffering from pathological diseases involving the V2R pathway.

[0158] The present invention also relates to the use of the V2R antagonistic peptide described herein, or selected compounds of formula (I) described herein, or nucleic acids or recombinant vectors encoding the thereof, for the treatment of subjects suffering from pathological diseases involving the V2R pathway.

[0159] In some embodiments, the active ingredient, namely the V2R antagonistic peptide or selected compound of formula (I) disclosed herein, may be administered as a single bolus, as a series of fractionated doses administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. For ease of administration and dose uniformity, formulation of the therapeutic agent in dose units may be particularly advantageous. As used herein, dose unit form refers to physically discrete units suitable as a single dose for use on a mammalian subject to be treated, each unit containing a predetermined amount of active compound calculated to combine with the desired drug carrier to produce the desired therapeutic effect. The specifications of the dose unit form of the present invention may be determined by and directly dependent on: (a) the unique characteristics of the active ingredient and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the art of combining such active ingredients to treat individual sensitivities.

[0160] Therefore, those skilled in the art will understand that, based on the disclosure provided herein, the dosage and administration regimen are adapted according to methods well known in the therapeutic field. That is, the maximum tolerable dose can be readily determined, as can the effective amount that provides a detectable therapeutic benefit to the subject, and the timing requirements for administering each agent to provide that detectable therapeutic benefit to the subject.

[0161] Determining the appropriate dosage and regimen for administering chemotherapeutic agents is well known in the relevant field and, once the teachings disclosed herein are provided, will be understood to be covered by those skilled in the art.

[0162] Symptoms involving the V2R pathway include, but are not limited to: (i) pathological diseases characterized by positive or hypovolemic hyponatremia, such as congestive heart failure (CHF), cirrhosis, syndrome of dysregulation of antidiuretic hormone secretion (SIADH), and cerebral edema; (ii) inappropriate antidiuretic nephrogenic syndrome (NSIAD); (iii) congenital nephrogenic diabetes insipidus (cNDI); (iv) polycystic kidney disease; (v) cancer, including renal cell carcinoma and lung cancer; (vi) thrombosis; (vii) Meniere's disease; (viii) refractory liver disease; and (ix) heart failure.

[0163] Diagnostic

[0164] According to another aspect, the present invention relates to the use of selected compounds of V2R antagonistic peptides or alternative formula (I) as diagnostic or imaging agents applicable to optical imaging, magnetic resonance imaging (MRI), and positron emission tomography (PET) for detecting V2R cell expression under physiological or pathological conditions or in response to endogenous or exogenous stimuli for diagnostic or research purposes. It can also be used as a drug screening tool for screening V2R ligands, including V2R agonists and antagonists.

[0165] In a preferred embodiment, the V2R antagonistic peptide is labeled by coupling to a detectable molecule.

[0166] The present invention also relates to labeled V2R antagonistic peptides as described herein.

[0167] Conventional detectable molecules (tags) capable of providing a detectable signal, alone or in combination with other compositions or compounds, can be used. Suitable detection methods include, for example, detection of agents directly or indirectly labeled with fluorescent tags by immunofluorescence microscopy (including confocal microscopy) or flow cytometry (FACscan); detection of radiolabeled agents by autoradiography; electron microscopy; immunostaining; subcellular fractionation, etc. In some embodiments, a radioactive element (e.g., a radioactive amino acid) is directly incorporated into the peptide chain; in other embodiments, the fluorescent tag may associate with the peptide via biotin / avidin interaction, associate with a fluorescein-conjugated peptide, etc.

[0168] In embodiments of the invention, the detection procedure includes visually inspecting color changes of the labeling agent or examining physicochemical changes in the labeled V2R peptide antagonist. Physicochemical changes may occur alongside oxidation or other chemical reactions. They can be detected with the eye, using a spectrophotometer, or the like.

[0169] Therefore, useful detectable molecules for labeling the V2R antagonistic peptides described herein are preferably labeling agents that generate detectable and / or quantifiable signals, particularly radioactive, magnetic, or luminescent (radioluminescence, chemiluminescence, bioluminescence, fluorescence, or phosphorescence) agents. The labeled protein can be labeled directly or indirectly via covalent or non-covalent bonds using standard conjugation techniques well known to those skilled in the art. Examples of labeling agents include radioactive isotopes such as technetium-99 (…). 99 Tc), Fluorine-18 ( 18 F), tritium ( 3 H) and iodine-125 ( 125 I); luminescent agents such as AlexaFluor, FITC and cyanine 3; paramagnetic contrast agents such as gadolinium compounds and superparamagnetic contrast agents such as iron oxide nanoparticles.

[0170] For clarity, the V2R antagonistic peptide as disclosed herein coupled to the detectable molecule may consist of a compound of formula (I) of the embodiments, wherein at least one of [Nt-EXT] or [Ct-EXT] (i.e., at least one of integers m or n represents 1), and includes or consists of the detectable molecule.

[0171] In some preferred embodiments, the V2R antagonistic peptide is covalently linked to a radioactive or fluorescent agent in the labeling agent.

[0172] Covalent coupling of a labeling agent (e.g., a fluorescent agent or a radioactive agent) to a V2R antagonistic peptide can be achieved by (i) incorporating the labeling agent at the N-terminus or C-terminus of the protein during its chemical synthesis, or (ii) incorporating a reactive group (free cysteine, biotinyl, or azide moiety) into a recombinant or synthetic protein and then covalently linking the labeling agent using that group.

[0173] Preferably, the labeler is covalently linked to the N- or C-terminus of the protein, since neither the N-terminus nor the C-terminus of the V2R antagonist peptide is involved in its binding to V2R.

[0174] This invention relates to the use of the labeled V2R antagonist peptide described herein for the detection of V2R-expressing cells in vitro or in vivo. The invention also relates to the use of the labeled V2R antagonist peptide described herein for the measurement of V2R expression levels in cells expressing V2R in vitro or in vivo.

[0175] The present invention also relates to an in vitro method for detecting regulatory abnormalities in the expression of vasopressin-2 receptor on cells, comprising the following steps:

[0176] a) Provide the cells to be tested.

[0177] b) Contact the cells provided in step a) with the labeled V2R antagonistic peptide according to the invention.

[0178] c) Measure the expression level of vasopressin-2 receptor in the cells provided at the end of step b).

[0179] d) Compare the expression level value obtained in step c) with the reference expression level value.

[0180] e) Identify abnormalities in the regulation of cellular expression of vasopressin g-2 receptor.

[0181] In some implementations, the reference expression level value used in step d) consists of the expected average V2R expression level value in cells of subjects who do not have abnormal V2R cell expression regulation (i.e., subjects who do not have a condition or disease involving the V2R pathway).

[0182] In some other embodiments, the reference expression level value used in step d) consists of known or otherwise determined or determinable V2R expression values ​​in the cells of subjects with abnormal regulation of V2R cell expression (i.e., subjects with a condition or disease involving the V2R pathway).

[0183] As is well known in the art, ectopic expression of AVP and its receptor has been reported in various cancers (North et al., 1999, Peptides, Vol. 20: 837-842; Sinha et al., 2019, Oncogene, Vol. 30(6): 1231-1245), among which V2R agonists have potential antiproliferative effects in breast cancer, pancreatic cancer, colorectal cancer, and lung cancer (Garona et al., 2015, Int J Oncol, Vol. 46: 2335-2345; Ripoll et al., 2013, Breast Cancer Res Treat, Vol. 142: 9-18; Iannucci et al., 2011, Future Med Chem, Vol. 3: 1987-1993; Garona et al., 2018, Cancer Res Treat, Vol. 51(2): 438-450; Pifano et al., 2017, Front Oncol, 7:11; Garona et al., 2020, Vitamins and Hormones, Vol. 113: 259-289), and V2R antagonists have antiproliferative effects in human renal cell carcinoma (Sinha et al., 2019, Oncogene, Vol. 30(6): 1231-1245). In this context, the V2R antagonistic peptide according to the invention can readily form a probe of interest for imaging cancer cells overexpressing V2R.

[0184] Another subject of the invention relates to the use of V2R antagonistic peptides as described herein in in vitro diagnostic methods.

[0185] Another subject of the present invention is the in vitro or in vivo use of the V2R antagonistic peptides described herein or the compounds of formula (I) selected herein for the diagnosis of conditions involving elevated or decreased V2R expression levels.

[0186] Another subject of this invention is the in vitro or in vivo use of the V2R antagonistic peptides or compounds of formula (I) described herein for the diagnosis of conditions involving elevated or decreased V2R expression levels.

[0187] For certain diagnostic applications, labeled V2R antagonist peptides are used to visualize V2R expression in patient tissues in vitro or in vivo and to evaluate expression levels by comparison with the same type of tissue from healthy individuals. V2R overexpression indicates pathological conditions such as cancer, while V2R low expression indicates pathological conditions such as congenital nephrogenic diabetes insipidus (cNDI). Once a diagnostic agent has been identified, effective treatments can be determined for diagnosed patients, including the use of V2R antagonists, for example, to treat cancer or cNDI.

[0188] One subject of this invention is the use of V2R antagonistic peptides as research tools for studying V2R.

[0189] Another subject of the present invention is a method for detecting V2R in vitro or in vivo, comprising the following steps:

[0190] a) Contact the cells to be analyzed with the labeled V2R antagonist peptide, and

[0191] b) Detect labeled cells.

[0192] Cell labeling, particularly fluorescent or magnetic labeling, is detected using any technique known to those skilled in the art (fluorescence microscopy, flow cytometry, magnetic resonance imaging).

[0193] In vivo detection (cell imaging), particularly real-time detection, of V2R receptors in mammals includes a preliminary step (parenteral injection, oral administration) of the labeled V2R antagonistic peptide to the mammal.

[0194] Another subject of this invention is the use of the V2R antagonistic peptides disclosed herein for screening V2R ligands.

[0195] Another subject of this invention is a method for screening V2R ligands, comprising:

[0196] a) Incubate V2R with the test molecule of the present invention and the labeled V2R antagonist peptide, and

[0197] b) Measure the signals obtained in the presence and absence of the test molecule, where the signal is lower in the presence of the molecule compared to the control in the absence of the test molecule, indicating that the test molecule is a V2R ligand.

[0198] The identified ligands act as agonists or antagonists of V2R, and are then used in V2R-expressing cells with pharmacological assays well known in the art, such as those disclosed in the embodiments of this application.

[0199] The present invention also provides a kit comprising: (a) a first container containing one or more of the following: a V2R antagonistic peptide as described herein, a nucleic acid or recombinant vector encoding the antagonist peptide, a modified host cell, a pharmaceutical composition, a diagnostic reagent or an imaging reagent, in solution or lyophilized form; (b) optionally, a second container containing a diluent or reconstitution solution for the lyophilized formulation; (c) optionally, a third container containing an isolated V2R receptor or host cell capable of expressing V2R (in solution or lyophilized form), and optionally instructions for use in one or more solutions and / or for reconstitution and / or use in one or more lyophilized formulations.

[0200] The present invention is further illustrated by the embodiments described herein, but is not limited in any way to the embodiments described herein.

[0201] Example

[0202] Example 1: Activity of V2R antagonistic peptide

[0203] A. Materials and Methods

[0204] Chemical synthesis of peptides.

[0205] Peptide synthesis was performed at a scale of 12.5 μmol on a Gyros Protein Technologies, Inc. Prelude synthesizer, and deprotection, purification and folding were carried out as described (Ciolek et al., 2017, Proc Natl Acad Sci USA, Vol. 114: 7154-7159).

[0206] Peptide batches with a purity higher than 95% were used throughout the experiment. Following automated synthesis of MQ1 and deprotection of the N-terminal amine functional groups, 6-azidohexanoic acid was coupled to a resin. Two equivalents of 6-azidohexanoic acid were coupled twice with the coupling agent HCTU (1.9 equivalents) in the presence of two equivalents of diisopropylethylamine for 60 minutes each time. 6-azidohexanoic acid-MQ1 was isolated from the resin, purified, and oxidized as with MQ1 V2R peptide antagonists. 10 equivalents of DFO-DBCO (p-isothiocyanate benzyl ferrochloride-diarylbicyclooctylene, dissolved in 200 μl DMF) or Cy5.5-DBCO (dissolved in 200 μl HEPES buffer) or AFDye-488-DBCO (dissolved in 200 μl HEPES buffer) were mixed with 0.3 μmol of 6-azidohexanoic acid-MQ1 dissolved in 200 μl HEPES buffer (pH 7.4), and the mixture was left to stand overnight at room temperature and purified by HPLC.

[0207] B. Result

[0208] The binding affinity of various MQ1-derived peptides to human angiotensin-2 receptor (V2R) has been tested.

[0209] The results are summarized in Table 1 below.

[0210] Table 1

[0211]

[0212] If there are differences between the sequences disclosed in Table 1 and those described in the sequence listing, then the correct sequences are those listed in Table 1.

[0213] The results depicted in Table 1 indicate that, compared to known MQ1 V2R antagonist peptides, it exhibits IC50. 50 The most effective peptides tested with a ratio of 0.5 or less are all peptides in which the lysine residue at amino acid position 39 in known MQ1 V2R antagonist peptides is replaced by an alanine residue (amino acid change is usually indicated as "K39A"). The most effective V2R antagonist peptides are as follows:

[0214] -The "K39A" peptide of the amino acid sequence of SEQ ID NO.2,

[0215] -The "K39A+I48L" peptide with the amino acid sequence of SEQ ID NO.3

[0216] -The amino acid sequence of SEQ ID NO.4 is the "N15A+K39A+I48L" peptide.

[0217] -The amino acid sequence of SEQ ID NO.5 is the "F4G+K39A+T27D" peptide.

[0218] The amino acid sequence of SEQ ID NO. 6, “F4G+K39A+T27D I48E” peptide, is also referred to herein as “MQ-LEAD”.

[0219] Example 2: Immunogenicity and selectivity of V2R antagonistic peptides

[0220] A. Materials and Methods

[0221] A.1. Immunogenicity

[0222] Immunogenicity of therapeutic proteins remains a major risk factor for development failure and is therefore a critical issue for the pharmaceutical industry. Immunogenicity is the ability of a molecule to induce an immune response. Antibodies generated from this response (anti-drug antibodies or ADAs) can inhibit the therapeutic activity of the protein or even induce allergic symptoms. Because immune responses are species-dependent, animals, such as mice and rats, are very poor models for predicting immunogenicity and do not allow for the evaluation of the immunogenicity of the molecule when injected into humans. Preclinical evaluation of immunogenicity is based on the mechanisms that regulate the human immune response.

[0223] The response of ADA to therapeutic proteins primarily involves three distinct cell types: i) antibody-producing B cells, ii) CD4 T cells, which provide the necessary assistance for B cells to secrete antibodies, and iii) dendritic cells, which activate T cells by presenting the protein as a peptide. When a protein is injected into the body, it is absorbed by dendritic cells and broken down into peptides. Some of the resulting peptides have appropriate anchor residues to bind to HLA class II molecules and are presented to T cells. Peptides recognized by T cells are called T epitopes. Because HLA class II molecules are polymorphic, T epitopes vary from molecule to molecule and therefore differ between individuals depending on the HLA class II molecule.

[0224] Given the role of T lymphocytes in the ADA response, the immunogenicity of therapeutic proteins is highly dependent on their ability to activate T lymphocytes, thus requiring the presence of T epitopes in their sequences. The evaluation of T epitope content can be performed using predictive software, with NETMHC being the most effective. The presence of potential T epitopes in U-Da2a and MQ-LEAD was tested using NETMHC software.

[0225] A.2. Selectivity

[0226] The general protocol for protease assay is as follows:

[0227] 1 Delivery of 2X enzyme

[0228] 2. Delivery buffer to enzyme-free wells

[0229] 3. Delivery of MQ-WT (U-Da2a) in water using Acoustic technology (Echo550; nanoliter range)

[0230] 4. Incubate for 5-15 minutes

[0231] 5. Delivery of 2X substrate to trigger application

[0232] 6. Rotation and oscillation, begin measurement in EnVision at room temperature; repeat 25 times at 5-minute intervals (2 hours).

[0233] 7. Analyze the data by taking the slope of the linear portion of the measurement multiplied by (signal / time).

[0234] 8. Calculate the slope using Excel and perform curve fitting using Prism software.

[0235] Specific specifications are disclosed in Table 2 at the end of this invention.

[0236] buffer solution :

[0237] A 25mM Tris pH 8.0, 100mM NaCl, 0.01% Brij35,

[0238] B'25mM MES pH 6, 50mM NaCl, 0.005% Brij35, 5mM DTT

[0239] B 75mM Tris pH 7.0, 0.005% Brij35, 3mM DTT

[0240] B+EDTA 75mM Tris pH 7.0, 1mM EDTA, 0.005% Brij35, 3mM DTT

[0241] C 100mM Tris-HCl, pH 8.0, 50mM NaCl, 10mM CaCl2, 0.025% CHAPS, 1.5mM DTT

[0242] D 25mM sodium acetate pH 5.5, 0.1M NaCl, 5mM DTT

[0243] E 25mM sodium acetate pH 3.5, 5mM DTT

[0244] F 25mM Tris pH 9, 150mM NaCl

[0245] L 400mM sodium acetate pH 5.5, 4mM EDTA, 8mM DTT

[0246] X (furin protease) 100mM Tris-HCl, pH 7.5, 1mM CaCl2, 0.5% TX-100, 1mM DTT

[0247] S 0.1M sodium acetate pH 3.5, 0.1M NaCl

[0248] TCN 25mM Tris pH 7.5, 10mM CaCl2, 150mM NaCl

[0249] TCNB 25mM Tris pH 7.5, 10mM CaCl2, 150mM NaCl, 0.05% Brij35

[0250] Z 25mM Tris pH 9, 2.5uM ZnCl2, 0.005% Brij

[0251] KLK 7 buffer: 50mM Tris, 150mM NaCl, pH 8.5

[0252] ACE2 buffer: 75mM Tris, pH 8.5, 1M NaCl

[0253] Enkephalin buffer, 50 mM Tris, pH 9.0

[0254] CTSD buffer: 100mM sodium acetate, pH 3.5, 200mM NaCl, 0.02% Brij35

[0255] CTSE buffer: 100mM sodium acetate, pH 3.5, 500mM NaCl, 0.005% Triton X-100

[0256] Elastase buffer 50mM Tris, pH 7.5, 1M NaCl, 0.05% Brij35

[0257] 1X Caspase Buffer: 1 50mM HEPES pH 7.4, 100mM NaCl, 0.01% CHAPS, 0.1mM EDTA, 10mM DTT

[0258] 1X Caspase Buffer 2: 50mM HEPES pH 7.4, 1M Sodium Citrate, 100mM NaCl, 0.01% CHAPS, 0.1mM EDTA, 10mM DTT

[0259] MMP buffer: 50 mM HEPES (pH 7.5), 10 mM CaCl2, 0.01% Brij-35, stored at 4°C. Add 0.1 mg / ml BSA to the buffer before use.

[0260] B. Result

[0261] B.1. Immunogenicity

[0262] The expected immunogenicity of the known MQ1 peptide of SEQ ID NO.7 and the MQ LEAD peptide of SEQ ID NO.6 has been calculated.

[0263] The comparison results are depicted in Figure 1 middle.

[0264] As it is Figure 1 As shown, the immunogenic regions present in the known MQ1 peptide are not evident in the MQ LEAD V2R antagonistic peptide.

[0265] On MQ1(U-Da2a), 15 potential epitopes were identified, including 6 epitopes with high scores (percentiles below 10%), compared to only 3 potential T epitopes identified in the MQ-LEAD sequence. These 3 potential T epitopes also had low scores (percentiles of 20% to 30%).

[0266] Then, the MQ-LEAD peptide will present a much lower risk of immunogenicity than the known MQ1 peptide.

[0267] B.2. Selectivity

[0268] The selectivity of the known MQ1 peptide for the angiotensin-2 receptor has been tested by measuring its binding to a large number of molecules.

[0269] The results are described in Table 3 at the end of this invention.

[0270] The results in Table 3 show the selectivity of the MQ1 peptide for the angiotensin-2 receptor.

[0271] Example 3: Pharmacology of V2R antagonistic peptides

[0272] A. Materials and Methods – V2R Combined Determination

[0273] Membranes from cells expressing angiotensin receptors were purchased from PERKINELMER (Cottage, France). 3 H-AVP (PERKINELMER, Cortabov, France) was used in binding assays in 96-well plates. The reaction mixture contained 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, and 1 g / L BSA, with a final volume of 100 μL. The plates were incubated at room temperature for 3 hours. The binding reaction was terminated by filtration through a GF / C filter pre-soaked in 0.5% polyethyleneimine on a cell collector (PERKINELMER, Cortabov, France), and the plates were dried. Ultimagold O (25 μl; PERKINELMER) was added to each well, and the samples were counted using a TopCount counter (PERKINELMER, Cortabov, France) (calculated yield 55%). Nonspecific binding was measured in the presence of 1 μM AVP. The unit point inhibition mass effect curve was fitted to the inhibition binding data using Kaleidagraph (Synergy software, Reading, PA, USA). IC50 was used to measure the binding mass effect. 50 The value was converted to K using the Cheng-Prusoff equation (Cheng et al., Biochem. Pharmacol., 1973, 22, 3099-3108). i Used for competitive experiments.

[0274] B. Result

[0275] The V2R antagonistic peptide according to the present invention binds to V2R

[0276] The results are depicted in Figure 2 middle.

[0277] Figure 2 The results showed that the MQ-LEAD peptide according to the present invention has a 5-fold increased affinity for the angiotensin-2 receptor compared to the known MQ1 (U-Da2a) peptide. Even better results were obtained using the V2R antagonist peptide MQ K39A.

[0278] These results show that the V2R antagonistic peptides MQ-LEAD and MQ K39A are strong V2R antagonistic peptides.

[0279] Example 4: In vivo activity of the V2R antagonist according to the present invention

[0280] A. Materials and Methods

[0281] A.1. In vivo assay of diuresis

[0282] Sprague Dawley rats aged 6 to 12 weeks were acclimatized for two days in metabolic cages (Techniplast France, Lyon, France) with free access to food and water, followed by intraperitoneal injections (1 ml fixed volume) of various MQs at 3 nmol / kg dissolved in 0.9% NaCl (French protocol number 2015082111349702v1). Urine was collected at different time points and centrifuged at 20,800g for 30 minutes. Urine osmolality was measured using an osmometer (Knauer, Berlin, Germany).

[0283] A.2. In vivo assays of a hyponatremia experimental model

[0284] A rat model of hyponatremia was established. Adult male Sprague-Dawley rats (weighing 445 to 525 grams) without specific pathogens were obtained from the Janvier laboratory (Le Genest St. Isle, 53941, Saint-Béte-Védex, France) and acclimatized to animal laboratory conditions for one week. An experimental protocol derived from a previous paper (Miyazaki T, Yamamura Y, Onogawa T, Nakamura S, Kinoshita S, Nakayama S et al., Therapeutic effects of tolvaptan, a potent, selective nonpeptide vasopressin V2 receptor antagonist, in rats with acute and chronic severe hyponatremia. Endocrinology. 2005; 146:3037–43.) was established to develop a rat model of hyponatremia, and was approved by the French Ministry of Education and Research (No. 18604-2019010915104191). A 2.2 mg / mL desmopressin (dDAVP) stock solution was prepared by dissolving 5 mg of the drug in physiological saline. dDAVP was administered using a subcutaneous ALZET osmotic micropump (model 2002, DURECT Corporation, Cupertino, CA95014, USA) pre-filled with dDAVP solution (pump rate 0.47 ± 0.02 μL / hour, average filling volume 233.6 ± 4.7 μL). The dDAVP dose was determined in the initial experiment (10 ng / hour). Buprenorphine (Centravet, 03120 Laparis, France) was administered subcutaneously once daily from day 0 to day 3 to suppress postoperative pain. Water was administered twice daily by gavage (30 mL / kg) for the first 3 days after ALZET pump implantation (9:00 AM and 4:00 PM) and at 9:00 AM on day 4. Body weight was recorded daily to adjust the volume of water to be administered. Rats had free access to standard rat food and water in their cages. MQ1 dissolved in physiological saline was administered at 10:00 AM on days 2-3-4 at a dose of 10 or 100 μg / kg (sc route, 0.8 mL / kg). At 9:00 AM on days 0, 2, 3, and 4, 400 μL of blood containing lithium heparin (Sanofi-Aventis, Gentilly, France) was collected from the tail vein of isoflurane-anesthetized rats. The samples were centrifuged (4°C, 2000 g, 5 min).Sodium quantification was performed at the Central Laboratory of the ENVT in Toulouse, France, using VITROS 250 / 350 / 950 / 5, 1FS, 4600 and the integrated system VITROS 5600 (Ortho-Clinical Diagnostics, Buckinghamshire, UK). P < 0.05, multivariate ANOVA followed by Tukey's test.

[0285] B. Result

[0286] B.1. The diuretic effect of the two V2R antagonistic peptides according to the present invention

[0287] The results are depicted in Figure 3. Figure 3A and 3B The results in Figure 3 show that both the MQ-LEAD and MQ K39A peptides of the present invention increased diuresis.

[0288] B.2. The effect of the V2R antagonistic peptide of the present invention on hyponatremia.

[0289] The aim of this study was to test the effect of a peptide derived from mambaquaretine (MQ-LEAD), a potent and selective antagonist of the V2 receptor, on DDAVP-induced hyponatremia in normal adult male CD strain rats (Sprague Dawley).

[0290] MQ-LEAD peptide has been validated in a rat model of hyponatremia. The in vivo assay timeline is depicted as follows: Figure 4 middle.

[0291] The results are depicted in Figure 5 middle.

[0292] Figure 5 The results described showed that on day 4, the effects of tolvaptan (10 mg / kg, oral route) differed significantly from those in the carcass-treated group. The mean sodium level in the tolvaptan treatment group was 137.6 ± 3.3 mM. On day 4, plasma sodium concentrations at the three doses of MQ-LEAD (20, 60, and 200 μg / kg, sc) were significantly higher than the corresponding values ​​in the carcass-treated group. A direct comparison with the tolvaptan dose (10 mg / kg, po) indicated that the potency of MQ-LEAD could be 500 times that of tolvaptan. While sodium levels increased after MQ-LEAD at 20 and 60 μg / kg on days 3 and 4, there was no statistically significant difference compared to baseline (day 0).

[0293] Example 5: Inhibition of cAMP production by the V2R antagonistic peptide of the present invention

[0294] A. Materials and Methods

[0295] A.1. The antagonistic effects of MQ-WT, MQ-18 (K39A), and MQ-232 (MQ-LEAD) in the U2OS AVPR2A Nomad cell line stably expressing human arginine angiotensin were examined.

[0296] The U2OS AVPR2 Nomad cell line contains U2OS cells that stably express untagged human arginine angiotensin receptor 2A (AVPR2A). This cell line was designed to assay compounds or analyze their ability to regulate arginine angiotensin receptor 2A (AVPR2A). When an agonist binds to AVPR2, the Gs protein is activated, which in turn triggers a cAMP-mediated cellular response. This cellular response can be measured by quantifying the increase in fluorescence intensity and its distribution within the cells.

[0297] A.2. Receptor 2A (V2R). Using 1 nM human Arg8-vasopressin as an agonist, the test items were measured at eight concentrations (in triplicate, from 1 μM to 1 nM) using a fluorescence-based assay.

[0298] - Day 1. Thaw Nomad AVPR2 U2OS cell line (2 x 10⁶ cells / T25).

[0299] - Day 2. Maintain the cells in DMEM-F12 supplemented with 10% FBS at 37°C under a humidified 5% CO2 atmosphere.

[0300] - Day 3. Spread cells at a concentration of 20,000 cells / well (+ / - 2000 cells) in 96-well plates. Maintain the cells in DMEM-F12 medium supplemented with 10% FBS at 37°C under a humidified atmosphere of 5% CO2 for 24 hours.

[0301] - Day 4. Cells were incubated for 24 hours with different concentrations of the test compound (1000, 333.33, 111.11, 37.04, 12.37, 4.12, 1.37, and 0.46 nM) and 1 nM of Arg8-vasopressin dissolved in Opti-MEM. Opti-MEM containing the medium (water) was added to the unstimulated control wells, 1 nM of Arg8-vasopressin was added to the positive control wells, and 1 nM of Arg8-vasopressin containing 100 nM conivatan was added to the antagonistic control wells. This experiment was performed in at least triplicate.

[0302] - Day 5. Activation of the Nomad biosensor was quantified after cell fixation with formaldehyde (3.7 wt.%, 20 min). Cell nuclei were stained with DAPI (2 μg / ml), and fluorescence was measured using Thermofisher's Cell Insight High-Content Bioimager. For DAPI detection, filters of 380 / 10 and 460 / 10 nm were used for excitation and emission, respectively, and for Nomad biosensor detection, filters of 548 / 20 and 645 / 75 nm were used, respectively. Images were obtained at 20X objectives, with nine images taken per well. Cell quantification was performed by delineating target regions of the nucleus (stained with DAPI), and the average of three replicates was taken after quantification. Particle quantification was also performed using Thermofisher CellomicsScan Viewer 6.1.1. Two target regions, the nucleus and cytoplasm, were delineated using the Spot Detector application from Cell Software. This software application quantified the number of particles per nucleus and calculated the average of the particle number / cytoplasm per well. The three copies of the experiment were then averaged. Excel 2003 and Sigmaplot 9.0 were used for data management.

[0303] B. Result

[0304] like Figure 5 As shown, the V2R antagonistic peptides of the present invention (i.e., K39A peptide and MQ-LEAD peptide) are more effective cAMP production inhibitors compared to the parental MQ1 peptide (MQ-WT peptide). The V2R antagonistic peptides inhibit cAMP production in a dose-dependent manner under conditions activated by Arg 8 angiotensin.

[0305] The cAMP production inhibition properties of the V2R antagonistic peptide of the present invention are further illustrated in Table 3 below.

[0306] Table 3: Inhibition of cAMP production

[0307] MQ1("MQ-WT") 192 81 U-Da2a K39A 46.1 23 MQ LEAD 9.27 4.6

[0308] *Kinac: Inactivation constant: measures the ability of a tested peptide to prevent receptor activation, specifically to prevent Arg8 angiotensin-activated cAMP production.

[0309]

[0310]

[0311]

[0312]

[0313]

[0314]

[0315]

[0316]

[0317] sequence list <110> CEA <120> Angiotensin-2 receptor antagonist peptide and its uses <130> PR89992 <150> EP20305696 <151> 2020-06-24 <160> 25 <170> BiSSAP 1.3.2 <210> 1 <211> 57 <212> PRT <213> Artificial sequence <223> X1, X2, X3, X4, X5, and X6 are as defined. <220> <223> X1, X2, X3, X4, X5, and X6 are as defined. <220> <221> variants <222> 4 <223> X1 represents amino acid residues F or G. <220> <221> variants <222> 15 <223> X2 represents any amino acid residue, except for basic amino acids. <220> <221> variants <222> 27 <223> X3 represents amino acid residues T or D. <220> <221> variants <222> 48 <223> X4 represents amino acid residues I, L, or E. <220> <221> variants <222> 56..57 <223> (i) X5 represents V and X6 represents G or (ii) X5 represents G and X6 represents V. <400> 1 Arg Pro Ser Xaa Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Xaa Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Xaa Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Ala Gly Asn Ala Asn Arg Phe Ser Thr Xaa 35 40 45 Glu Lys Cys Arg Arg Thr Cys Xaa Xaa 50 55 <210> 2 <211> 57 <212> PRT <213> Artificial sequence <223> K39A <220> <223> K39A <400> 2 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Ala Gly Asn Ala Asn Arg Phe Ser Thr Ile 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 3 <211> 57 <212> PRT <213> Artificial sequence <223> K39A+I48L <220> <223> K39A+I48L <400> 3 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Ala Gly Asn Ala Asn Arg Phe Ser Thr Leu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 4 <211> 57 <212> PRT <213> Artificial sequence <223> N15A+K39A+I48L <220> <223> N15A+K39A+I48L <400> 4 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Ala Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Ala Gly Asn Ala Asn Arg Phe Ser Thr Leu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 5 <211> 57 <212> PRT <213> Artificial sequence <223> F4G K39A T27D <220> <223> F4G K39A T27D <400> 5 Arg Pro Ser Gly Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Asp Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Ala Gly Asn Ala Asn Arg Phe Ser Thr Ile 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 6 <211> 57 <212> PRT <213> Artificial sequence <223> MQ-LEAD F4G+K39A+T27D+I48E <220> <223> MQ-LEAD F4G+K39A+T27D+I48E <400> 6 Arg Pro Ser Gly Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Asp Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Ala Gly Asn Ala Asn Arg Phe Ser Thr Glu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 7 <211> 57 <212> PRT <213> Artificial sequence <223> WT MQ1 peptide <220> <223> WT MQ1 peptide <400> 7 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Lys Gly Asn Ala Asn Arg Phe Ser Thr Ile 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 8 <211> 57 <212> PRT <213> Artificial sequence <223> K10A <220> <223> K10A <400> 8 Arg Pro Ser Phe Cys Asn Leu Pro Val Ala Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Lys Gly Asn Ala Asn Arg Phe Ser Thr Leu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 9 <211> 57 <212> PRT <213> Artificial sequence <223> K10E <220> <223> K10E <400> 9 Arg Pro Ser Phe Cys Asn Leu Pro Val Glu Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Lys Gly Asn Ala Asn Arg Phe Ser Thr Leu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 10 <211> 57 <212> PRT <213> Artificial sequence <223> F17A <220> <223> F17A <400> 10 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Ala Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Lys Gly Asn Ala Asn Arg Phe Ser Thr Leu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 11 <211> 57 <212> PRT <213> Artificial sequence <223> F18A <220> <223> F18A <400> 11 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Ala Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Lys Gly Asn Ala Asn Arg Phe Ser Thr Leu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 12 <211> 57 <212> PRT <213> Artificial sequence <223> N41A <220> <223> N41A <400> 12 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Lys Gly Ala Ala Asn Arg Phe Ser Thr Ile 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 13 <211> 57 <212> PRT <213> Artificial sequence <223> R44A <220> <223> R44A <400> 13 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Lys Gly Asn Ala Asn Ala Phe Ser Thr Leu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 14 <211> 57 <212> PRT <213> Artificial sequence <223> R44E <220> <223> R44E <400> 14 Arg Pro Ser Phe Cys Asn Leu Pro Val Lys Pro Gly Pro Cys Asn Gly 1 5 10 15 Phe Phe Ser Ala Phe Tyr Tyr Ser Gln Lys Thr Asn Lys Cys His Ser 20 25 30 Phe Thr Tyr Gly Gly Cys Lys Gly Asn Ala Asn Glu Phe Ser Thr Leu 35 40 45 Glu Lys Cys Arg Arg Thr Cys Val Gly 50 55 <210> 15 <211> 9 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> chain <222> 1 <223> (7-Methoxycoumarin-4-yl)acetyl <220> <223> enzyme substrate <220> <221> chain <222> 9 <223> 2,4-Dinitrophenyl <400> 15 Arg Pro Pro Gly Phe Ser Ala Phe Lys 1 5 <210> 16 <211> 7 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> chain <222> 1 <223> (7-Methoxycoumarin-4-yl)acetyl <220> <223> enzyme substrate <220> <221> chain <222> 7 <223> 2,4-Dinitrophenyl <400> 16 Tyr Val Ala Asp Ala Pro Lys 1 5 <210> 17 <211> 12 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> chain <222> 1 <223> (7-Methoxycoumarin-4-yl)acetyl <220> <223> enzyme substrate <220> <221> NON_STD <222> 7 <223> X is N-3-(2,4-dinitrophenyl)-L-α,β-diaminopropionyl] <400> 17 Pro Leu Ala Gln Ala Val Xaa Arg Ser Ser Ser Arg 1 5 10 <210> 18 <211> 13 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> chain <222> 1 <223> (7-Methoxycoumarin-4-yl)acetyl <220> <223> enzyme substrate <220> <221> chain <222> 11 <223> 2,4-Dinitrophenyl <400> 18 Ser Glu Val Asn Leu Asp Ala Glu Phe Arg Lys Arg Arg 1 5 10 <210> 19 <211> 4 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> MOD_RES <222> 1 <223> Acetylation <220> <223> enzyme substrate <220> <221> chain <222> 4 <223> 7-Amino-4-methylcoumarin <400> 19 Leu Glu His Asp 1 <210> 20 <211> 4 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> MOD_RES <222> 1 <223> Acetylation <220> <223> enzyme substrate <220> <221> chain <222> 4 <223> 7-Amino-4-methylcoumarin <400> 20 Asp Glu Val Asp 1 <210> twenty one <211> 4 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> MOD_RES <222> 1 <223> Acetylation <220> <223> enzyme substrate <220> <221> chain <222> 4 <223> 7-Amino-4-methylcoumarin <400> twenty one Trp Glu His Asp 1 <210> twenty two <211> 7 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> chain <222> 1 <223> (7-Methoxycoumarin-4-yl)acetyl <220> <223> enzyme substrate <220> <221> NON_STD <222> 5 <223> [N-3-(2,4-dinitrophenyl)-L-α,β-diaminopropionyl group attached to 2,4-dinitrophenyl] <400> twenty two Pro Leu Gly Leu Xaa Ala Arg 1 5 <210> twenty three <211> 4 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> NON_STD <222> 1 <223> Succinyl <220> <223> enzyme substrate <220> <221> chain <222> 4 <223> 7-Amino-4-methylcoumarin <400> twenty three Ala Ala Pro Phe 1 <210> twenty four <211> 4 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> NON_STD <222> 1 <223> Methoxysuccinyl <220> <223> enzyme substrate <220> <221> chain <222> 4 <223> 7-Amino-4-methylcoumarin <400> twenty four Ala Ala Pro Val 1 <210> 25 <211> 10 <212> PRT <213> Artificial sequence <223> enzyme substrate <220> <221> chain <222> 1 <223> (7-Methoxycoumarin-4-yl)acetyl ` <220> <223> enzyme substrate <220> <221> NON_STD <222> 7 <223> X is pentylamine. <220> <221> chain <222> 10 <223> 2,4-Dinitrophenyl <400> 25 Arg Pro Lys Pro Val Glu Xaa Trp Arg Lys 1 5 10

Claims

1. A peptide comprising the amino acid sequence of SEQ ID NO. 1: in, - X1 represents amino acid residue F or G, - X2 represents amino acid residue N or A. - X3 represents amino acid residues T or D. - X4 represents amino acid residues I, L, or E. - X5 represents V and X6 represents G, and - The amino acid located at position 39 is A.

2. The peptide according to claim 1, wherein X1 represents the amino acid residue G.

3. The peptide according to claim 1, wherein X3 represents amino acid residue D.

4. The peptide according to claim 1, wherein X4 represents the amino acid residue E or L.

5. The peptide according to claim 1, comprising the following: The amino acid sequence selected from the group consisting of SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5 and SEQ ID NO.

6.

6. The peptide according to any one of claims 1 to 5, wherein it is labeled with a detectable molecular marker.

7. A nucleic acid encoding a peptide according to any one of claims 1 to 5.

8. The nucleic acid according to claim 7, wherein it is contained in an expression vector.

9. A pharmaceutical composition comprising a peptide as described in any one of claims 1 to 5 and a physiologically acceptable carrier.

10. Use of the peptide according to any one of claims 1 to 5 in the preparation of a medicament for treating hyponatremia or autosomal dominant polycystic kidney disease.

11. Use of the labeled peptide according to claim 6 in the preparation of a formulation for in vitro detection of cells expressing angiotensin-2 receptor.

12. Use of the labeled peptide according to claim 6 in the preparation of a formulation for in vitro detection of regulatory abnormalities in vasopressin-2 receptor cell expression, wherein the detection comprises the following steps: a) Provide the cells to be tested. b) Contact the cells provided in step a) with the labeled peptide. c) Measure the expression level of vasopressin-2 receptor in cells. d) Compare the expression level value obtained in step c) with the reference expression level value. e) Identify abnormalities in the regulation of angiotensin-2 receptor expression in cells.

Images